WO2023198201A1 - 适配体、缀合物与组合物及制备方法和用途 - Google Patents

适配体、缀合物与组合物及制备方法和用途 Download PDF

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WO2023198201A1
WO2023198201A1 PCT/CN2023/088466 CN2023088466W WO2023198201A1 WO 2023198201 A1 WO2023198201 A1 WO 2023198201A1 CN 2023088466 W CN2023088466 W CN 2023088466W WO 2023198201 A1 WO2023198201 A1 WO 2023198201A1
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group
alkyl
conjugate
groups
nucleotide sequence
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WO2023198201A9 (zh
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王珊
梁子才
张鸿雁
高山
胡欣
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苏州瑞博生物技术股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the present disclosure relates to an aptamer, as well as conjugates and pharmaceutical compositions containing a delivery group based on the aptamer.
  • the present disclosure also relates to preparation methods and uses of aptamers, conjugates and pharmaceutical compositions.
  • Tumors refer to new organisms formed by the proliferation of local tissue cells in the body under the action of various tumorigenic factors. Among them, the situation in which tumor cells metastasize and invade surrounding tissues is called malignant tumor. According to the classification of tissue cells from which tumors originate, they are generally divided into malignant tumors (cancer) produced by epithelial cells, malignant tumors produced by mesenchymal cells (sarcoma), malignant tumors produced by blood stem cells (leukemia, etc.), and glial cells. Malignant tumors (glioma) produced, etc. Among them, glioma is the most common primary intracranial malignant tumor, accounting for approximately 40%-50% of brain tumors. The global annual incidence rate is 3-8 cases per 100,000 people.
  • gliomas are neuroepithelial tumors, including a variety of pathological types, including but not limited to pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, glioblastoma tumors, oligodendroglioma, anaplastic oligodendroglioma, etc.
  • Aptamers, or nucleic acid aptamers are oligonucleotide molecules that can bind to a variety of target molecules, such as small molecule compounds, proteins, nucleic acids, and even cells, tissues, and organs. Aptamers can provide the important property of "recognition of specific molecules" and are therefore often used in biotechnology and therapy, similar to antibodies. Aptamers can be designed in test tubes and can be quickly synthesized using chemical methods. They also have the excellent properties of being easy to preserve and having low or no immunogenicity. Therefore, they have gradually attracted the attention of researchers in this field in recent years. However, aptamers suitable for tumor-targeted delivery still require further development and application in this field.
  • the inventors of the present disclosure unexpectedly discovered a method that can specifically target tumor cells, especially An aptamer for glioma cells, which shows high specificity for tumor cells, especially glioma cells, and thus can be effectively enriched in tumor cells, especially glioma cells. Furthermore, by covalently linking and conjugating the aptamer with various functional groups, the obtained conjugate can effectively deliver diagnostic and/or therapeutic agents suitable for various types of tumors to tumor cells. , especially glioma tissues and/or cells, show excellent diagnostic and/or therapeutic effects. Therefore, the inventor made the following invention:
  • the present disclosure provides an aptamer, comprising a continuous nucleotide sequence, and the group connecting two adjacent nucleotides is independently a phosphate group or a phosphate group with a modifying group.
  • each nucleotide is selected from one of modified or unmodified A, U, C or G, and the continuous nucleotide sequence has the sequence represented by the following formula (1):
  • T 1 is a motif composed of 1-3 nucleotides
  • T 2 is a motif composed of 0-15 nucleotides
  • T 2 does not contain a completely reverse complementary motif to T 1 ;
  • S 1 and S 4 are each a motif composed of 3-7 nucleotides. S 1 and S 4 are the same length and completely reverse complementary;
  • Na and Nc are each a motif consisting of 1-4 nucleotides, each nucleotide in Na is not complementary to every nucleotide in Nc , and in Na and Nc The total number of U accounts for more than 50% of the total number of nucleotides in N a and N c ;
  • S 2 and S 3 are each a motif composed of 1-4 nucleotides. S 2 and S 3 are the same length and completely reverse complementary;
  • N b is a motif composed of 3-6 nucleotides, and the nucleotides at both ends of N b do not form AU or GC complementarity.
  • the present disclosure also provides a conjugate comprising one or more delivery groups and one or more functional groups, the delivery group removing one hydrogen atom from the aptamer provided by the present disclosure.
  • a functional group is formed, each of the delivery groups is independently connected to the functional group through a covalent bond, or connected through a linking group, and each of the functional groups is independently a diagnostic agent group, One of a group consisting of a small molecule therapeutic agent group that has a therapeutic effect on tumors, especially gliomas, a functional oligonucleotide group that has a therapeutic effect on tumors, especially gliomas, and a delivery auxiliary group.
  • the present disclosure also provides a pharmaceutical composition comprising the conjugates and pharmaceutically acceptable carriers.
  • the present disclosure also provides the use of the aptamers and/or conjugates and/or pharmaceutical compositions provided by the present disclosure in the preparation of medicaments for the diagnosis and/or treatment of tumors and tumor-related diseases or symptoms. .
  • the present disclosure also provides a method for diagnosing and/or treating tumors and tumor-related diseases or symptoms, the method comprising administering to a subject in need an effective amount of a conjugate provided by the present disclosure and /or pharmaceutical compositions.
  • the present disclosure also provides a kit comprising the conjugates and/or pharmaceutical compositions provided by the present disclosure.
  • aptamer can be specifically delivered to various tumor cells, especially glioma tissues and cells.
  • the conjugates and pharmaceutical compositions provided by the present disclosure have excellent ability to target tumors, especially glioma tissues and cells, and can significantly improve the diagnostic accuracy and/or significantly treat or alleviate tumor-related diseases and/or symptoms. Tumor-related diseases and/or symptoms.
  • the present disclosure provides conjugates that can efficiently and specifically enter tumor cells in vitro.
  • the conjugates provided by the present disclosure all showed significantly higher average fluorescence intensity in U118MG glioma cells, and the R value basically remained above 2, indicating that the conjugates provided by the present disclosure provide
  • the conjugate has excellent ability to enter U118MG glioma cells.
  • the conjugate provided by the present disclosure has a strong ability to enter various cancer cells such as U118MG glioma cells, U251 human glioma cells, A549 human non-small cell lung cancer cells, and MCF-7 human breast cancer cells.
  • the conjugates provided by the present disclosure have a strong ability to enter the interior of U118MG and A549 tumor spheres.
  • conjugates provided by the disclosure of different lengths and sequences can effectively target U118MG glioma tissue.
  • the present disclosure provides aptamers capable of delivering various diagnostic agent groups, such as different fluorescent groups, to tumor tissue.
  • different conjugates containing fluorescent groups provided by the present disclosure can display strong fluorescence signals at the site of tumor inoculation in mice, indicating that the aptamer provided by the present disclosure can specifically target U118MG glioma;
  • the conjugate provided by the present disclosure still shows a strong fluorescence signal from 24h to 48h after administration, indicating that it can stably target glioma tissue for a longer period of time.
  • the conjugate provided by the present disclosure can still reach and target brain glioma when administered through the tail vein, suggesting that the aptamer provided by the present disclosure also has the ability to penetrate the blood-brain-barrier (BBB). ) and excellent ability to deliver diagnostic agent groups across the blood-brain barrier to gliomas.
  • BBB blood-brain-barrier
  • the conjugates provided by the present disclosure with various modifications can stably target U118MG glioma, A549 human non-small cell lung cancer tumors, and PAN02 pancreatic cancer tumor cells, showing a wide range of specificity for targeting tumors. ability.
  • the aptamer provided by the present disclosure can efficiently and specifically deliver different diagnostic agent groups (such as different fluorescent groups Cy3 or Cy5) to U118MG glioma, and can perform Remain stable over long periods of time.
  • diagnostic agent groups such as different fluorescent groups Cy3 or Cy5
  • the results show that the conjugate provided by the present disclosure can efficiently and specifically deliver the diagnostic agent group into the interior of the tumor cells of U118MG glioma, showing excellent targeting effect and Potential diagnostic capabilities.
  • the conjugates provided by the present disclosure can specifically deliver various small molecule drug groups, such as small molecule toxin groups, to tumor tissues and show excellent tumor inhibitory effects.
  • the conjugate provided by the present disclosure can effectively deliver MMAE to tumor tissue. While showing tumor targeting ability, it also reduces the risk of toxicity caused by the distribution of MMAE molecules in other tissues, and various administration methods All can effectively inhibit the increase rate of tumor volume and tumor weight, indicating that the conjugate of the present disclosure can effectively inhibit tumor proliferation.
  • further increasing the dosage of the conjugate resulted in almost no increase in tumor volume during the test period, showing a more excellent anti-tumor effect.
  • the conjugates provided by the present disclosure can specifically deliver various functional oligonucleotide groups, such as siRNA groups, to tumor tissues and show excellent tumor cell viability inhibition effects.
  • the aptamer provided by the present disclosure can not only efficiently target tumor cells and inhibit the content of target mRNA, but also does not significantly affect the corresponding expression in normal cells. mRNA level, with excellent targeted delivery efficiency and high safety.
  • various conjugates containing siRNA groups of the present disclosure can effectively target and enrich U118MG tumor tissue at different times after administration.
  • the conjugate of the present disclosure can be freely taken up into PANC1 human pancreatic cancer cells in in vitro experiments, and shows a significant inhibitory effect on hSTAT3 mRNA.
  • laser confocal imaging and imaging results in high-content imaging systems show that the conjugate containing siRNA groups of the present disclosure can effectively enter U118MG tumor cells, thereby facilitating the efficient production of RNAi in tumor cells.
  • the inventors provided by the present disclosure unexpectedly discovered that the conjugates and/or pharmaceutical compositions provided by the present disclosure can efficiently pass through the blood-brain barrier and can be targeted to the brain in the case of systemic administration. into tumors, thereby further improving the delivery efficiency of functional groups, saving costs and reducing undesirable side reactions.
  • the conjugate provided by the present disclosure can cross the blood-brain barrier under subcutaneous administration, effectively target into orthotopic U118MG glioma, and significantly inhibit the increase in tumor volume or even reduce it to 1/10 of the initial volume.
  • the conjugate of the present disclosure can effectively penetrate the blood-brain barrier and efficiently target into brain glioma, and has a good tumor growth inhibition effect, showing It shows good treatment compliance and the ability to effectively suppress tumors.
  • the aptamer provided by the present disclosure has excellent ability to target tumors, especially glioma tissues and cells, and includes the aptamer provided by the present disclosure and a diagnostic agent group (such as a fluorescent developer group)
  • the conjugate can significantly improve the success rate of tumor diagnosis, and the conjugate containing the aptamer and therapeutic agent group provided by the present disclosure can significantly and effectively inhibit tumor growth, or reduce the expression level of cancer-related genes in tumor cells, It has good application prospects.
  • Figures 1A and 1B are high-content imaging images showing the entry of AP1 and contrasting AP10 into U118MG glioma cells and SVGp12 normal astrocytes, respectively.
  • Figures 1C-1G respectively show the high-content imaging of AP1 and contrast AP10 entering U251 human glioma cells, A549 human non-small cell lung cancer cells, MCF-7 human breast cancer cells and 293T human kidney epithelial cells.
  • Figures 2A and 2B show high-content imaging of AP1 and contrast AP10 entering U118MG glioma tumor spheres and A549 human non-small cell lung cancer tumor spheres, respectively.
  • Figures 3A-3D respectively show the results of different administrations at 1h, 4h, 24h and 48h after administration. Pictures of in vivo imaging and tumor tissue imaging of the U118MG subcutaneous tumor model mouse after conjugation.
  • Figures 4A and 4B respectively show fluorescence imaging images of the brain tissue of mice establishing the U118MG orthotopic tumor model after administration of different conjugates at 24h and 48h after administration.
  • Figures 5A-5E respectively show fluorescence imaging in mice at different time points after administration of different sequences of conjugates and comparative conjugates provided by the present disclosure
  • Figures 5F-5H respectively show tumor tissue imaging at different time points. picture.
  • Figure 6 is a diagram showing the fluorescence image of the organs of one mouse in each group 1 hour after administration of the conjugate.
  • Figure 7 is a diagram showing fluorescence imaging of various organs and tissues in mice establishing a PAN02 subcutaneous tumor model after administration of the conjugate provided by the present disclosure.
  • Figures 8A-8E are diagrams respectively showing fluorescence images of organs of mice administered the conjugate provided by the present disclosure at 48h or 96h after administration.
  • Figure 9 is a graph showing tumor fluorescence images of mice in each group on D9 after administration of the conjugate.
  • Figure 10 is a line graph showing changes in tumor volume over time in each group of mice after administration of conjugates or control compounds provided by the present disclosure.
  • Figures 11A and 11B are graphs showing the effects of different concentrations of conjugate AP1 and conjugate 19 on the cell viability of U118-MG glioma cells and SVGp12 human astrocytes, respectively.
  • Figure 12 is a graph showing fluorescence imaging results in mice after administration of conjugates provided by the present disclosure containing different linking groups.
  • Figures 13A-13C are graphs respectively showing the fluorescence imaging results in mice at 1 hour, 24 hours and 48 hours after administration of different conjugates.
  • Figure 13D is a diagram showing fluorescence signal imaging of tumor tissues and kidneys of mice in each group after the mice were sacrificed on D5.
  • Figures 14A-14C are graphs respectively showing the fluorescence imaging results in mice at 1 hour, 24 hours and 48 hours after administration of different conjugates.
  • Figure 14D is a diagram showing fluorescence signal imaging of tumor tissues and kidneys of mice in each group after the mice were sacrificed on D6.
  • Figure 15 is a graph showing laser confocal imaging results within U118MG glioma when different conjugates were administered for 24 hours.
  • 16A and 16B are graphs showing fluorescence imaging results in mice after administration of different conjugates.
  • Figure 17 is a graph showing the effect of conjugates of the present disclosure on PANC-1 human pancreatic cancer cells. Bar graph of relative inhibition rates of hSTAT3 mRNA.
  • Figure 18 is a graph showing laser confocal imaging results of conjugates of the present disclosure in U118MG glioma cells.
  • Figure 19 is a bar graph showing relative fluorescence intensity results for conjugates of the present disclosure within U118MG glioma.
  • Figure 20 is a line graph showing changes in tumor fluorescence intensity over time in U118MG orthotopic tumor model mice after administration of conjugates or control compounds provided by the present disclosure.
  • Figure 21 is a line graph showing changes in tumor volume over time in U118MG subcutaneous tumor model mice after administration of conjugates or control compounds provided by the present disclosure.
  • Figure 22 is a line graph showing changes in tumor volume over time in U118MG subcutaneous tumor model mice after administration of different concentrations of conjugates or control compounds provided by the present disclosure.
  • Figure 23 is a line graph showing changes in tumor volume over time in U118MG subcutaneous tumor model mice after administration of different concentrations of conjugates or control compounds provided by the present disclosure.
  • Figure 24 is a line graph showing the changes in tumor volume over time in A549 subcutaneous tumor model mice after administration of different concentrations of conjugates or control compounds provided by the present disclosure.
  • A, U, C, G and T refer to adenine nucleotides, uracil nucleotides, cytosine nucleotides, guanine nucleotides and thymine nucleotides, respectively.
  • 2-Methylcytosine nucleotide refers to a nucleotide in which the hydrogen at the 2' position of the cytosine base in the cytosine nucleotide is replaced by a methyl group. The structure of these nucleotides is well known to those skilled in the art.
  • nucleic acid motif refers to a fragment of a nucleic acid sequence in an aptamer, consisting of 1 or more nucleotides.
  • a motif is a fragment of a nucleic acid sequence that has a biological function.
  • alkyl refers to straight and branched chain saturated hydrocarbon radicals having a specified number of carbon atoms, typically from 1 to 20 carbon atoms, such as from 1 to 10 carbon atoms, Such as 1 to 8 or 1 to 6 carbon atoms.
  • C 1 -C 6 alkyl refers to straight and branched chain alkyl groups containing 1 to 6 carbon atoms.
  • alkyl residue having a particular number of carbons it is intended to encompass all branched and straight chain forms having that number of carbons; thus, for example, "butyl” is meant to include n-butyl, sec-butyl , isobutyl and tert-butyl; “propyl” includes n-propyl and isopropyl.
  • Alkylene is a subset of alkyl and refers to the same residue as alkyl but with two points of attachment.
  • alkenyl refers to an unsaturated branched or linear alkyl group having one or more carbon-carbon double bonds formed by adjacent atoms from the parent alkyl group. Obtained by removing a molecule of hydrogen from a carbon atom. The group can be in the cis or trans configuration of the double bond.
  • alkenyl groups include, but are not limited to: vinyl; propenyl, such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl base), prop-2-en-2-yl; butenyl, such as but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-1-en-1-yl base, but-2-en-1-yl, but-2-en-2-yl, but-1,3-dien-1-yl, but-1,3-dien-2-yl, etc.
  • alkenyl groups have 2 to 20 carbon atoms, and in other embodiments 2 to 10, 2 to 8, or 2 to 6 carbon atoms.
  • Alkenylene is a subset of alkenyl and refers to the same residue as alkenyl but with two points of attachment.
  • alkynyl refers to an unsaturated branched or linear alkyl group having one or more carbon-carbon triple bonds formed by adjacent atoms from the parent alkyl group. Obtained by removing two molecules of hydrogen from a carbon atom.
  • Typical alkynyl groups include, but are not limited to: ethynyl; propynyl, such as prop-1-yn-1-yl, prop-2-yn-1-yl; butynyl, such as but-1-yn- 1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.
  • alkynyl groups have 2 to 20 carbon atoms, while in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbon atoms.
  • Alkynylene is a subset of alkynyl and refers to the same residue as alkynyl but with two points of attachment.
  • heterocyclyl refers to a stable 3- to 18-membered non-aromatic cyclic group containing 2-12 carbon atoms and 1-6 heteroatoms selected from nitrogen , oxygen and sulfur. Unless otherwise stated in the specification, heterocyclyl is a monocyclic, bicyclic, tricyclic or tetracyclic ring system and may include fused or bridged ring systems. The heteroatoms in the heterocyclyl group may be oxidized heteroatoms. One or more nitrogen atoms, if present, may be quaternized nitrogen atoms. Heterocyclyl groups are partially or fully saturated. A heterocyclyl group can be attached to the rest of the molecule through any ring atom.
  • heterocyclyl groups include, but are not limited to: dioxanyl, thienyl[1,3]dithianyl, decahydroisoquinolinyl, imidazolinyl, imidazolidine base, isothiazolidine base, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxapiperazinyl, 2-oxapiperidinyl, 2-oxapyrrolidinyl, oxa Azolidinyl, piperidinyl, piperazinyl, 4-piperidinonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, trithianyl, tetrahydropyridyl Pyryl, thiomorpholinyl, thiamorpholinyl, 1-oxxany
  • aryl refers to a group derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removal of a hydrogen atom from a ring carbon atom.
  • the aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and 6 to 18 carbon atoms of carbon, wherein one or more rings in the ring system are completely unsaturated, i.e., contain according to Hückel's theory Cyclic, delocalized (4n+2) ⁇ -electron system.
  • Aryl groups include, but are not limited to, phenyl, fluorenyl, and naphthyl groups.
  • Arylene is a subset of aryl and refers to the same residue as aryl but with two points of attachment.
  • Heteroaryl refers to a group derived from a 3- to 18-membered aromatic ring free radical, containing 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur.
  • a heteroaryl group may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, wherein one or more rings in the ring system are fully unsaturated, i.e., contain cyclic ionization according to Hückel's theory. Domain (4n+2) ⁇ -electron system.
  • Heteroaryl groups include fused or bridged ring systems. The heteroatoms in the heteroaryl group may be oxidized heteroatoms.
  • heteroaryl group is attached to the rest of the molecule through any ring atom.
  • heteroaryl groups include, but are not limited to: azepantrienyl, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzobisoxazolyl, benzofuranyl, benzene Oxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[ b][1,4]oxazinyl (benzo[b][1,4]oxazinyl), 1,4-benzodioxanyl (1,4-benzodioxanyl), benzonaphthofuranyl, benzo Oxazolyl, benzodioxolyl (benzodioxolyl), benzodioxin
  • the present disclosure provides an aptamer, comprising a continuous nucleotide sequence, and the group connecting two adjacent nucleotides is independently a phosphate group or a phosphate group with a modifying group.
  • each nucleotide is selected from one of modified or unmodified A, U, C or G, and the continuous nucleotide sequence has a sequence represented by formula (1):
  • T 1 is a motif consisting of 1-3 nucleotides. The inventors found that the presence of T 1 is beneficial to the aptamer provided by the present disclosure showing high tumor targeting effect.
  • T 1 consists of 2 nucleotides. In this case, the aptamer provided by the present disclosure has more excellent tumor targeting ability.
  • T1 consists of 2 nucleotides and contains at least one C. In some embodiments, in the 5'-3' direction, T 1 is CU, UC, or AC.
  • T 2 is a motif consisting of 0-15 nucleotides. The inventors found that T2 with these nucleotide numbers and various nucleotide sequences did not significantly affect the tumor targeting ability of the aptamer.
  • T2 consists of 0-10 nucleotides. In some embodiments, according to the 5'-3' direction, T2 consists of 1-9 nucleotides starting from U. In this case, the aptamer may have better stability.
  • T 2 does not contain a motif that is completely reverse complementary to T 1 .
  • reverse complementarity refers to the formation of hydrogen bonds between two nucleotide sequences or motifs according to the rules of nucleic acid base pairing, and a nucleotide sequence or motif according to the 5' Each nucleotide in the -3' direction can form base pairing with each nucleotide in the 3'-5' direction of the nucleotide sequence or motif at the other end.
  • "reverse complement” includes one or more of AU, GC, and UG complement.
  • S 1 and S 4 are each a motif composed of 3-7 nucleotides, and S 1 and S 4 are the same length and completely reverse complementary. Aptamers with the above S 1 and S 4 motifs have better stability and can target tumor tissues and cells for a longer period of time.
  • S 1 and S 4 each consist of 3-5 nucleotides and are the same length.
  • GC complementation accounts for more than 40% of the total complementation amount. At this time, the aptamer provided by the present disclosure has further better stability and tumor targeting. ability.
  • S 1 is GCU and S 4 is AGC, or S 1 is GAGU and S 4 is GCUC, or S 1 is GGAGU and S 4 is GCUCU, or S 1 is UAUGG and S 4 is CCAUG.
  • Na and Nc are each a motif composed of 1-4 nucleotides, and each nucleotide in Na is different from each nucleotide in Nc .
  • the total number of Us in Na and N c accounts for more than 50% of the total number of nucleotides in Na and N c .
  • Aptamers with the above Na and N c motifs show excellent tumor tissue targeting ability. When the Na and /or N c motifs are missing, or the total number of Us in Na and N c is insufficient, Aptamers basically do not show tumor tissue targeting effect.
  • the sum of the numbers of nucleotides in Na and Nc is an integer from 2 to 4.
  • the sum of the number of nucleotides in Na and Nc is 3 or 4, and the sum of the number of Us in Na and Nc is 2 or 3.
  • Na and/or Nc are U, UU, UC, or CU in the 5'-3' direction.
  • S 2 and S 3 are each a motif composed of 1-4 nucleotides, and S 2 and S 3 have the same length and are completely reverse complementary.
  • the aptamer provided by the present disclosure shows good stability and excellent tumor targeting ability.
  • S2 and S3 each consist of 2-3 nucleotides and are the same length.
  • the reverse complement formed by S 2 and S 3 includes at least one GC complement, in which case the reverse complement has better stability.
  • S2 is CA and S3 is UG, or S2 is AC and S3 is GU, or S2 is GCC and S3 is GGU.
  • N b is a motif consisting of 3-6 nucleotides, and the nucleotides at both ends of N b are not AU or GC complementary.
  • aptamers with the above-mentioned N b motif can maintain a specific configuration in space, thereby enabling the aptamers provided by the present disclosure to target tumor tissues and cells stably and efficiently.
  • Nb consists of 4-5 nucleotides.
  • N b in the 5'-3' direction, N b is GACG, GACGU, GACCG, UACU, GUUG, or GAUCU.
  • the inventor of the present disclosure unexpectedly discovered that the aptamer provided by the present disclosure having the sequence represented by the above formula (1) can effectively target tumors, especially glioma tissue, thereby allowing the aptamer provided by the present disclosure to be used as Delivery carriers deliver functional groups with diagnostic/therapeutic effects on tumors to tumors. Furthermore, the aptamers provided by the present disclosure can specifically enter tumor cells, thereby more effectively delivering diagnostic/therapeutic agent groups at the cellular level or even the genetic level.
  • the length of the continuous nucleotide sequence is 18-50 nucleotides, or 20-40 nucleotides, or 21-36 nucleosides. acid, or 24-32 nucleotides. Aptamers with these contiguous nucleotide sequence lengths can be more easily targeted to tumors and have a good balance between synthesis cost and targeting effectiveness.
  • the continuous nucleotide sequence has the sequence shown in the following SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3:
  • T 1 is CU
  • S 1 is GCU
  • Na is U
  • S 2 is CA
  • N b GACG
  • S 3 is UG
  • N c is UU
  • S 4 is AGC
  • T 2 is UU;
  • T 1 is CU
  • S 1 is GAGU
  • Na is U
  • S 2 is CA
  • N b is GACG
  • S 3 is UG
  • N c is UU
  • S 4 is GCUC
  • T 2 is U;
  • T 1 is CU
  • S 1 is GAGU
  • Na is U
  • S 2 is CA
  • N b is GACG
  • S 3 is UG
  • N c is UU
  • S 4 is GCUC
  • T 2 is U.
  • the continuous nucleotide sequence has the nucleotide sequence shown in SEQ ID NO: 4:
  • N 1 , N 2 and N 3 are each independently one of A, U, C and G, and N 4 is U, C or G or a motif composed of two of U, C or G; N 5 is U, CU or UU; N 6 is CU, UC or AC; N 7 is U, UU or UUN 8 , and N 8 is a motif composed of 1-15 nucleotides.
  • T 1 is the motif represented by N 6
  • S 1 is the motif represented by GGAGU
  • Na is U
  • S 2 is CA
  • N b is N 1
  • N 2 , N 3 and N 4 constitute the motif N 1 N 2 N 3 N 4
  • S 3 is UG
  • N c is the motif represented by N 5
  • S 4 is GCUC and the first core in N 7
  • T 2 is a motif composed of the remaining nucleotides in N 7 .
  • the aptamer containing the nucleotide sequence shown in SEQ ID NO: 4 above can more effectively target tumors, especially gliomas, and be enriched in tumor tissues.
  • the motif N 1 N 2 N 3 N 4 composed of N 1 , N 2 , N 3 and N 4 is one of GACG, GACGU, GACCG, UACU, GUUG or GAUCU, including these motifs
  • the aptamer has higher tumor-specific targeting effect.
  • N 5 is U or UU.
  • the aptamers provided by the present disclosure all have excellent targeting effects on tumors.
  • the continuous nucleotide sequence has the nucleotide sequence shown in any one of SEQ ID Nos: 5-11:
  • the aptamer provided by the present disclosure having the above nucleotide sequence shows a high targeting effect on tumors.
  • motif N8 consists of 1-15 nucleotides. In some embodiments, N8 consists of 1-8 nucleotides.
  • the presence of the motif N 8 makes the aptamer provided by the present disclosure more stable to exonucleases in the body, thereby enabling it to exert its tumor-targeting effect for a longer period of time in the body.
  • N 8 can increase or maintain the tumor targeting effect of the aptamers provided by the present disclosure.
  • the motif N 8 consists of 8 nucleotides.
  • the nucleotide sequence of motif N 8 is CCGAUCUC in the 5'-3' direction.
  • the contiguous nucleotide sequence has the nucleotide sequence shown in any one of SEQ ID NOs: 12-14:
  • the end groups of the ribose 5' end of the 5' end nucleotide and the ribose 3' end of the 3' end nucleotide are independently hydroxyl or phosphate groups, and the selection of these end groups does not matter. would alter the targeting ability of the aptamers provided by the present disclosure.
  • the end groups of the ribose 5' end of the 5' terminal nucleotide and the ribose 3' end of the 3' terminal nucleotide are both hydroxyl groups.
  • each nucleotide can be a modified or unmodified nucleotide.
  • modification of nucleotides may alter the stability of the aptamers provided by the present disclosure and/or the ability to target tumors.
  • at least one nucleotide in the aptamer provided by the present disclosure is a modified nucleotide.
  • at least one of the groups connecting two adjacent nucleotides in the aptamer provided by the present disclosure has a phosphate group of a modifying group.
  • each of the modified nucleotides is independently a 2'-halogen modified nucleotide, a 2'-alkoxy modified nucleotide, 2′-alkyl modified nucleotides, 2′-substituted alkyl modified nucleotides, 2′-amino modified nucleotides, 2′-substituted amino modified nucleotides, 2′ - One of deoxynucleotides, base-modified nucleotides and nucleotide analogs.
  • fluoro-modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribosyl group of the nucleotide is substituted with fluorine and has a structure represented by the following formula (7).
  • Non-fluorinated modified nucleotides refers to nucleotides or nucleotide analogs in which the hydroxyl group at the 2' position of the ribosyl group of the nucleotide is replaced by a non-fluorinated group.
  • each non-fluorinated modified nucleotide is independently selected from nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2' position of the ribosyl group of the nucleotide with a non-fluorinated group. A sort of.
  • Nucleotides formed by replacing the hydroxyl group at the 2' position of the ribosyl group with a non-fluorine group are well known to those skilled in the art. These nucleotides can be selected from 2'-alkoxy modified nucleotides, 2'- Alkyl-modified nucleotides, 2′-substituted alkyl-modified nucleotides, 2′-amino-modified nucleotides, 2′-substituted amino-modified nucleotides, 2′-deoxynucleoside One of the glycosides.
  • the 2'-alkoxy modified nucleotide is a methoxy-modified nucleotide (2'-OMe), as shown in formula (8).
  • the 2'-amino modified nucleotide (2'- NH2 ) is represented by formula (9).
  • the 2'-deoxynucleotide (DNA) is represented by formula (10):
  • base modification includes, but is not limited to, adding one or more methyl groups to the base.
  • thymine (T) is considered a type of base-modified uracil (U).
  • 2-methylcytosine is considered a type of base-modified cytosine (C).
  • Nucleotide analogues are those that can replace nucleotides in nucleic acids, but are structurally different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides or thymine deoxyribonucleotides Ribonucleotide group.
  • nucleotide analogs may be isonucleotides, bridged nucleic acid (BNA), or acyclic nucleotides.
  • BNA refers to constrained or inaccessible nucleotides.
  • BNA may contain a five-membered ring, a six-membered ring, or a seven-membered ring bridged structure with a "fixed"C3'-endoglycocondensation. The bridge is typically incorporated into the 2',4'-position of the ribose sugar to provide a 2',4'-BNA nucleotide.
  • BNA can be LNA, ENA, cET BNA, etc., wherein LNA is represented by formula (12), ENA is represented by formula (13), and cET BNA is represented by formula (14):
  • Acyclic nucleotides are a type of nucleotide formed by opening the sugar ring of the nucleotide.
  • the acyclic nucleotide can be unlocked nucleic acid (UNA), glycerol nucleic acid (GNA) or peptide nucleic acid (PNA), wherein UNA is represented by formula (15) and GNA is represented by formula (16) :
  • R is selected from H, OH or alkoxy (O-alkyl).
  • Peptide nucleic acids are a class of nucleotide analogs formed by replacing the glycoside-phosphate backbone with a polypeptide backbone.
  • the peptide nucleic acid may be, for example, a nucleotide analog formed by substituting a 2- aminoethylglycine bond for a glycoside-phosphate unit.
  • Isonucleotides are compounds formed by changing the positions of the bases on the ribose ring in nucleotides.
  • the isonucleotide can be a compound formed by moving the base from the 1′-position of the ribose ring to the 2′-position or the 3′-position, as shown in formula (17) or (18).
  • Base represents a nucleic acid base, such as A, U, G, C or T; R is selected from H, OH, F or a non-fluorine group as described above.
  • the nucleotide analog is selected from one of isonucleotides, LNA, ENA, cET, UNA, and GNA.
  • each non-fluoro-modified nucleotide is a methoxy-modified nucleotide, above and below, the methoxy-modified nucleotide refers to the 2' of the ribosyl group -Nucleotides formed by replacing the hydroxyl group with a methoxy group.
  • each cytosine nucleotide in the continuous nucleotide sequence in the aptamer provided by the present disclosure is a fluoro-modified cytosine nucleotide, and/or the Each uracil nucleotide in the contiguous nucleotide sequence is a fluorinated modified uracil nucleotide.
  • each nucleotide in the contiguous nucleotide sequence in the aptamer provided by the present disclosure is a 2'-methoxy modified nucleotide.
  • one or more uracil nucleotides in the aptamers provided by the present disclosure have modified bases. .
  • the group connecting two adjacent nucleotides can be a phosphate group or a modified phosphate group.
  • the modification of the phosphate group is, for example, replacing at least one non-bridging oxygen atom in the phosphate group with a sulfur atom to form a phosphorothioate group or a phosphorodithioate group.
  • at least one group connecting two adjacent nucleotides in the aptamer provided by the present disclosure is a phosphorothioate group.
  • at least one of the three groups connecting two adjacent nucleotides between the first four nucleotides at the 5' end of the aptamer provided by the present disclosure is a phosphorothioate. base.
  • At least 2 of the 3 groups connecting two adjacent nucleotides between the first four nucleotides at the 5' end of the aptamer provided by the present disclosure are phosphorothioates. base. In some embodiments, at least one of the groups connecting two adjacent nucleotides between the first four nucleotides at the 3' end of the aptamer provided by the present disclosure is a phosphorothioate group. In some embodiments, at least 2 of the 3 groups connecting two adjacent nucleotides between the first four nucleotides at the 5' end of the aptamer provided by the present disclosure are phosphorothioates. base. In some embodiments, each group connecting two adjacent nucleotides in the aptamer provided by the present disclosure is a phosphorothioate group.
  • Aptamers with the above modifications are not only low in cost, but also make it difficult for ribonucleases in the body to cleave the aptamers, thereby increasing the stability of the aptamers and making them more resistant to nuclease hydrolysis.
  • the above-mentioned modified aptamers have higher activity in targeting tumor tissues and/or cells.
  • the contiguous nucleotide sequence has a nucleotide sequence set forth in one of SEQ ID NOs: 15-39:
  • the capital letters C, G, U, and A represent the base composition of nucleotides;
  • the small letter m means that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide;
  • the small letter f means The adjacent nucleotide to the left of the letter f is a fluorinated modified nucleotide;
  • the lowercase letter s indicates that the two nucleotides to the left and right of the letter s are connected by a phosphorothioate group.
  • the aptamers provided by the present disclosure can be obtained by conventional oligonucleotide preparation methods in the art (such as solid phase synthesis and liquid phase synthesis methods). Among them, solid-phase synthesis already has commercial customization services. Modified nucleotide groups can be introduced into the aptamers provided by the present disclosure by using nucleoside monomers with corresponding modifications, methods of preparing nucleoside monomers with corresponding modifications, and modifying nucleotide groups. Methods for introducing aptamers are also well known to those skilled in the art. All modified nucleoside monomers are commercially available or prepared using known methods.
  • the present disclosure also provides a conjugate comprising one or more delivery groups and one or more functional groups, the delivery group removing one hydrogen atom from the aptamer provided by the present disclosure.
  • a functional group is formed, each of the delivery groups is independently connected to the functional group through a covalent bond, or connected through a linking group, and each of the functional groups is independently a diagnostic agent group, It is one of a small molecule therapeutic agent group that has a therapeutic effect on tumors, a functional oligonucleotide group that has a therapeutic effect on tumors, and a delivery auxiliary group.
  • the aptamers provided by the present disclosure are capable of conjugated delivery of functional groups to tumors.
  • the delivery group is formed by the removal of one or more hydrogen atoms or functional groups by the aptamers provided by the present disclosure.
  • the ribose 5′ group of the 5′ terminal nucleotide and the ribose 3′ group of the 3′ terminal nucleotide of the aptamer provided by the present disclosure are both hydroxyl groups, and the delivery group is provided by the present disclosure.
  • the aptamer is formed by removing a hydrogen atom from the 5' hydroxyl group of the 5' terminal nucleotide; in some embodiments, the delivery group is removed from the 3' hydroxyl group of the 3' terminal nucleotide by an aptamer provided by the present disclosure a hydrogen source In some embodiments, the delivery group is formed by removing the 5' hydroxyl group in the 5' terminal nucleotide by the aptamer provided by the present disclosure; in some embodiments, the delivery group is formed by the aptamer provided by the present disclosure in Formed by removing the 3' hydroxyl group from the 3' terminal nucleotide. In some embodiments, the delivery group is formed by the aptamer provided by the present disclosure removing the ribose 2'-hydroxyl group from the nucleotide it contains.
  • the present disclosure provides a conjugate having a structure represented by formula (101):
  • each R AP group is independently a group having a structure shown in formula (102):
  • each AP group is the same or different and independently represents one of the delivery groups; each A 0 group is the same or different and independently represents one of the functional groups; R j , each R k Or each R i is the same or different, each independently represents a covalent bond or a connecting group, and both R i and R k are different at the same time as a covalent bond; m 0 is an integer from 1 to 6; n 0 is 1- An integer of 6, each n 1 independently represents an integer of 0-4; Indicates the site at which a group is covalently attached.
  • m 0 is an integer of 1-6, that is, the conjugate represented by formula (101) contains 1-6 functional groups A 0 .
  • m 0 is an integer of 1-4, that is, the conjugate represented by formula (101) contains 1-4 of the functional groups A 0 .
  • m 0 is 1, that is, the conjugate represented by formula (101) contains 1 functional group A 0 .
  • n 0 is an integer from 1 to 6, that is, the conjugate represented by formula (101) contains 1 to 6 R AP groups. From the perspective of delivery efficiency and cost, in some embodiments, n 0 is an integer of 1-3, that is, the conjugate represented by formula (101) contains 1-3 R AP groups. In some embodiments, n 0 is 1, that is, the conjugate represented by formula (101) contains 1 R AP group.
  • each n 1 independently represents an integer from 0 to 4, and both R i and R k are not covalently bonded at the same time. Therefore, each R AP group contains 1 to 5 of the present invention.
  • publicly available The aptamer derives the delivery group AP.
  • each n 1 independently represents an integer from 0 to 1, such that each R AP group contains 1 to 2 delivery groups AP.
  • n 0 is 1 and n 1 is 0. In this case, the conjugate represented by formula (101) contains 1 delivery group AP.
  • the function of R k and R i is to covalently connect the delivery group AP to the R j group and to the functional group A 0 via the R j group. Therefore, any R k or R i that can achieve the above connection without negatively affecting the effects of the delivery group AP and the functional group A 0 can be used in the present invention.
  • each n 1 is 0, and each R i is independently a covalent bond, or one of the following linking groups
  • One or more connection combinations C 1 -C 20 alkylene, phosphate bond, phosphorothioate bond, amide bond, ester bond, ether bond, thioether bond, disulfide bond, 1, 2, 3- Triazole subunit, polyethylene glycol subunit, pyrrolidine subunit, 2-oxopyrrolidine subunit, phenylene, cyclohexylene, 2-succinimide subunit, 2-thiobutanediyl Imide subunit, amino acid subunit, Nucleotide subunits.
  • the linking group Rj includes linking groups known to those skilled in the art to be useful in antibody drug conjugates.
  • the linking group R j may be cleavable or non-cleavable.
  • linking group Rj may be cleavable.
  • "cleavable" means that upon targeting of the conjugate of the present disclosure to a tumor, the linker group Rj undergoes covalent cleavage in the intratumoral environment and/or within the tumor cells, releasing a separate function Sexual groups produce therapeutic or diagnostic effects.
  • the linker R j comprises an activating enzyme linker, a sulfatase-cleavable linker, a galactose-cleavable linker, a lysosomal protease-sensitive linker, a peptidyl linker group, a glucuronide linking group, an acid-sensitive cleavable linking group, or a glutathione-sensitive disulfide linking group.
  • linking group R j comprises a peptidyl linking group.
  • the peptidyl linking group is selected from the group consisting of valine-citrulline dipeptide linker (Val-Cit), alanine-alanine dipeptide linker (Ala-Ala), valine - One or more of the alanine dipeptide linker (Val-Ala) and the glycine-glycine-phenylalanine-glycine tetrapeptide linker (Gly-Gly-Phc-Gly).
  • linking group R j is selected from N-succinimide 4-(2-dithiopyridine)butyrate (SPDB), N-succinimide-4-(2-thiopyridine) Subunit) valerate (SPP), (S)-2-((S)-2-amino-3-methylbutanamide)-5-ureidopentanoic acid (Val-Cit-PAB-OH), N -Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) or 2-(phosphate-(CH 2 ) 6 -S-)- One of the maleimidocaproyl-valine-citrulline-p-aminobenzyl subunits.
  • SPDB N-succinimide 4-(2-dithiopyridine)butyrate
  • SPP N-succinimide-4-(2-thiopyridine) Subunit) valerate
  • SPP S)-2-((S)-2-amino
  • the linking group R j comprises a linking group enumerated in McKertish CM, Kayser V. Advances and Limitations of Antibody Drug Conjugates for Cancer. Biomedicines. 2021 Jul 23;9(8):872., cite The entire content of this document is incorporated into this article in its entirety.
  • the linking group R j includes a valine-citrulline dipeptide linker (Val-Cit), a polyethylene glycol subunit, an iminohexyl subunit, an N-succinimidyl group, One or more of the GAU trinucleotide linking groups.
  • each R i is independently a covalent bond, a disulfide bond, a dodecylene group, a valine-citrulline dipeptide linker (Val-Cit), a polyethylene glycol subunit , iminohexyl subunit, N-succinimidyl group or GAU trinucleotide subunit or a connection combination of two.
  • the function of the R j group is to connect the R AP group with the functional group A 0 , thereby through the R AP
  • the tumor targeting effect of the delivery group AP in the group specifically delivers the functional group A 0 to tumor tissues and/or cells. Therefore, any R j group that can achieve the above connection without affecting the tumor targeting effect of the delivery group AP and the effect of the functional group A 0 can achieve the purpose of the present invention and solve the technology to be solved by the present invention. question.
  • the conjugate represented by formula (101) after the conjugate represented by formula (101) reaches the tumor tissue and/or enters the tumor cell, the R j is cleaved, releasing the pharmaceutically active molecule corresponding to the separate functional group A 0 .
  • the R j does not undergo cleavage in vivo, and the presence of the R j group and the R AP group in the conjugate will not affect the diagnostic and/or therapeutic effects of the functional group A 0 .
  • R j is a covalent bond
  • m 0 is 1.
  • the conjugate represented by formula (101) contains 1 functional group A 0 and 1 R AP group, each R AP groups are directly linked to the functional group A 0 .
  • each R AP group is linked to the same atom of functional group A0 .
  • each R AP group is attached to a different atom of functional group A0 .
  • R j is a linking group comprising a backbone moiety, a side chain moiety , and a conjugation linkage.
  • each side chain moiety is independently a covalent bond, or a linear alkylene group of 1 to 70 carbon atoms in length, or one or more of the linear alkylene groups
  • each conjugation linkage is independently a covalent bond or a combination of one or more of the following linkage structures: C 1 -C 10 linear alkylene, phosphate ester bond, phosphorothioate Ester bond, amide bond, ester bond, ether bond, disulfide bond, 1,2,3-triazole subunit, polyethylene glycol subunit, pyrrolidine subunit, 2-oxopyrrolidine subunit, phenylene base, cyclohexylene group, 2-succinimide subunit, 2-thiosuccinimide subunit, amino acid subunit, nucleotide subunit.
  • each of the conjugated linking moieties in linking group Rj is individually associated with the The main chain part is connected to one of the functional groups A 0 ; the number of side chain parts is n 0 , and each side chain part is connected to the main chain part and one of the R AP groups respectively.
  • each functional group A 0 and R AP group is each independently linked to the linking group R j .
  • all side chain moieties are connected to the same atom in the backbone moiety; alternatively, each side chain moiety is connected to a different atom in the backbone moiety.
  • m 0 is 1, and the linking group R j includes a structure shown in formula (301):
  • k is an integer from 1 to 3; LC is the main chain part, L A is the side chain part, LB is the conjugation connection part, Indicates the site at which a group is covalently attached.
  • the length of LC is 5-30 atoms, wherein the length of LC refers to the longest atomic chain in LC formed from the atoms directly connected to LA to the atoms directly connected to LB The number of atoms in the chain. To simplify the structure, in some embodiments, the length of LC is 8-25 atoms.
  • the conjugated linker LB is a connection combination of one or more of the following 1-5 linkages: phosphate bond, phosphorothioate bond, amide bond, ester bond, ether bond, and disulfide bond.
  • k is an integer of 1-3;
  • L C contains any one of the groups represented by formula (L1)-(L3), through the group represented by formula (L1)-(L3) The ether bond in the group is connected to the L A part:
  • L C contains a group represented by formula (L1), and the O atom in group (L1) is directly connected to L A.
  • L B is a phosphate bond or disulfide bond
  • Each L A is a covalent bond, or each L A is selected from the group consisting of groups (L4)-(L23) and their connecting combinations:
  • each j1 is an integer from 1 to 10;
  • Each R' is C 1 -C 10 alkyl
  • Each Ra is a hydrogen atom, a C 1 -C 10 alkyl group, or selected from the group consisting of groups (L24) - (L37):
  • each LA is a connected combination of at least 2 of the groups (L4)-(L9), (L13), (L14), (L18). In some embodiments, each LA is a connected combination of at least 2 of the groups (L4), (L5), (L7), (L9), (L13), (L14), (L18).
  • L A has a structure containing an amide bond as shown in Formula (302)
  • L B has an N-acylpyrrolidine-containing structure as shown in formula (303), containing a carbonyl group and an oxygen atom
  • L C is based on hydroxymethylaminomethane, dihydroxymethylaminomethane or trimethylolaminomethane.
  • n 302 , q 302 and p 302 are each independently an integer from 2 to 6.
  • n 302 , q 302 and p 302 are each independently an integer from 2 or 3;
  • n 303 is an integer from 4 to 16.
  • Select the location, n 303 is an integer from 8 to 12, Indicates the site at which a group is covalently attached.
  • each of the side chain portions L A is connected to one R AP group through a phosphate bond, an ether bond or an ester bond, and is connected to the oxygen atom of the hydroxyl group in the main chain portion L C through a phosphate bond, an ether bond or an ester bond.
  • the main chain part LC is connected by forming an ether bond;
  • the conjugated connection part LB is connected by forming an amide bond between the carbonyl group in the formula (303) and the nitrogen atom of the amino group in the main chain part LC , and is connected by
  • the oxygen atom in the formula (303) is connected to the functional group A 0 by forming a phosphate bond, an ether bond or an ester bond.
  • the backbone moiety LC is a linking group based on hydroxymethylaminomethane, dihydroxymethylaminomethane or trimethylolaminomethane via the oxygen atom of the hydroxyl group.
  • Each of the side chain moieties L A is connected by an ether bond and is connected to the conjugate linker L B by an amide bond via the nitrogen atom of the amino group.
  • 1-3 side chains in the linking group Rj are connected to the carbon atoms of the same aminomethyl group and are connected to the R AP group containing the delivery group through the conjugated linker LB.
  • the conjugate has the structure shown in formula (305):
  • the linking group R j comprises a structure shown in formula (306):
  • n 306 is an integer from 0 to 3
  • each p 306 is independently an integer from 1 to 6,
  • the linkage combination formed by all pyrrolidin subunits and any possible phosphodiester groups constitutes the backbone part, consisting of the carbonyl group attached to the nitrogen atom of the pyrrolidin subunit and the one marked by *
  • a chain of atoms between the oxygen atoms shown constitutes each side chain moiety, and the side chain part is connected to the R AP group by an ether bond formed by the oxygen atom marked by *;
  • at least one of the oxygen atoms marked by # To conjugate the linking part and form an ether bond, ester bond or phosphate bond with the functional group A 0 , and the remaining oxygen atoms marked by # are connected to hydrogen atoms to form hydroxyl groups, or with C 1 -C 3 alkyl groups The connection forms a C 1 -C 3 alkoxy group.
  • the present disclosure provides conjugates having a structure shown in formula (307a), (307b) or (307c):
  • the present disclosure provides conjugates having a structure shown in formula (308):
  • n 308 can be an integer from 1 to 10; in some embodiments, n 308 can be an integer from 2 to 6, taking into account various aspects such as ease of synthesis, structure/process cost, and tumor cell specificity. In some embodiments, n 308 is 3 or 4.
  • Each R 3 is independently a functional group A 0 , or a R AP group including a delivery group AP;. In some embodiments, at least one R3 is the functional group A0 , and at least one R3 is RAP . In some embodiments, one R 3 is a functional group A 0 and the remaining R 3 are R AP groups.
  • each m 308 when each m 308 is independently selected from an integer of 2-10, it is considered possible to make the spatial positions between the multiple delivery groups AP in the conjugate more suitable for contact with the surface of tumor cells. The corresponding receptors interact.
  • each m 308 is independently an integer of 2-5, In some embodiments, each m 308 is equal.
  • each R 308 is independently selected from H, C 1 -C 10 alkyl, C 1 -C 10 haloalkyl, and C 1 -C 10 alkoxy, formula (308) does not change Indicates the nature of the conjugate quality, can achieve the purpose of this disclosure.
  • each R 308 is independently selected from H, methyl, or ethyl.
  • each R 308 is H.
  • Each L 1 connected to the functional group A 0 represents the conjugation linkage, and each L 1 connected to the R AP represents the side chain moiety.
  • one R 3 is the functional group A 0 and the remaining R 3 are the R AP group.
  • one or more L 1 serves as the side chain moiety connecting the R AP group to an N atom on the nitrogen-containing backbone; and an additional one or more L 1 serves as the conjugation linker , connect the functional group A 0 to the N atom on the nitrogen-containing skeleton.
  • the nitrogen-containing skeleton together constitutes the main chain part of the connecting group Rj .
  • nitrogen-containing backbone refers to the chain structure in the structure represented by formula (308), in which the carbon atom to which R 308 is attached and the N atom are interconnected.
  • each L 1 is independently 3-25 atoms.
  • each L1 is independently 4-15 atoms in length.
  • L1 is defined as a linear alkylene group for convenience, it may not be a linear group or have a different name, such as an amine or alkenyl group resulting from the above substitutions and/or substitutions.
  • the length of L is the number of atoms in the chain connecting two junction points.
  • the ring obtained by replacing the carbon atoms of the linear alkylene group (such as heterocyclylene or heteroarylene) is calculated according to the minimum number of atoms between the connection points on the ring corresponding to the ring in the chain. The length of the section.
  • L 1 is selected from the group consisting of the groups represented by the above formulas L4-L23 and any connected combinations thereof. In some embodiments, each L 1 is independently selected from the group consisting of linked combinations of at least 2 of groups L4-L9, L13, L14, L18. In some embodiments, each L 1 is independently a connected combination of at least 2 of the groups L4, L5, L7, L9, L13, L14, and L18.
  • each L 1 simultaneously contains a connection site connected to an N atom on the nitrogen-containing skeleton and a functional group A 0 or the R
  • the connection site where the AP group is connected to the N atom on the nitrogen-containing skeleton forms an amide bond with the N atom.
  • one or more L1 is selected from B5, B6, B5' or B6':
  • q 2 is an integer from 1 to 10. In some embodiments, q2 is an integer from 1 to 5.
  • each R AP group contains multiple functional groups.
  • each functional group in the compound of formula (308) is the same functional group.
  • each functional group in the compound of formula (308) is a functional group serving the same purpose and function.
  • the compound of formula (308) contains different types of functional groups for different purposes and functions.
  • the compound represented by formula (308) has formula (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421), (422), (423 ), (424), (425), (426) or (427):
  • linking group R j comprises nucleotide sequence I and nucleotide sequence II, each of said nucleotide sequence I and said nucleotide sequence II comprising 5-25 modified or unmodified Nucleotides, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary, the delivery group is connected to the nucleotide sequence I, and the functional group is connected to the Nucleotide sequence II, and said nucleotide sequence I and said nucleotide sequence II do not trigger an immune response or toxic reaction in a subject.
  • the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary or completely reverse complementary; or, the nucleotide sequence I and the nucleotide sequence II are The lengths are equal and both are 10-20 modified or unmodified nucleotides; alternatively, the nucleotide sequence I and the nucleotide sequence II are both composed of 17 nucleotides and are completely reverse complementary.
  • the 3' end of the delivery group is connected to the ribose 5' position of the 5' terminal nucleotide of the nucleotide sequence I via a phosphate bond
  • the functional group is connected to the The ribose 5' position of the 5' terminal nucleotide of the nucleotide sequence II;
  • the functional group includes a nucleotide sequence, the 3' end of the nucleotide sequence is connected to the The ribose sugar 5' position of the 5' terminal nucleotide of the nucleotide sequence I.
  • the nucleotide sequence I and the nucleotide sequence II have the sequences shown in SEQ ID NO: 40 and SEQ ID NO: 41 respectively:
  • nucleotide sequence I and the nucleotide sequence II respectively have Sequences shown in SEQ ID NO:42 and SEQ ID NO:43:
  • Conjugates provided by the present disclosure may contain one or more functional groups.
  • a functional group refers to a group consisting of an active agent molecule with corresponding functionality (e.g., a diagnostic agent compound, a small molecule therapeutic agent group compound having a therapeutic effect on tumors, especially glioma, and /or functional oligonucleotide compounds that regulate the expression level of cancer-related genes in tumor cells) a group containing free radicals formed by removing one or more atoms, functional groups or part of a structural group (for example, removing a hydrogen atom) , and the functional group forms a covalent connection with other parts of the molecule of the conjugate provided by the present disclosure through the free radical.
  • an active agent molecule with corresponding functionality e.g., a diagnostic agent compound, a small molecule therapeutic agent group compound having a therapeutic effect on tumors, especially glioma, and /or functional oligonucleotide compounds that regulate the expression level of cancer-related genes in tumor cells
  • At least one functional group in the conjugates provided by the present disclosure is a diagnostic agent group, each of the diagnostic agent groups is independently selected from a contrast agent group or a fluorescent imaging group (fluorescence imaging group). group).
  • all functional groups in the conjugates provided by the present disclosure are diagnostic agent groups as described above.
  • the conjugate provided by the present disclosure can deliver the diagnostic agent group to the tumor, thereby specifically, efficiently and accurately detecting relevant disease and/or symptom information such as the presence of the tumor and the disease progression status. Make a diagnosis.
  • the diagnostic agent group can be used to diagnose tumor cells/tissues in a test sample in in vitro experiments.
  • the diagnostic agent group can be used to diagnose the presence and/or nature of tumor cells/tissue in a subject.
  • At least one functional group in the conjugate provided by the present disclosure is a small molecule therapeutic agent group that has a therapeutic effect on tumors, especially glioma, and each of the small molecule therapeutic agent groups The group is independently selected from a cytotoxic group, an antibiotic group, an angiogenesis inhibitor, or an antibody drug group.
  • all functional groups in the conjugates provided by the present disclosure are the small molecule therapeutic agent groups described above.
  • Or symptoms can be treated and/or alleviated, for example, by specifically delivering cytotoxic groups to tumors through the conjugates provided by the present disclosure, causing cancer cells in the tumors to specifically die, thereby reducing cytotoxicity. While reducing the side effects caused by the low targeting of the hormone itself, it can significantly reduce the number of cancer cells in the tumor, thereby treating the tumor.
  • At least one functional group in the conjugate provided by the present disclosure is a functional oligonucleotide group capable of regulating the expression of cancer-related genes in tumor cells.
  • all functional groups in the conjugates provided by the present disclosure are functional oligonucleotide groups as described above.
  • the conjugates provided by the present disclosure are able to specifically deliver the functional oligonucleotide group to the tumor, thereby through the action of the functional oligonucleotide group , such as RNAi effects, regulating the expression levels of cancer-related genes in tumor cells, such as inhibiting the expression of oncogenes, thereby treating and/or alleviating tumor-related disease processes or symptoms.
  • the functional oligonucleotide is siRNA.
  • the functional oligonucleotide is an siRNA targeting STAT3 mRNA, RRM2 mRNA, or PLK1.
  • the functional oligonucleotide is an siRNA with a sense strand as shown in SEQ ID NO: 44, an antisense strand as shown in SEQ ID NO: 45, or a sense strand as shown in SEQ ID NO: 67 siRNA with an antisense strand as shown in SEQ ID NO: 68, or one or more siRNAs with a sense strand as shown in SEQ ID NO: 69 and an antisense strand as SEQ ID NO: 70:
  • the functional groups may be included in the conjugates provided by the present disclosure by any suitable means.
  • the functional group A 0 can be connected to the main chain moiety through the aforementioned conjugation linker.
  • At least one functional group in the conjugate provided by the present disclosure is a delivery aid group, and the delivery aid group is selected from the group consisting of C 10 -C 30 hydrocarbon groups, cholesterol groups, and phospholipid groups. one or more of the groups.
  • the present disclosure provides conjugation
  • the conjugates may be better compatible with the in vivo environment in the central nervous system, may have better bioavailability, and/or enable more efficient delivery of the conjugates provided by the present disclosure to tumors.
  • the synthesis method of the conjugate provided by the present disclosure includes contacting the protected conjugate with a deprotection reagent in a solvent under deprotection reaction conditions, and isolating to obtain the conjugate provided by the present disclosure.
  • a protected conjugate is a compound in which any active functional group in the conjugate provided by the present disclosure is protected by a protecting group.
  • the reactive functional groups include, but are not limited to, hydroxyl, amino and/or phosphate groups, and the protecting groups are, respectively, hydroxyl protecting groups, amino protecting groups and/or phosphate hydroxyl protecting groups (e.g., cyanoethyl base protecting group).
  • the solvent, deprotection reaction conditions and deprotection reagents used are selected and determined.
  • the deprotection reaction conditions, solvents and deprotection reagents are those used in solid phase synthesis of nucleic acids.
  • the method includes adding the protected conjugate into a mixed solution of methylamine aqueous solution and ammonia water, and the deprotection reaction conditions include reaction at normal temperature and pressure for 1-5 h.
  • the methylamine aqueous solution and the saturated concentrated ammonia solution are mixed in equal volumes to obtain the mixed solution, and the dosage of the solution relative to the protected conjugate is 0.1-10 ml/ ⁇ mol.
  • the separation includes purification by column chromatography, collecting the product eluate and removing the solvent.
  • the purification conditions may be, for example, using a preparative ion chromatography purification column and eluting with a gradient eluent of sodium chloride aqueous solution and sodium phosphate aqueous solution.
  • the conjugate provided by the present disclosure has a structure represented by formula (101), and the synthesis method of the protected conjugate includes in an organic solvent, under coupling reaction conditions, will contain active
  • the compound of the group R The body is formed by removing one or more hydrogen atoms or one or more functional groups, and each of the functional groups is independently a diagnostic agent group, a small molecule therapeutic agent group that has a therapeutic effect on tumors, and a small molecule therapeutic agent group that has a therapeutic effect on tumors.
  • One of the functional oligonucleotide groups Any reactive group in the delivery group and functional group is protected by a protecting group, and the reactive group R x1 and the reactive group R x2 are capable of forming a covalent bond or linking group through reaction.
  • the group of group R j The group of group R j .
  • the molar ratio of the delivery group connected to the active group R x1 and the functional group connected to the active group R x2 is m 0 :n 0 .
  • the active groups in the delivery group and functional group include, but are not limited to, one or more of hydroxyl, amino, and phosphate groups.
  • the compound containing the reactive group R x1 and the delivery group can obtain compounds containing the reactive group R x1 and the delivery group through various methods.
  • the compound containing the reactive group R Obtained through synthesis.
  • the compound containing the reactive group R The sequence of nucleotides in the chain is used to connect the nucleoside monomers in sequence, where at least one nucleoside monomer is a nucleoside monomer with an active group R
  • the phosphoramide solid-phase synthesis method connects a phosphoramidite monomer or a protected phosphoramidite monomer with an active group R x1 , and then removes the protecting group to form an active group R x1 .
  • the coupling reaction conditions are condensation reaction conditions or thiol-disulfide exchange reaction conditions.
  • the coupling reaction conditions are condensation reaction conditions, and the condensation reaction conditions are acylation condensation reaction conditions, dehydration condensation reaction conditions or click chemical reaction conditions, and the reactive group R x1 and the reactive group R x2 is a group capable of undergoing the aforementioned condensation reaction.
  • the condensation reaction conditions are conditions for an acylation condensation reaction, and the active groups R x1 and R x2 are groups capable of undergoing an acylation condensation reaction to form R I .
  • the condensation reaction conditions are conditions for dehydration condensation reaction, one of the active groups R x1 and R x2 is a group containing an acid halide group or a carboxyl group, and the other is a group containing an amino group or a hydroxyl group. group.
  • the condensation reaction conditions are click chemistry conditions, one of the active groups R x1 and R x2 is a group containing an alkynyl group, and the other is a group containing an azide group. The group of the group.
  • the condensation reaction conditions are conditions of a Michael addition reaction, one of the active groups R x1 and R x2 is a group containing a thiol group, and the other is a group containing a succinimide group. group.
  • the condensation reaction conditions are N-hydroxysuccinimide-carbodiimide (NHS-EDC) coupling reaction conditions, and one of the active groups R x1 and R x2 One is a group containing N-hydroxysuccinimide (NHS), and the other is a group containing a carbodiimide group (EDC).
  • the compound containing the reactive group R x1 and the delivery group is obtained by contacting the aptamer provided by the present disclosure with the reactive group R Prepared, the cross-linking agent contains click chemical active groups and acylation groups.
  • the reactive group R is an active ester group, for example, it can be one of an NHS ester group, an imide ester group, and a pentafluorophenyl ester group.
  • the cross-linking agent can be done as follows: Michael E., et al.
  • the reactive group RxO is amino.
  • the coupling conditions are basic conditions.
  • the alkaline condition is a condition in which a weak alkali aqueous solution is present, such as a sodium bicarbonate aqueous solution.
  • the aptamer with the active group R x0 is synthesized by synthesizing the aptamer. It is prepared by using phosphoramidite monomers containing active groups at corresponding positions in the process. Those skilled in the art can obtain phosphoramidite monomers containing reactive groups in various ways.
  • the reactive group R The monomer may be 6-(trifluoroacetylamino)-hexyl- (2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite monomer, wherein the reactive group R x0 is an amino group, and the reactive group R x0 can be synthesized by solid phase phosphoramidite After connecting the phosphoramidite monomer to the single chain of the oligonucleotide, the trifluoroacetyl protecting group is removed through a deprotection reaction (such as ammonolysis of concentrated ammonia) that is easily implemented by those skilled in the art.
  • a deprotection reaction such as ammonolysis of concentrated ammonia
  • the coupling reaction condition is one of sulfhydryl-disulfide bond exchange reactions, one of the reactive groups R x1 and R Sulfur bonded leaving group.
  • R x1 in the above-mentioned phosphoramidite monomer containing reactive group R x1 exists in the form of protected R x1 ', and the preparation method also includes deprotection reaction conditions Next, the step of isolating the compound containing the active group R x1 and the delivery group by contacting the prepared compound containing the protected active group R x1 ' and the delivery group with a deprotecting reagent.
  • the disulfide bond activator is a disulfide pyridine.
  • phosphorus acid amide monomer containing the reactive group R Amide monomers are commercially available, for example, phosphoramidite monomers represented by formula (105) can be obtained commercially.
  • n 105 and m 105 are each independently an integer from 1 to 10.
  • the coupling reaction conditions are thiol- disulfide bond exchange reaction conditions
  • the reactive group R The skilled person can obtain it in various known ways, for example, by using a phosphoramidite monomer containing a thiol group, prepared by a phosphoramidite solid phase synthesis method, or obtained commercially.
  • the functional group is a functional oligonucleotide group
  • the coupling reaction conditions are phosphoramidite solid phase synthesis reaction conditions
  • the active group R Sequence connect nucleoside monomers sequentially according to the phosphoramidite solid-phase synthesis reaction.
  • the functional group is a diagnostic agent group or a small molecule therapeutic agent group
  • the coupling reaction conditions are phosphoramidite solid phase synthesis reaction conditions
  • the active group R Phosphoramidite group, the compound containing the reactive group R x2 and the functional group can be, for example, an easily commercially available compound containing a phosphoramidite group and a fluorescent group or a small molecule therapeutic agent group.
  • the coupling reaction conditions are Michael addition reaction conditions
  • the reactive group R x2 is an N-succinimide group
  • the reactive group R x2 is included
  • the compound with the functional group may be, for example, a readily commercially available compound containing an N-succinimide group and a small molecule therapeutic agent group.
  • the functional group is a functional oligonucleotide group
  • the reactive group R x1 and the reactive group R x2 are nucleotide sequence I and nucleotide sequence II respectively
  • the nucleotide sequence I and the nucleotide sequence II each comprise 5-25 modified or unmodified nucleotides, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary.
  • the delivery group is connected to the nucleotide sequence I
  • the functional group is connected to the nucleotide sequence II
  • the nucleotide sequence I and the nucleotide sequence II are tested
  • the coupling reaction conditions are the reaction conditions for annealing to form nucleic acid double strands.
  • the functional oligonucleotide group is siRNA
  • the 5′ end of the nucleotide sequence I is connected to the delivery group through a phosphodiester bond
  • the nucleotide sequence I The 3' end of sequence II is connected to the 5' end of the siRNA via a phosphodiester bond.
  • the nucleotide sequence I and the nucleotide sequence II each consist of 17 nucleotides and are completely reverse complementary.
  • the nucleotide sequence I and the nucleotide sequence II have the sequences shown in SEQ ID NO: 40 and SEQ ID NO: 41, respectively.
  • the nucleotide sequence I and the nucleotide sequence II have the sequences shown in SEQ ID NO: 42 and SEQ ID NO: 43, respectively.
  • the conjugates may also be used in the present disclosure in the form of pharmaceutically acceptable salts or precursor compounds thereof.
  • pharmaceutically acceptable salts refer to the formation of corresponding salts of a drug that do not produce additional pharmaceutical side effects in the human body in order to increase the stability, solubility and/or bioavailability of the drug, e.g. Potassium salt, sodium salt, carboxylate, etc.
  • Precursor compound refers to a compound that, although structurally and functionally different from the conjugate itself, is capable of reacting after entering the body or in a body fluid environment to form the compound containing the present compound.
  • the conjugates provided by the disclosure are disclosed to thereby exert effects and achieve the purposes provided by the present disclosure.
  • these precursor compounds can increase the stability of the drug, extend the sustained release time, increase bioavailability and other effects.
  • the precursor compound includes a precursor group that reacts in humans to form all functional groups A0 in the conjugate.
  • the precursor compound includes a compound in which all active hydroxyl groups in the conjugate are replaced with acetoxy groups.
  • the precursor compound includes a prodrug group that is a diagnostic agent, therapeutic agent, and/or functional oligomeric agent corresponding to the functional group in the conjugate. Residues from which nucleotide precursor compounds are formed.
  • the prodrug group may be, for example, a group formed by replacing the active hydrogen in the hydroxyl or amino functional group in the functional group with an acyl group, an alkyl group, or a phosphoryl group.
  • the present disclosure also provides a pharmaceutical composition comprising the conjugate provided by the present disclosure and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier can be a carrier commonly used in the art, such as but not limited to water, physiological saline, magnetic nanoparticles (such as nanoparticles based on Fe 3 O 4 or Fe 2 O 3 ), carbon Nanotubes (carbon nanotubes), mesoporous silicon (mesoporous silicon), calcium phosphate nanoparticles (calcium phosphate nanoparticles), polyethylenimine (PEI), polyamide dendrimer (polyamidoamine (PAMAM) dendrimer), Poly(L-lysine), PLL, chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) , poly(D&L-lactic/glycolic acid)copolymer (PLGA), poly(2-aminoethyl ethylene phosphate) (poly(2-aminoethyl ethylene phosphate), PPEEA) and one or more of poly(2-dimethylaminoethy
  • the pharmaceutically acceptable carrier contains a physiologically acceptable compound that acts, for example, to stabilize the pharmaceutical composition or to increase or decrease the absorption of the conjugate and/or the pharmaceutical composition.
  • the physiologically acceptable compound is selected from the following compounds One or more of: carbohydrates, such as glucose, sucrose and/or dextran; antioxidants, such as ascorbic acid and/or glutathione; chelating agents; low molecular weight proteins; reducing the activity of any co-administered substance Clear or hydrolyzable compositions; excipients; stabilizers and buffers.
  • Detergents may also be used to stabilize the composition or to increase or decrease absorption of the pharmaceutical composition.
  • the physiologically acceptable compound may also include one or more wetting agents, emulsifiers, dispersants or preservatives, especially to prevent the growth or action of microorganisms.
  • the physiologically acceptable compounds are known to those skilled in the art and will not be described in detail in this disclosure. As will be readily understood by those skilled in the art, the selection of pharmaceutically acceptable carriers and physiologically acceptable compounds depends, for example, on the route of administration and the specific physiochemical properties of any co-administered substances.
  • pharmaceutically acceptable carriers are sterile and generally free of undesirable materials.
  • the pharmaceutical composition provided by the present disclosure may further contain pharmaceutically acceptable auxiliary substances as needed to approximate physiological conditions, such as pH regulators and buffers, toxicity regulators, etc., such as sodium acetate, sodium chloride, potassium chloride, chloride Calcium, sodium lactate, etc., the concentration of the conjugate provided by the present disclosure in the pharmaceutical composition can vary within a wide range, and is mainly selected according to a specific administration method based on fluid volume, viscosity, body weight, etc.
  • the pharmaceutical composition has no special requirements on the content of the conjugate and the pharmaceutically acceptable carrier.
  • the weight ratio of the conjugate to the pharmaceutically acceptable carrier can be It is 1: (1-500). In some embodiments, the above weight ratio is 1: (1-50).
  • the pharmaceutical composition may also contain other pharmaceutically acceptable auxiliary materials, which may be one or more of various preparations or compounds commonly used in the art.
  • the other pharmaceutically acceptable excipients may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.
  • the pH buffer can be a trishydroxymethylaminomethane hydrochloride buffer with a pH of 7.5-8.5 and/or a phosphate buffer with a pH of 5.5-8.5, for example, it can be a phosphate with a pH of 5.5-8.5. Buffer.
  • the protective agent may be at least one of myo-inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose and glucose. Based on the total weight of the pharmaceutical composition, the content of the protective agent may be 0.01-30% by weight.
  • the osmotic pressure regulator may be sodium chloride and/or potassium chloride.
  • the content of the osmotic pressure regulator is such that the osmotic pressure of the pharmaceutical composition is 200-700 milliosmole/kg (mOsm/kg).
  • the content of the osmotic pressure regulator can be easily determined by those skilled in the art based on the desired osmotic pressure.
  • the dosage of the preparation made from the pharmaceutical composition during administration will be adjusted due to different administration methods.
  • the pharmaceutical composition can be a liquid preparation, such as an injection; it can also be a freeze-dried powder for injection, which is mixed with liquid excipients during administration to prepare a liquid preparation.
  • the liquid preparation may be used for, but is not limited to, subcutaneous, intramuscular or intravenous injection.
  • the pharmaceutical composition may also be delivered by, but is not limited to, puncture injection, oropharyngeal inhalation, or nasal administration.
  • the pharmaceutical composition is for subcutaneous, intramuscular, intravenous or intrathecal administration.
  • the pharmaceutical composition may be in the form of a liposome formulation.
  • the pharmaceutically acceptable carrier used in the liposome formulation includes an amine-containing transfection compound (hereinafter also referred to as an organic amine), a helper lipid, and/or a pegylated Lipids.
  • an organic amine, auxiliary lipid and pegylated lipid can be respectively selected from the amine-containing transfection compounds described in Chinese patent application CN103380113A (which is incorporated by reference into this disclosure in its entirety) or One or more of its pharmaceutically acceptable salts or derivatives, auxiliary lipids and pegylated lipids.
  • the organic amine can be a compound represented by formula (201) described in Chinese patent application CN103380113A or a pharmaceutically acceptable salt thereof:
  • X 101 and X 102 are each independently O, S, NA or CA, where A is hydrogen or C 1 - C 20 hydrocarbon chain;
  • R 101 , R 102 , R 103 , R 104 , R 105 , R 106 and R 107 are each independently hydrogen, cyclic or acyclic, substituted or unsubstituted, branched or linear aliphatic group Group, cyclic or acyclic, substituted or unsubstituted, branched or linear heteroaliphatic group, substituted or unsubstituted, branched or linear acyl group, substituted or unsubstituted Substituted, branched or linear aryl, substituted or unsubstituted, branched or linear heteroaryl;
  • x is an integer from 1 to 10;
  • n is an integer from 1 to 3
  • m is an integer from 0 to 20
  • R 103 and nitrogen in formula (201) form a structure shown in formula (202) or formula (203):
  • g, e and f are each independently an integer from 1 to 6
  • HCC represents a hydrocarbon chain
  • each *N represents a nitrogen atom in formula (201).
  • R 103 is a polyamine. In other embodiments, R 103 is a ketal. In some embodiments, each of R 101 and R 102 in formula (201) is independently any substituted or unsubstituted, branched or linear alkyl or alkenyl group, the alkyl The radical or alkenyl group has 3 to about 20 carbon atoms, such as 8 to about 18 carbon atoms, and 0 to 4 double bonds, such as 0 to 2 double bonds.
  • R 103 may be any of the following formulas (204) to (213):
  • g, e and f are each independently an integer from 1 to 6
  • each "HCC” represents a hydrocarbon chain
  • each * shows that R 103 is the same as in formula (201)
  • Possible attachment points to the nitrogen atoms in where each H at any * position can be replaced to effect attachment to the nitrogen atom in formula (201).
  • the compound represented by formula (201) can be prepared according to the description in Chinese patent application CN103380113A.
  • the organic amine is an organic amine represented by formula (214) and/or an organic amine represented by formula (215):
  • the auxiliary lipid is cholesterol, cholesterol analogs and/or cholesterol derivatives
  • the pegylated lipid is 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine-N-[methoxy (polyethylene glycol)]-2000.
  • the molar ratio between the organic amine, the auxiliary lipid and the PEGylated lipid is (19.7-80): (19.7-80 ):(0.3-50), for example, it can be (50-70):(20-40):(3-20).
  • composition particles formed from the conjugates provided by the present disclosure and the above-described amine-containing transfection reagents have an average diameter of about 30 nm to about 200 nm, typically about 40 nm to about 135 nm, and more typically, the The average diameter of the liposome particles is about 50 nm to about 120 nm, about 50 nm to about 100 nm, about 60 nm to about 90 nm, or about 70 nm to about 90 nm.
  • the average diameter of the liposome particles is about 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150 or 160nm.
  • the conjugate in a pharmaceutical composition formed from a conjugate provided by the present disclosure and the above-mentioned amine-containing transfection reagent, is PEGylated with all lipids (e.g., organic amines, auxiliary lipids, and/or
  • the weight ratio (weight/weight ratio) of lipids) ranges from about 1:1 to about 1:50, from about 1:1 to about 1:30, from about 1:3 to about 1:20, from about 1:4 to about 1:18, from about 1:5 to about 1:17, from about 1:5 to about 1 In the range of: 15, from about 1:5 to about 1:12, from about 1:6 to about 1:12, or from about 1:6 to about 1:10, for example, the conjugates provided by the present disclosure are The weight ratio of lipids is about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15 , 1:16, 1:17 or 1:18.
  • each component of the pharmaceutical composition may exist independently when sold, and may exist in the form of a liquid preparation when used.
  • the pharmaceutical composition formed by the conjugate provided by the present disclosure and the above-mentioned pharmaceutically acceptable carrier can be prepared according to various known methods, except that the existing adapter is replaced by the conjugate provided by the present disclosure.
  • the body or conjugate is sufficient; in some embodiments, it can be prepared as follows:
  • the organic amine, auxiliary lipid and PEGylated lipid are suspended in alcohol according to the above molar ratio and mixed to obtain a lipid solution; the amount of alcohol is such that the total mass concentration of the obtained lipid solution is 2-25 mg/mL, For example, it can be 8-18 mg/mL.
  • the alcohol is selected from pharmaceutically acceptable alcohols, such as alcohols that are liquid near room temperature, for example, ethanol, propylene glycol, benzyl alcohol, glycerin, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400 One or more, for example, it can be ethanol.
  • the conjugate provided by the present disclosure is dissolved in a buffered salt solution to obtain an aqueous conjugate solution.
  • concentration of the buffer salt solution is 0.05-0.5M, for example, it can be 0.1-0.2M.
  • the pH of the buffer salt solution is adjusted to 4.0-5.5, for example, it can be 5.0-5.2.
  • the amount of the buffer salt solution is such that the concentration of the conjugate does not exceed 0.6mg/mL, for example, it can be 0.2-0.4mg/mL.
  • the buffer salt is selected from one or more of soluble acetate and soluble citrate, for example, it can be sodium acetate and/or potassium acetate.
  • the volume ratio of the lipid solution and the aqueous conjugate solution is 1:(2-5), for example, it can be 1:4.
  • the incubated liposome preparation is concentrated or diluted, impurities are removed, and sterilization is performed to obtain the pharmaceutical composition provided by the present disclosure.
  • Its physical and chemical parameters are a pH value of 6.5-8, an encapsulation rate of not less than 80%, and a particle size of 40-200nm, polydispersity index not higher than 0.30, osmotic pressure 250-400mOsm/kg; for example, physical and chemical parameters can be pH value 7.2-7.6, encapsulation rate not less than 90%, particle size 60- 100nm, the polydispersity index is not higher than 0.20, and the osmotic pressure is 300-400mOsm/kg.
  • concentration or dilution can be performed before, after or at the same time as impurities are removed.
  • Various existing methods can be used to remove impurities, such as using a tangential flow system, a hollow fiber column, ultrafiltration at 100K Da, and the ultrafiltration exchange solution is phosphate buffer saline (PBS) with pH 7.4.
  • PBS phosphate buffer saline
  • the sterilization method can adopt various existing methods, for example, filtration sterilization can be performed on a 0.22 ⁇ m filter.
  • the present disclosure also provides the use of the aptamers and/or conjugates and/or pharmaceutical compositions provided by the present disclosure in the preparation of medicaments for the diagnosis and/or treatment of tumors and tumor-related diseases or symptoms.
  • the present disclosure also provides a method for diagnosing and/or treating tumors and tumor-related diseases or symptoms, the method comprising administering to a subject in need a conjugate provided by the present disclosure and/or or pharmaceutical compositions.
  • the methods provided by the present disclosure can effectively diagnose and/or treat tumors and tumor-related diseases or symptoms; and, in the high specificity of the conjugates provided by the present disclosure, Under targeting, the distribution of diagnostic and/or therapeutic agents in undesirable other organs/tissues of the body can be reduced, reducing potential side effects.
  • the drug can be used to diagnose tumor cells/tissues in a test sample in in vitro experiments.
  • the medicaments can be used to diagnose tumor cells/tissues in a subject.
  • the medicaments may be used to treat tumors or tumor-related diseases and symptoms in a subject.
  • administering/administering refers to the administration of a conjugate and/or pharmaceutical composition by a method or pathway that at least partially localizes the conjugate and/or pharmaceutical composition to a desired site to produce the desired effect. or the pharmaceutical composition is placed into a subject.
  • Routes of administration suitable for the methods of the present disclosure include local administration and systemic administration. Generally speaking, local administration results in the delivery of more conjugates and/or pharmaceutical compositions to a specific site compared to the subject's entire body; whereas systemic administration results in the delivery of the conjugates and/or pharmaceutical compositions in combination with The substance is delivered to substantially the entire body of the subject.
  • the inventors provided by the present disclosure unexpectedly discovered that the conjugates provided by the present disclosure and/or the pharmaceutical composition can efficiently pass through the blood-brain barrier and be targeted to tumors in the brain when administered systemically, thereby further improving the delivery efficiency of functional groups, saving costs and reducing undesirable side effects. side effects.
  • Administration to the subject may be by any suitable route known in the art, including, but not limited to, oral or parenteral routes, such as intravenous, intramuscular, subcutaneous, or transdermal. medicine, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration and topical administration (including buccal administration and sublingual administration).
  • the frequency of administration may be once or more daily, weekly, every two weeks, every three weeks, monthly or yearly.
  • the dosage of the conjugates and/or pharmaceutical compositions provided by the present disclosure can be conventional dosages in the art, and the dosage can be determined according to various parameters, especially the age, weight and gender of the subject. Toxicity and efficacy can be measured in cell culture or experimental animals by standard pharmaceutical procedures, such as determining the LD50 (the dose that causes 50% of the population to be lethal) and the ED50 (the dose that causes 50% of the maximum response intensity in a quantitative response, and in a qualitative response Medium refers to the dose that causes a positive reaction in 50% of the experimental subjects). Dosage ranges for humans can be derived based on data from cell culture assays and animal studies.
  • the conjugated When administering the conjugates and/or pharmaceutical compositions provided by the present disclosure, for example, to male or female C57BL/6J or C3H/HeNCrlVr mice aged 6-12 weeks and weighing 18-25g, the conjugated
  • the amount of the conjugate in the drug and/or pharmaceutical composition can be 0.001-100 mg/kg body weight, in In some embodiments, it is 0.01-50 mg/kg body weight, in further embodiments, it is 0.05-20 mg/kg body weight, in still further embodiments, it is 0.1-15 mg/kg body weight, and in still further embodiments, it is 0.1 -10mg/kg body weight.
  • the above amounts may be preferred when administering the conjugates and/or pharmaceutical compositions provided by the present disclosure.
  • the present disclosure provides a kit comprising a conjugate and/or pharmaceutical composition provided by the present disclosure.
  • kits that provide the conjugate and/or pharmaceutical composition in one container.
  • kits provided by the present disclosure may include a container providing a pharmaceutically acceptable excipient.
  • the kit Other ingredients may also be included, such as stabilizers or preservatives.
  • the kits provided by the present disclosure can include at least one additional therapeutic agent in a container other than the container in which the conjugates and/or pharmaceutical compositions provided by the present disclosure are provided.
  • the kit may contain instructions for mixing the conjugate and/or pharmaceutical composition with pharmaceutically acceptable carriers and/or excipients or other ingredients, if any.
  • the conjugate and the pharmaceutically acceptable carrier and/or excipients and the pharmaceutical composition, and/or the pharmaceutically acceptable excipients can be provided in any form, such as liquid form, Dry or lyophilized form.
  • the conjugate and pharmaceutically acceptable carrier and/or excipients and the pharmaceutical composition and optional pharmaceutically acceptable excipients are substantially pure and/or sterile.
  • sterile water can be provided in the kits provided by the present disclosure.
  • the reagents and culture media used in the following examples are all commercially available products.
  • the nucleic acid electrophoresis, real-time PCR and other operations used are all described in Molecular Cloning (Cold Spring Harbor LBboratory Press (1989)). method to proceed.
  • the conjugates numbered AP1-AP8 and AP12 in Table 1A were synthesized respectively by solid-phase synthesis method, and the nucleotide sequences corresponding to AP1-AP8 and AP12 in Table 1A were respectively synthesized from the 3′-5′ direction.
  • After connecting all the nucleoside monomers connect the Cy5 phosphoramidite monomer (purchased from Suzhou Jima Company, batch number CY5P21H1B) according to the solid-phase synthesis method to connect the nucleoside phosphoramidite monomers.
  • the nucleotide sequence was added to a mixed solution of equal volumes of methylamine aqueous solution and ammonia water. The dosage of the solution relative to the conjugate was 0.5 ml/ ⁇ mol.
  • the reaction was carried out at 25°C for 2 hours. The solid was removed by filtration, and the supernatant was concentrated in vacuum. To dryness.
  • eluent A 20mM sodium phosphate (pH 8.1)
  • eluent B 1.5M sodium chloride, 20mM sodium phosphate (pH 8.1)
  • the specific conditions include using a Sephadex column for desalting.
  • the filler is Sephadex G25 and eluting with deionized water.
  • the obtained eluate was concentrated to remove the solvent and lyophilized to obtain conjugates AP1-AP8 in which the 5' position of the ribose sugar of the 5' terminal nucleotide was connected to the fluorescent group Cy5 through a phosphate linking group.
  • the prepared conjugates are diluted to a concentration of 0.2 using ultrapure water (made by Milli-Q ultrapure water instrument, resistivity 18.2M ⁇ *cm (25)) mg/mL, use liquid chromatography-mass spectrometry (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters Company, model: LCT Premier) for molecular weight detection.
  • ultrapure water made by Milli-Q ultrapure water instrument, resistivity 18.2M ⁇ *cm (25)
  • LC-MS liquid chromatography-mass spectrometry
  • comparative AP10 and comparative AP11 are negative control conjugates with different sequences or modification schemes, and their aptamer sequences have a certain degree of randomness and have almost no sequence homology with the aptamers provided by the present disclosure.
  • Comparative AP13 and Comparative AP14 are comparative conjugates whose sequences differ only by a few nucleotides from conjugates AP2 and AP4, respectively. Specifically, comparing AP13 with AP2, the 5′ end has two more nucleotides G and A, the 3′ end has one less nucleotide C, and the 17th to 18th nucleotides in the chain correspond to the 5′ end of AP2.
  • a nucleotide U is missing at the position of 3 nucleotides; comparing AP14 with AP4, there are two more nucleotides G and A at the 5' end and one less nucleotide U at the 3' end, and they correspond within the chain.
  • a nucleotide U is missing at the 17th to 18th nucleotide position from the 5′ end of AP2.
  • the capital letters C, G, U, and A indicate the base composition of nucleotides; the small letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; the small letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; f indicates that the nucleotide adjacent to the left side of the letter f is a fluorinated modified nucleotide; the lowercase letter s indicates that the two nucleotides to the left and right of the letter s are connected by a phosphorothioate group; CY3 and CY5 respectively Indicates the connection sites of the fluorescent dye groups Cy3 (Cyanine 3) and Cy5 (Cyanine 5) groups on the aptamer.
  • conjugate 15 was prepared according to the following steps.
  • the oligonucleotide sequence in conjugate 15 in Table 1B was synthesized by the method of Preparation Examples 1-8 and 12. The only difference is that the sense strand sequence contains the sense strand of siRNA, and the sense strand of the siRNA The 3′ end of the sense chain is connected to the delivery group contained in the conjugate AP2 via Spacer18-GfAfUf as a linking group, where Spacer18 refers to 6 ethoxy subunits connected in sequence and a length of 18 atoms. of six polyethylene glycol subunit linking groups.
  • HEG-phosphoramidite monomer After the linking group is connected to the nucleoside monomer GfAfUf sequentially from the 3'-5' direction, the hexapolyethylene glycol (HEG)-phosphoramidite monomer is connected according to the solid phase synthesis method.
  • HEG-phosphoramidite monomer has the structure shown in formula (15-a).
  • the supernatant and precipitation are the crude single-stranded C1 oligonucleotide required.
  • the solvent was concentrated and removed to obtain 43.4 mg of oligonucleotide single-chain C2 with Cy5 fluorescent group connected through -NH-(CH 2 ) 6 - group and phosphate linking group.
  • the sense strand of conjugate 16 was synthesized. The only difference was that the nucleotides were connected sequentially according to the nucleotide sequence of the sense strand of conjugate 16 in Table 1B.
  • the siRNA sense strand of conjugate 4 is connected to conjugate AP2 via a Spacer18-GfAfUf connection as a linking group at the 5′ end.
  • the 3′ end of the delivery group, and the ribose 5′ position of the 5′ terminal nucleotide of the delivery group is connected to a fluorescent group through a -NH-(CH 2 ) 6 - group and a phosphate linking group Cy5.
  • the antisense strand sequences of the conjugates numbered as conjugates 15 and 16 in Table 1B were synthesized.
  • Conjugate 15 and Conjugate 16 are conjugates comprising a double-stranded siRNA and a delivery group contained in conjugate AP2, respectively, wherein the delivery group is connected to the 5′ end of the siRNA sense strand via a Spacer18-GfAfUf linker.
  • the capital letters C, G, U, A, and T represent the base composition of nucleotides;
  • the small letter m represents that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide;
  • the lowercase letter d indicates that the nucleotide adjacent to the right side of the letter d is a deoxynucleotide;
  • the lowercase letter f indicates that the nucleotide adjacent to the left side of the letter f is a fluorinated modified nucleotide;
  • the lowercase letter s Indicates that the two nucleotides on the left and right of the letter s are connected by phosphorothioate groups;
  • CY3 and CY5 respectively represent the fluorescent dye groups Cy3 (Cyanine 3) and Cy5 (Cyanine 5) groups in the aptamer
  • the connection site on the siRNA; CY5-(NH-C6H12) represents the site in siRNA that is connected
  • conjugates numbered as conjugate 17 and conjugate 18 in Table 1B were synthesized according to the same method as in Preparation Examples 15-16. The only difference is that the conjugates 17 and 18 in Table 1B correspond to each other.
  • the nucleic acid sequence is connected to the nucleoside monomers in sequence; and, when preparing the sense strand, C3-spacer phosphoramidite monomer (purchased from Shanghai Zhaowei Company, Cat. No. OP-005) replaces the Spacer18-GfAfUf linking group for connection.
  • the ligation was repeated four times, and then the nucleoside monomers were continued to be ligated according to the nucleic acid sequence in Table 1B.
  • the obtained conjugate 17 is a conjugate comprising a double-stranded siRNA and an aptamer provided by the present disclosure, wherein the aptamer is connected to the siRNA via four sequentially connected propylene phosphate groups as a linking group.
  • the 5′ end of the sense strand, and the Cy5 fluorescent group is connected to the 5′ end of the aptamer through the NH-(CH 2 ) 6 -group and the phosphate group;
  • conjugate 18 is the adapter in conjugate 17
  • the first two nucleotides at each end of the body have a 2'-O-hexadecyl modified conjugate.
  • conjugate numbered as conjugate 19 in Table 1B was synthesized according to the same method as in Preparation Examples 15-16. The only difference is that the nucleoside monomers are connected sequentially according to the nucleic acid sequence corresponding to conjugate 19 in Table 1B. Three nucleotide sequences are obtained; and, during annealing, the three obtained nucleotide sequences are dissolved together in water for injection and mixed in an equal molar ratio.
  • the obtained conjugate 19 is a conjugate comprising double-stranded siRNA and the conjugate AP2 provided by the present disclosure, wherein the aptamer is connected to the siRNA via a linking group, and the linking group has SEQ ID NO: 28 and the complementary nucleotide sequence of the sequence shown in SEQ ID NO: 29, the aptamer is connected to the 5' end of the sequence shown in SEQ ID NO: 28, and the siRNA is connected to the sequence shown in SEQ ID NO: 29
  • the 3' end of the sequence, and the NH-(CH 2 ) 6 -group and phosphate group at the 5' end of the aptamer are connected to the Cy5 fluorophore.
  • conjugate 20 was prepared according to the following steps.
  • the conjugate 20 includes the aptamer provided by the present disclosure and the small molecule drug group MMAE.
  • the MMAE group is connected to the aptamer through a linking group.
  • the 5' end of the body, the connecting group is 2-(phosphate-(CH 2 ) 6 -S-)-maleimidocaproyl-valine-citrulline-p-aminobenzyl subunit.
  • the aptamer sequence in conjugate 20 in Table 1C was synthesized by solid-phase synthesis method. The only difference is that the nucleoside monomers were connected in sequence according to the aptamer sequence corresponding to conjugate 20 in Table 1C; in solid-phase synthesis In the process, after connecting the last nucleoside monomer at the 5' end, follow the method of connecting the nucleoside phosphoramidite monomer, and additionally connect the group containing HO-(CH 2 ) 6 -SS-(CH 2 ) 6 - The group of phosphoramidite monomer (purchased from Hongene Biotech Company) was cleaved from the solid phase carrier to obtain the aptamer single chain S1 (70.00 mg, 6.42 ⁇ mol):
  • the obtained crude conjugate 20 was diluted with 0.5 ml of purified water and filtered with a 0.45 ⁇ m membrane.
  • the filtrate was purified using Agilent semi-preparative reversed-phase column chromatography.
  • the product peak eluate was collected, and the solvent was evaporated to remove conjugate 20 (55 mg, yield 56.7%).
  • the molecular weight was detected by LC-MS, the theoretical value: 12092.67, the measured value: 12091.68, the measured value is consistent with the theoretical value, indicating that the conjugate 20 has the structure shown in S3, and contains the aptamer and small molecule drug provided by the present disclosure.
  • Group MMAE the MMAE group is connected to the 5′ end of the aptamer via a linking group, the linking group is 2-(phosphate-(CH 2 ) 6 -S-)-maleimide Aminocaproyl-Valine-citrulline-p-aminobenzyl subunit (2-(phosphate-(CH 2 ) 6 -S-)-MC-Val-Cit-PAB).
  • the capital letters C, G, U, and A represent the base composition of nucleotides;
  • the small letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide;
  • the small letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide;
  • f means that the nucleotide adjacent to the left side of the letter f is a fluorinated modified nucleotide;
  • MMAE means the connection position of the small molecule drug group MMAE (monomethyl auristatin E) group on the aptamer point.
  • the conjugates numbered AP21-AP26 in Table 2 were respectively synthesized and the molecular weights were detected to confirm the synthesized conjugates. The only difference was that the conjugates numbered AP21-AP26 in Table 2 were respectively synthesized.
  • the sequences of AP21-AP26 connect the nucleoside monomers in sequence.
  • conjugates AP21-AP26 with the fluorescent group Cy5 connected to the 5' end were obtained respectively.
  • AP21 has one nucleotide less at the 3′ end; compared with the aforementioned AP4, AP22 corresponds to the S 1 and S 4 motifs in the sequence shown in formula (1) each having one less nucleotide.
  • the structure shown by the nucleotide; AP23 is the structure shown by AP22 corresponding to the S 1 motif in the sequence shown in formula (1) and then reduced by 1 nucleotide;
  • AP24 is obtained by changing the first 2 nucleotides of the 5' end of AP4 Conjugates; AP25 and AP26 are conjugates obtained by changing the N b motif in the sequence shown in formula (1) compared with the aforementioned AP4.
  • the prepared conjugates are diluted to a concentration of 0.2mg/ After mL, use liquid chromatography-mass spectrometry (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters Company, model: LCT Premier) for molecular weight detection.
  • LC-MS Liquid Chromatography-Mass Spectrometry
  • the N b motif is missing in the sequence shown; comparing AP33 with AP4, it corresponds to the missing Na and N c motifs in the sequence shown in formula (1); comparing AP34 with comparing AP35, it is between S2 and Control conjugates that each added multiple nucleotides to the S3 motif portion and altered part of the sequence.
  • the capital letters C, G, U, and A indicate the base composition of nucleotides; the small letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; the small letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; f indicates that the nucleotide adjacent to the left side of the letter f is a fluorinated modified nucleotide; CY5 indicates the connection site of the fluorescent dye group Cy5 (Cyanine 5) group on the aptamer.
  • the prepared conjugate was diluted to a concentration of 0.2mg/mL using ultrapure water (made by Milli-Q ultrapure water instrument, resistivity 18.2M ⁇ *cm (25))
  • ultrapure water made by Milli-Q ultrapure water instrument, resistivity 18.2M ⁇ *cm (25)
  • LC-MS Liquid Chromatography-Mass Spectrometry, purchased from Waters Company, model: LCT Premier
  • the conjugate numbered as conjugate 36 in Table 1B was synthesized according to the same method as in Preparation Examples 1-8 and 12. The only difference is that the nucleoside monomers were connected sequentially according to the nucleic acid sequence corresponding to conjugate 36 in Table 1B. body; and, when preparing the sense strand, the 3' end of the siRNA sense strand is connected to the delivery group contained in the conjugate AP2 via 6 dT phosphate groups as the linking group; the 5' end of the siRNA sense strand passes through A Cy5 fluorophore is attached to the phosphate linker.
  • the purity of the obtained conjugate 36 sense strand sequence and antisense strand sequence were detected using ion exchange chromatography (IEX-HPLC), and the molecular weight was analyzed using liquid mass spectrometry chromatography (LC-MS).
  • IEX-HPLC ion exchange chromatography
  • LC-MS liquid mass spectrometry chromatography
  • conjugates numbered as conjugate 37 and conjugate 38 in Table 1B were synthesized according to the same method as in Preparation Examples 1-8 and 12. The only difference is that the conjugates 37 and 38 in Table 1B were synthesized according to the same method.
  • the nucleotide sequences of the sense strand or antisense strand of 38 are connected in sequence from the 3′-5′ direction to all nucleoside monomers to obtain the sense strand and antisense strand sequences of the conjugate. in:
  • the sense strand sequence contains the sense strand of siRNA, and the 3′ end of the sense strand of siRNA is connected to the 5′ end of the delivery group contained in conjugate AP2 via Spacer18-GfAfUf as a linking group, which The fluorescent group Cy5 is connected to the 5' position of the ribose sugar of the 5' terminal nucleotide of the antisense strand through a phosphate linking group.
  • the sense strand sequence contains the sense strand of siRNA, and the 5′ end of the sense strand of siRNA is connected to the delivery group 3′ of conjugate AP2 via 4 propylene-phosphate groups connected in sequence.
  • the fluorescent group Cy5 is connected to the 5' position of the ribose sugar of the 5' terminal nucleotide of the antisense chain through a phosphate linking group.
  • the purity of the obtained sense strand sequence and antisense strand sequence of conjugate 37 and conjugate 38 is detected by ion exchange chromatography (IEX-HPLC), and liquid mass spectrometry (LC-MS) is used.
  • IEX-HPLC ion exchange chromatography
  • LC-MS liquid mass spectrometry
  • Conjugate 39 was prepared according to the same method as preparation 20, and the molecular weight of conjugate 39 was detected by LC-MS.
  • the theoretical value of conjugate 39 was 9473.03, and the measured value was 9472.06.
  • the measured value was consistent with the theoretical value, indicating that the conjugate 39 was consistent with the sequence structure of Table 1C.
  • the molecular weights of conjugate 40, conjugate 41, conjugate 42, comparative conjugate 43 and comparative conjugate 44 were all detected by LC-MS, and the measured values were consistent with the theoretical values.
  • the conjugate 39, conjugate 40, conjugate 41, conjugate 42, comparative conjugate 43 and comparative conjugate 44 numbered in Table 1C were synthesized respectively, and the molecular weight was detected to Confirm the synthesized conjugates, the only difference is that they are conjugate 39, conjugate 40, conjugate 41, conjugate 42, comparative conjugate 43 and comparative conjugate corresponding to Table 1C.
  • the sequence of substance 44 is connected to the nucleoside monomers in sequence.
  • U118MG gliomas were cultured in DMEM medium (Thermo Fisher Company) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Company) at 37°C in an incubator containing 5% CO 2 /95% air.
  • FBS fetal bovine serum
  • U118MG cells were seeded in 96-well plates at 5 ⁇ 10 5 cells/well.
  • the previously prepared conjugate AP1 and yeast tRNA were prepared into conjugate solutions 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H and 1I respectively using DMEM medium.
  • the conjugate solutions 1A-1I contained The final concentration was 100 ⁇ g/mL of yeast tRNA, and each contained conjugate AP1 at final concentrations of 5 nM, 10 nM, 20 nM, 40 nM, 60 nM, 80 nM, 100 nM, 150 nM, 200 nM and 500 nM.
  • the conjugate solutions 1A′-1I′ contain yeast tRNA with a final concentration of 100 ⁇ g/mL, and respectively contain final concentrations of 5nM, 10nM, 20nM, 40nM, 60nM, 80nM, 100nM, 150nM, 200nM and 500nM. Control conjugate versus AP10.
  • MFI AP1 represents the average fluorescence intensity of conjugate AP1 in U118-MG glioma cells
  • MFI CAP10 represents the average fluorescence intensity of AP10 in U118-MG glioma cells.
  • the R value reflects the relative ability of the conjugate AP1 to enter U118-MG glioma cells. When R>2, it indicates that the corresponding conjugate has a stronger ability to enter the cells (see Engineering of Targeted Nanoparticles for Cancer Therapy Using Internalizing Aptamers Isolated by Cell-Uptake Selection. ACS Nano. 2012 January 24; 6(1) is recorded in Table 2, the entire disclosure of which is incorporated herein by reference).
  • the conjugate AP1 provided by the present disclosure showed significantly higher average fluorescence intensity in U118MG glioma cells compared with the comparison AP10 of the random sequence, indicating that the conjugate AP1 provided by the present disclosure provides The conjugate has excellent ability to enter U118MG glioma cells.
  • the PerkinElmer high-content imaging system (Operatta Company) was used to observe the entry of the conjugate AP1 and the comparative conjugate AP10 provided in the present disclosure into different cells.
  • DPBS buffer purchased from Thermo Fisher Company
  • conjugate AP1 and yeast tRNA were prepared into conjugate solution 2A using DMEM medium.
  • the conjugate solution 2A contained yeast at a final concentration of 100 ⁇ g/mL.
  • tRNA and conjugate AP1 at a final concentration of 100 nM;
  • control conjugate solution 2B contains yeast tRNA with a final concentration of 100 ⁇ g/mL and a final concentration of 100 nM. Comparison of AP10;
  • test group 2X For each cell line, add 200 ⁇ L of conjugate solution 2A to 1 culture well, incubate in the dark at 37°C for 30 minutes, aspirate the supernatant, add DPBS buffer and wash twice, 100 ⁇ L each time, to obtain the test group 2X;
  • control conjugate solution 2B For each cell line, add 200 ⁇ L of control conjugate solution 2B to another culture well, incubate at 37°C in the dark for 30 minutes, aspirate the supernatant, add DPBS buffer and wash twice, 100 ⁇ L each time, to get Control group 2Y.
  • Figures 1A and 1B are high-content imaging images showing the entry of AP1 and contrasting AP10 into U118MG glioma cells and SVGp12 normal astrocytes, respectively.
  • Figures 1A and 1B only the U118MG cells treated with AP1 showed strong Cy5 fluorescence signals, while there was no fluorescence signal in the SVGp12 cells in the test group and the control group, indicating that compared with the comparative AP10, this
  • the publicly provided conjugates have a strong ability to enter U118MG glioma cells, while basically not entering SVGp12 normal astrocytes.
  • the above results also show that the conjugate provided by the present invention can selectively target and deliver functional groups to U118MG glioma cells.
  • Figures 1C-1G respectively show the high-content imaging images of AP1 and comparative AP10 entering U251 human glioma cells, A549 human non-small cell lung cancer cells, MCF-7 human breast cancer cells and 293T human kidney epithelial cells.
  • U251 human glioma cells, A549 human non-small cell lung cancer cells, and MCF-7 human breast cancer cells treated with AP1 all showed strong Cy5 fluorescence signals.
  • Comparative AP10-treated tumor cells and 293T human kidney epithelial cells did not have any fluorescence signals in the test group and the control group, indicating that compared with the comparative AP10, the conjugate provided by the present disclosure has a strong ability to enter various tumor cells. While not entering normal cells, the conjugate provided by the invention can selectively target and deliver functional groups to various tumor cells. .
  • the PerkinElmer high-content imaging system (Operatta Company) was used to examine the entry of the conjugate AP1 provided by the present disclosure and the comparison conjugate AP10 into U118MG glioma tumor spheres and A549 human non-small cell lung cancer tumor spheres. observe.
  • each cell line was inoculated into 2 culture wells, with 200 ⁇ L of cell suspension in each well, cultured in DMEM medium supplemented with 10% FBS and 1% penicillin double antibody, and incubated at 37°C in a 5% CO 2 /95 % air incubator. Aspirate half of the supernatant every 48 hours, and add new DMEM medium to the wall of the culture well. After 7 days of culture, it was confirmed that complete cell spheres were obtained under the microscope, indicating that the tumor spheres were successfully cultured.
  • Figures 2A and 2B show high-content imaging of AP1 and contrast AP10 entering U118MG glioma tumor spheres and A549 human non-small cell lung cancer tumor spheres, respectively.
  • the AP1-treated U118MG tumor spheres and A549 tumor spheres showed strong Cy5 fluorescence signals in the middle and inner parts, while in the AP10-treated tumor spheres, only the edges of the tumor spheres were obvious.
  • the weaker fluorescence signal indicates that the conjugate provided by the present disclosure has a strong ability to enter the interior of U118MG and A549 tumor spheres compared with comparative AP10.
  • the above results illustrate that the aptamer provided by the present disclosure can effectively deliver functional groups into tumors and has high drug-making ability.
  • U118MG human colloid was cultured in DMEM complete medium (MACGENE Company, Cat. No. CM15019) supplemented with 10% fetal bovine serum (FBS, RMBIO Company) at 37 in an incubator containing 5% CO 2 /95% air.
  • Tumor cells purchased from Guangzhou Genio Biotechnology Co., Ltd.).
  • NOD-SCID mice purchased from Spefford (Beijing) Biotechnology Co., Ltd.
  • the above cell culture medium was inoculated into the subcutaneous position of the right forelimb of NOD-SCID mice.
  • the inoculation volume was 100 ⁇ L per mouse, that is, 1 ⁇ 10 7 cells were inoculated into each mouse.
  • the mice were kept for 20 days after injection.
  • mice vaccinated with U118MG were randomly divided into 3 groups, with 4 mice in each group; for the 4 mice in each group, the dosage volume was calculated according to 10 ⁇ L/g body weight, and the mice in different groups were injected into the tail vein.
  • AP1, AP2 and comparison AP11 were administered respectively, and were recorded as test group 4A1, test group 4A2 and control group 4A3 respectively. Therefore, each mouse was administered a dose of 3 mg/kg (based on aptamer).
  • Another mouse inoculated with U118MG was injected into the tail vein with 1 ⁇ DMEM medium at a volume of 10 ⁇ L/g body weight, which was recorded as the blank control group 4Y.
  • mice in each group were placed in the small animal in vivo optical imaging system IVIS Lumina Series III (PerkinElmer).
  • the mice were anesthetized with isoflurane gas, and the anesthetized mice were placed in the small animal with their abdomen facing up.
  • In vivo imaging is performed in an in vivo optical imaging system for animals.
  • 1h, 4h, 24h and 48h after administration one mouse in each group was sacrificed, and the tumor tissue was taken for fluorescence imaging (no blank control group was used for the 24h and 48h results). The results are shown in Figures 3A, 3B, and Shown in 3C and 3D.
  • Figures 3A-3D respectively show the in vivo imaging and tumor tissue imaging at 1h, 4h, 24h and 48h after administration, where blank indicates the blank control group 4Y.
  • the blank control group 4Y and the control group 4A3 administered with comparative AP11 with random sequences showed basically no or only weak fluorescence signals in mice; different from this, the modified ones provided by the present disclosure Conjugates AP1 and AP2 showed strong fluorescence signals at the site of tumor inoculation in mice, indicating that the aptamer provided by the present disclosure can specifically deliver fluorescent groups to U118MG glioma; further, the conjugates AP2 still showed a strong fluorescence signal from 24h to 48h after administration, indicating that this The publicly provided conjugates are able to stably target glioma tissue over an extended period of time.
  • NOD-SCID mice 16 NOD-SCID mice (purchased from Spefford (Beijing) Biotechnology Co., Ltd.), male, 12 weeks old.
  • the above cell culture fluid was inoculated into NOD-SCID mice, and the cell culture fluid was injected into the right striatum of the mouse using the lateral cerebral ventricle injection method.
  • the position was AP (anteroposterior anteroposterior/anterior position): 1 mm
  • ML medial lateral
  • DV diorsal ventral
  • injection volume 10 ⁇ L that is, each mouse is inoculated with 4 ⁇ 10 5 cells. Mice were kept for 14 days after injection.
  • AP1, AP2 and comparative AP11 were dissolved into conjugate solutions with a concentration of 0.3 mg/mL (based on aptamer) using 1 ⁇ DMEM medium. Take another 6 mice mentioned above and administer them through tail vein injection. Inject AP1, AP2 and comparative AP11 solutions respectively. The dosage of all animals is calculated according to their body weight. The dosage volume is 10 ⁇ L/g, based on the amount of aptamer. , the dosage of each animal was 3 mg/kg, and 2 mice were administered to each group, which were recorded as test group 5D, 5E and control group 5F respectively.
  • mice in each group were killed and the brain tissue was taken.
  • mice were killed and the brain tissue was taken.
  • the mouse brain tissue was analyzed in IVIS Lumina Series III. Fluorescence imaging. The results are shown in Figures 4A and 4B respectively.
  • FIGS 4A and 4B show respectively that at 24h and 48h after administration, the blank control group was given 5Y, Fluorescence imaging of the brain tissue of mice with U118MG orthotopic tumor model established in test groups 5A, 5B, 5D and 5E and control groups 5C and 5F.
  • Test groups 5A and 5B administered the conjugates provided by the present disclosure showed strong fluorescence signals at the site of inoculated tumors, indicating that they can effectively target tumor tissues, and indicating that the aptamers provided by the present disclosure can combine diagnostic agent groups such as The fluorescent group was effectively delivered to the tumor tissue; further, test group 5E showed obvious fluorescence signals at both 24h and 48h, indicating that the conjugate AP2 could still reach and target brain glioma when administered through the tail vein.
  • the conjugates provided by the present disclosure can also penetrate the blood-brain-barrier (BBB) and enter glioma.
  • BBB blood-brain-barrier
  • U118MG subcutaneous tumor model mice were prepared according to the method described in Experimental Example 4.
  • AP2, AP3, and AP12 were dissolved into conjugate solutions with a concentration of 0.3 mg/mL (based on aptamer) using 1 ⁇ DMEM medium.
  • Each mouse in each group is injected with the above-mentioned AP2, AP3 and AP12 solutions through the tail vein respectively.
  • the dosage of all animals is calculated according to their body weight, and the dosage volume is 10 ⁇ L. /g, based on the amount of aptamer, the dosage per animal is 3mg/kg, and the three groups of mice are respectively designated as test group 6A, test group 6B and test group 6C.
  • mice were given DMEM medium with a dosage volume of 10 ⁇ L/g, which was recorded as the blank control group 6Y.
  • One mouse in each group was placed in the small animal in vivo optical imaging system IVIS Lumina Series III (PerkinElmer) at 30 min, 1 h, 24 h, and 48 h after administration, and the mice were anesthetized with isoflurane gas.
  • the mouse was placed belly-up in the small animal in vivo optical imaging system for in vivo imaging. The results are shown in Figures 5A-5D.
  • Figures 5A-5E respectively show fluorescence imaging in mice at different time points after administration of different sequences of conjugates and comparative conjugates provided by the present disclosure
  • Figures 5F-5H respectively show tumor tissue imaging at different time points. picture.
  • the results in Figures 5A-5H show that 30 minutes after administration, each test group and Fluorescence signals were visible in the subcutaneous tumors of the control group; 4h-24h after administration, mice in test groups 6A, 6B and 6C that were administered AP2, AP3 and AP12 provided by the present disclosure still showed higher fluorescence intensity; 48h after administration - On day 10, the test group 6A mice administered AP2 still showed strong fluorescence signals.
  • the conjugate provided by the present disclosure can stably target tumor tissue for a long time, and illustrate that the aptamer provided by the present disclosure can stably target the diagnostic agent for a long time. Targeted delivery of groups to tumors.
  • A549 human non-small cell lung cancer cell subcutaneous tumor model mice were prepared according to the method described in Experimental Example 4. The only difference was that A549 human non-small cell lung cancer cells were used instead of U118MG glioma cells to prepare the cell culture medium.
  • mice Eleven 6- to 8-week-old NOD-SCID male mice (purchased from Spefford (Beijing) Biotechnology Company) were selected and randomly divided into groups, including 2 mice in the blank control group and 3 mice in each group.
  • the above culture medium containing A549 tumor cells was inoculated into the subcutaneous position of the right forelimb of mice.
  • the volume of A549 tumor cells inoculated into each mouse was 100 ⁇ L. From the calculation, the number of cells inoculated into each mouse was 1 ⁇ 10 7 .
  • the dosage for all animals was calculated based on body weight, and the dosage volume for each mouse was 10 ⁇ L/g. That is, except for the blank control group, the dosage of each mouse in the other groups was 3 mg/kg (based on the amount of aptamer); the blank control group was only given 10 ⁇ L/g DMEM medium.
  • Figure 6 shows the fluorescence image of the organs of one mouse in each group 1 hour after administration. From Figure 7 It can be seen that, except for the blank control group, strong fluorescence signal intensity can be detected in the A549 tumors of the AP2 group, AP3 group and AP12 group, and the fluorescence signal intensity of the AP3 group is stronger than that of other groups. .
  • the PAN02 tumor cell model was used to verify the ability of the conjugate provided by the present disclosure to enter PAN02 tumor cells.
  • PAN02 tumor cells growing in the logarithmic phase (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were digested and resuspended in DMEM medium supplemented with 10% fetal calf serum until the cell density of PAN02 tumor cells reached 1 ⁇ 10 8 cells/mL. .
  • mice were sacrificed, and the heart, liver, spleen, lung and tumor tissues of the sacrificed mice were removed, fluorescence imaging was performed in IVIS Lumina Series III, and photos were taken under the same field of view. The results are shown in As shown in Figure 7.
  • Figure 7 is a diagram showing fluorescence imaging of various organs and tissues in mice establishing a PAN02 subcutaneous tumor model after administration of the conjugate provided by the present disclosure. As can be seen from Figure 7, the fluorescence signal in PAN02 tumor cells is significantly stronger than that in other organs, indicating that the conjugate provided by the present disclosure can specifically target PAN02 pancreatic cancer tumor cells.
  • the U118MG glioma model was used to verify that the methods provided by this disclosure include Entry ability of CY5-fluorophore and CY3-fluorophore conjugates into U118MG glioma.
  • U118MG glioma cells (purchased from Guangzhou Genio Biotechnology Co., Ltd.) growing in the logarithmic phase were digested and resuspended in DMEM medium supplemented with 10% fetal calf serum until the cell density of U118MG glioma cells reached Reach 1 ⁇ 10 8 cells/mL.
  • mice Eleven 6-8 week old NOD-SCID male mice (purchased from Spefford (Beijing) Biotechnology Company) were selected and randomly divided into groups, including 3 mice in the first group and 2 mice in the second group. There were 3 mice in the third group and 3 mice in the fourth group.
  • the above-mentioned medium containing U118MG glioma cells was inoculated into the subcutaneous position of the right forelimb of mice.
  • the volume of cells inoculated per mouse was 100 ⁇ L. From the calculation, the number of cells inoculated per mouse was 1 ⁇ 10 7 .
  • 1 ⁇ DMEM (purchased from Zhongke Maichen (Beijing) Technology Co., Ltd., batch number K1902200) culture medium to dissolve AP2, AP9 and AP12 into conjugate solutions with a concentration of 0.3mg/mL (based on aptamer). .
  • 1 ⁇ DMEM and the above-prepared AP2, AP9 and AP12 conjugate solutions were injected into the tail vein of each mouse.
  • mice in the first group were injected with AP2 conjugate solution
  • 2 mice in the second group were injected with AP9 conjugate solution
  • 3 mice in the third group were injected with AP12 conjugate solution
  • 3 mice in the fourth group were injected with AP12 conjugate solution.
  • Three mice were injected with 1 ⁇ DMEM as a blank control group.
  • the dosage for all animals was calculated based on body weight, and the dosage volume for each mouse was 10 ⁇ L/g. That is, the dose administered to each mouse in the first group to the third group was 3 mg/kg (based on the amount of aptamer).
  • Figure 8A shows the fluorescence images of organs of one mouse in the first group, one mouse in the third group, and one mouse in the fourth group 48 hours after administration. As can be seen from Figure 8A, no fluorescence signal was detected in either the third or fourth group. At the same time, significant fluorescent signals could be detected in the tumor tissues of mice injected with AP2, and no fluorescent signals were detected in other tissues except the metabolic organ kidney, indicating that AP2 can be effectively targeted compared with the comparison conjugate. delivered to tumor tissue, and There was no significant targeted delivery in other tissues.
  • Figure 8B shows the fluorescence images of organs of one mouse in the first group, one mouse in the third group, and one mouse in the fourth group 96 hours after administration. As can be seen from Figure 8B, AP2 still showed a significant fluorescence signal in tumor tissue after 96 hours.
  • Figure 8C shows the fluorescence images of organs of one mouse in the second group and one mouse in the fourth group 48 hours after administration.
  • Figure 8C compared with the control group, significant fluorescence signals can be detected in the tumor tissues of mice injected with Cy3-labeled AP9, indicating that the aptamer provided by the present disclosure can efficiently combine different diagnostic agent groups. (such as fluorescent groups) for targeted delivery to tumor tissue, and the conjugates containing diagnostic agent groups provided by the present disclosure can stably target to tumor tissue.
  • diagnostic agent groups such as fluorescent groups
  • Figure 8D shows the fluorescence images of organs of one mouse in the second group and one mouse in the fourth group 96 hours after administration.
  • significant fluorescent signals can be detected in the tumor tissues of mice injected with Cy3-labeled AP9, and no fluorescent signals were detected in other tissues except the metabolic organ kidney.
  • the aptamer provided by the present disclosure can efficiently deliver different diagnostic agent groups (such as different fluorescent groups) to U118MG glioma, and can remain stable for a long time.
  • Figure 8E shows the fluorescence images of organs of one mouse in the first group, one mouse in the third group, and one mouse in the fourth group 11 days after administration.
  • AP2 could still detect significant fluorescence signals in tumor tissues on the 11th day after administration, and no fluorescence signals were detected in other tissues except the metabolic organ kidney.
  • the conjugate provided by the present disclosure can still stably and highly specifically target U118MG glioma tissue over a long period of time.
  • the aptamer provided by the present disclosure can not only specifically target glioma tissue, but also deliver different diagnostic agent groups to glioma. Therefore, the aptamer provided by the present disclosure shows excellent delivery ability; at the same time, the conjugate containing a diagnostic agent group provided by the present disclosure can target glioma efficiently and stably, thereby being able to rapidly and effectively target glioma. The presence of tumor can be diagnosed, and the diagnostic effect can remain stable for a long time.
  • the U118MG glioma model was used to examine the targeting effects of conjugates of different sequences and different lengths on U118MG glioma.
  • U118MG glioma cells growing in logarithmic phase (purchased from Guangzhou Genio Biotechnology Co., Ltd. Company), digested and resuspended in DMEM medium supplemented with 10% fetal calf serum until the cell density of U118MG glioma cells reached 1 ⁇ 10 8 cells/mL.
  • mice Fourteen 6-8 week old NOD-SCID male mice (purchased from Spefford (Beijing) Biotechnology Company) were selected and randomly divided into groups, with 2 mice in each group.
  • the above culture medium containing U118MG glioma cells was inoculated into the subcutaneous position of the right forelimb of mice.
  • the volume of U118MG glioma inoculated into each mouse was 100 ⁇ L. From the calculation, it can be seen that the number of cells inoculated into each mouse was 1 ⁇ 10 7 .
  • each group of mice injected with AP2, AP4, AP5, AP6, AP7, AP8 and AP12 were respectively named AP2 group, AP4 group, AP5 group, AP6 group, AP7 group, AP8 group and AP12 group.
  • the dosage of all animals was calculated based on their body weight. Based on the amount of aptamer, the dosage volume for each mouse was 10 ⁇ L/g. That is, the dose administered to each mouse was 3 mg/kg.
  • mice in each group were placed in the small animal in vivo optical imaging system IVIS Lumina Series III. Mice were anesthetized with halothane gas, and the anesthetized mice were placed with their abdomen facing up in a small animal in vivo optical imaging system for in vivo imaging. The fluorescence signal of Cy5 was dynamically detected and the distribution of different Cy5-labeled conjugates in the living animals was tracked. Condition. Analysis of the observation results showed that 1 hour after administration, fluorescence signals were visible in the subcutaneous tumors in each mouse, and there was no significant difference in fluorescence signal intensity between mice in each group.
  • Figure 9 is a diagram showing tumor fluorescence images of mice in each group on D9 after administration. As can be seen from Figure 9, Cy5 fluorescence signals can be detected in the tumor tissues of mice in each group, indicating that the present disclosure does not Conjugates of the same length and sequence can stably and effectively target tumor tissues over a long period of time.
  • This experimental example examined the anti-tumor activity of the prepared conjugate 20 in mice.
  • mice in this experiment were purchased from Spefford Company.
  • the germ line is NOD-SCID, the grade is SPF, both genders are female, and the age is 6-8 weeks.
  • U118MG glioma cells were purchased from Genio.
  • U118MG cells growing in the logarithmic phase were digested and resuspended in DMEM complete medium (MACGENE Company, Cat. No. CM15019) supplemented with 10% fetal bovine serum (FBS, GIBCO Company) and cultured until the cell density was 1 ⁇ 10 8 cells/mL to obtain a culture medium containing U118MG cells.
  • the above culture medium containing U118MG cells was inoculated into the subcutaneous position of the right forelimb of each mouse, with an injection volume of 100 ⁇ L. Thus, each mouse was inoculated with 1 ⁇ 10 7 U118MG glioma cells.
  • the conjugate AP2 prepared above was prepared into a 1.94 mg/mL solution using PBS.
  • Conjugate 20 was prepared with PBS into solutions of 0.625 mg/mL, 1.25 mg/mL and 2.06 mg/mL (all calculated based on the amount of aptamer); use 10% DMSO + 90% PBS (volume ratio) Mix the solution to dissolve MMAE (purchased from Shanghai McLean Company, batch number C12886583) into a solution of 0.038 mg/mL.
  • the day of cell inoculation was recorded as D1, and the cells were administered once each on D8, D12, D16 and D20.
  • mice 36 mice were randomly divided into the following 6 groups, with 6 mice in each group:
  • PBS was administered by tail vein injection, with a single administration volume of 10 ⁇ L/g;
  • control group 2 the above-mentioned conjugate AP2 solution was administered by tail vein injection, with a single administration volume of 10 ⁇ L/g and a single administration dose of 15.5 mg/kg;
  • MMAE solution was administered by tail vein injection, with a single administration volume of 10 ⁇ L/g and a single administration dose of 0.3 mg/kg;
  • the conjugate 20 solution with the above concentration of 0.625 mg/mL was administered by tail vein injection, with a single administration volume of 10 ⁇ L/g and a single administration dose of 5 mg/kg (based on the mass of the aptamer). (calculated), which contains MMAE at a dose equivalent to 0.3 mg/kg;
  • Conjugate 20 solution has a single administration volume of 10 ⁇ L/g and a single administration dose of 16.5 mg/kg (based on aptamer mass), in which the dose containing MMAE is equivalent to 1 mg/kg;
  • the conjugate 20 solution with the above concentration of 1.25 mg/mL was administered by subcutaneous injection, with a single administration volume of 5 ⁇ L/g and a single administration dose of 5 mg/kg (based on aptamer mass). ), which contains MMAE at a dose equivalent to 0.3 mg/kg.
  • the long diameter and short diameter of the tumor were determined by in vitro measurement. Tumor volume was calculated according to the formula 1/2 (long diameter ⁇ short diameter 2 ). Before the first administration on D8, the tumor volume of each group was measured and the average tumor volume was recorded. The tumor volume of each group was measured and recorded on D16, twice a week.
  • Figure 10 is a line graph showing changes in tumor volume over time in each group of mice. It can be seen from the results in Figure 10 that in the blank control group 1 and control group 2 that were only given PBS and AP2, the tumor volume increased rapidly; in the control group 3 that was only given MMAE, the tumor volume increase rate was reduced, indicating that MMAE itself has an effect on tumor proliferation. Shows inhibitory effect. Furthermore, test groups 4 and 6, whose MMAE content was equivalent to that of control group 3, had significantly smaller tumor volumes during the test period than control group 3, showing more excellent anti-tumor activity than control group 3, which was administered MMAE alone. This shows that the conjugate provided by the present disclosure can effectively deliver MMAE to tumor tissue.
  • the above results show that the conjugates provided by the present disclosure can effectively deliver small molecule drug groups with inhibitory effects on tumors to tumor tissues.
  • the conjugates containing small molecule drug groups provided by the present disclosure show good performance. antitumor activity and dose-dependent effects.
  • This experimental example examined the effect of the prepared conjugate 19 on the viability of normal cells and tumor cells.
  • the U118-MG glioma cells used in this experiment were purchased from Genio; SVGp12 human astrocytes were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences; the CCK8 kit was purchased from Tongren Chemical Research Institute.
  • U118-MG cells and SVGp12 cells were selected respectively, and 5,000 cells per well were seeded in a 96-well plate of the CCK8 kit. They were cultured at 37°C with 5% CO2 for 24 hours. When the cell confluence was 80%, the cells to be obtained were obtained. Detect U118-MG cells and SVGp12 cells to be detected.
  • AP1 solution 1 (concentration is 0 nM); AP1 solution 2 (concentration is 50 nM); AP1 solution 3 (concentration is 200 nM); AP1 solution 4 (concentration is 800 nM); and the following conjugate solutions: Conjugate 19 solution 1 (concentration is 0nM); Conjugate 19 solution 2 (concentration is 25nM); Conjugate 19 solution 2 (concentration is 25nM); Conjugate 19 solution 3 (concentration is 50nM); Conjugate 19 solution 4 (concentration is 100nM) ; Conjugate 19 solution 5 (concentration is 200nM); Conjugate 19 solution 6 (concentration is 400nM); Conjugate 19 solution 7 (concentration is 800nM), and each of the above conjugate solutions also contains
  • Conjugate 19 solution 1 conjugate 19 solution 2, conjugate 19 solution 3, conjugate 19 solution 4, conjugate 19 solution 5, conjugate 19 solution 6 and conjugate 19 solution 7 respectively.
  • Conjugate 19 solution 1 conjugate 19 solution 2, conjugate 19 solution 3, conjugate 19 solution 4, conjugate 19 solution 5, conjugate 19 solution 6 and conjugate 19 solution 7 respectively.
  • Add to the SVGp12 cells to be detected and incubate at 37°C in the dark for 72h. Each group of experiments was repeated 5 times, and was recorded as the blank control group 12D1 and the test group 12D2-12D7 respectively.
  • Cell viability% (test group OD450-blank group OD450)/(control group OD450-blank group OD450) ⁇ 100%
  • test group OD450 refers to the absorbance value of each test group at 450nm
  • control group OD450 is the absorbance value of the blank control group corresponding to each test group at 450nm
  • blank group OA450 is the blank colorimetric cell used in UV detection. Absorbance value at 450nm.
  • Figure 11A shows the effects of conjugate AP1 at different concentrations on the cell viability of U118-MG glioma cells and SVGp12 human astrocytes
  • Figure 11B shows the effects of conjugate 19 at different concentrations on U118- Effects on cell viability of MG glioma cells and SVGp12 human astrocytes.
  • conjugate AP1 containing only the diagnostic agent group CY5 has no effect on the cell viability of normal cells and tumor cells at different concentrations.
  • conjugate 19 containing siRNA can significantly reduce the cell viability of tumor cells, and the inhibitory efficacy increases with the increase in dosage.
  • the cell viability was only slightly reduced and remained basically stable, indicating that the aptamer provided by the present disclosure, after being conjugated with siRNA, can not only efficiently target tumor cells and inhibit the content of target mRNA, but also It does not significantly affect the corresponding mRNA levels in normal cells, and has excellent targeted delivery efficiency and high safety.
  • the above results indicate that the conjugates containing functional oligonucleotides provided by the present disclosure can effectively deliver functional oligonucleotides with inhibitory effects on tumors, such as siRNA, to tumor tissues, showing good Antitumor activity and dose-dependent effects.
  • This experimental example examines the targeting properties of the prepared conjugate 15, conjugate 16, conjugate 17 and conjugate 18 in mice.
  • Conjugates 15-18 contained the same aptamer sequence.
  • the sense strand sequence includes the sense strand of siRNA, the 5′ end of the sense strand of siRNA is connected to the fluorescent group Cy5, and the 3′ end is connected to the GAU trinucleotide subunit as a linking group.
  • Aptamers provided by the present disclosure.
  • the sense strand sequence contains the sense strand of siRNA, which is The 5' end of the sense chain is connected to the aptamer provided by the present disclosure via the GAU trinucleotide subunit as a linking group, and the 5' end of the aptamer is connected to the fluorescent group Cy5.
  • Conjugate 17 is a conjugate comprising a double-stranded siRNA and an aptamer provided by the present disclosure, wherein the aptamer is connected to the 5′ sense strand of the siRNA via four sequentially connected propylene phosphate groups as a linking group. end, and the Cy5 fluorophore is connected to the 5′ end of the aptamer.
  • Conjugate 18 has a similar structure to conjugate 17, but the aptamer structure is slightly different from the aptamer in conjugate AP2, see Table 1B and Preparation Example 18.
  • U118MG cells were cultured according to the method of Experimental Example 4 and inoculated subcutaneously into mice.
  • Conjugate 15 conjugate 16, conjugate 17 and conjugate 18 prepared above were prepared into 0.3 mg/mL solutions using PBS.
  • Administration began 30 days after U118MG cells were inoculated, and the day of administration was recorded as D1. The experiment adopted tail vein injection, once a day, for a total of three administrations.
  • mice 12 mice (all weighing about 25g) were randomly divided into 4 groups, with 3 mice in each group:
  • test group 1 conjugate 15 was used, the single administration volume was 250 ⁇ L, and the single administration dose was 3 mg/kg;
  • test group 2 conjugate 16 was used, the single administration volume was 250 ⁇ L, and the single administration dose was 3 mg/kg;
  • conjugate 17 was used, the single administration volume was 250 ⁇ L, and the single administration dose was 3 mg/kg;
  • conjugate 18 was used, with a single administration volume of 250 ⁇ L and a single administration dose of 3 mg/kg.
  • each mouse was imaged in vivo using the small animal in vivo optical imaging system IVIS Lumina Series III.
  • Figure 12 is a graph showing fluorescence imaging results in mice after administration of different conjugates. It can be seen from Figure 12 that each conjugate using different linking groups, different connection methods, and different aptamers is enriched in tumor cells. The above results indicate that although the included aptamers The sequences linked to siRNA or aptamers in different ways vary, but the various conjugates provided by the present disclosure are all able to effectively target tumor tissue. Furthermore, compared with other conjugates, the fluorescence signal of conjugate 18 is more intense and displays a diffuse distribution in the body, indicating that conjugate 18 lasts longer in the circulation system without being rapidly metabolized and excreted from the body. Therefore, it may have a more durable targeting effect.
  • This experimental example examines the targeting properties of the prepared conjugates AP2, AP21-26 and comparison AP27-contrast AP30 in mice.
  • U118MG cells were cultured according to the method of Experimental Example 4 and subcutaneously inoculated into 24 mice (all female) to obtain mice inoculated with U118MG subcutaneous tumors.
  • the above-prepared conjugates AP2, AP21-26, and comparison AP27-contrast AP30 were prepared into 0.3 mg/mL solutions using DMEM medium.
  • Administration began 14 days after U118MG cells were inoculated, and the day of administration was recorded as D1.
  • mice inoculated with U118MG subcutaneous tumors were randomly divided into 12 groups, with 2 mice in each group:
  • mice For 7 groups of mice, AP2, AP21, AP22, AP23, AP24, AP25 or AP26 were administered to each mouse in each group respectively.
  • the volume of single administration was 10 ⁇ L/g mouse body weight. Calculation shows that single administration The dose is 3mg/kg, which are recorded as test groups 14A-14G respectively;
  • each mouse in each group was given contrast AP27, contrast AP28, contrast AP29, or contrast AP30.
  • the single administration volume was 10 ⁇ L/g mouse body weight.
  • the single administration dose was calculated. is 3mg/kg, which are recorded as control group 14H-14K respectively;
  • each mouse was given DMEM culture medium with a dosage volume of 10 ⁇ L/g mouse body weight, which was recorded as the blank control group 15Y.
  • each mouse was imaged in vivo using the small animal in vivo optical imaging system IVIS Lumina Series III. On D5, mice in each group were sacrificed and tumor tissues and kidneys were harvested for fluorescence imaging.
  • FIGs 13A-13C are respectively diagrams showing the fluorescence imaging results in mice at 1h, 24h and 48h after administration of different conjugates, in which the leftmost one of the three mice in each small picture is It is the blank control group of 15Y mice.
  • the blank control group did not show any fluorescence signal; unlike this, 1 hour after administration, the mice in each test group and the control group showed fluorescence signals at the subcutaneous tumors; as can be seen from Figures 13B and 13C, At 24h and 48h after administration, only the mice in the test group 14A-14G showed strong fluorescence signals at the subcutaneous tumors, while the mice in the control group 14H-14K showed almost no fluorescence signals or only very weak ones. Fluorescent signal.
  • Figure 13D is a diagram showing the fluorescence signal imaging of tumor tissues and kidneys of mice in each group after the mice were sacrificed on D5, where Blank represents the blank control group 14Y.
  • Blank represents the blank control group 14Y.
  • the tumor tissues of the mice in the blank control group 14Y and the control group 14H-14K showed almost no fluorescence signal or only a very weak fluorescence signal; in contrast, the test group given the conjugate provided by the present disclosure
  • the tumor tissues of 14A-14G mice all showed strong fluorescence signals, while only weak fluorescence signals were shown in the metabolic organ kidney, indicating that compared with the control conjugates, various aptamers provided by the present disclosure can Stable and efficient targeted delivery of fluorescent groups to tumor tissue; various conjugates containing diagnostic agent groups provided by the present disclosure can stably and efficiently target tumor tissue, thus contributing to the successful detection of the presence of tumors. Diagnosis and monitoring.
  • This experimental example examines the targeting properties of the prepared conjugates AP2, AP12 and comparison AP31-contrast AP35 in mice.
  • U118MG cells were cultured according to the method of Experimental Example 4 and subcutaneously inoculated into 16 mice (all male) to obtain mice inoculated with U118MG subcutaneous tumors.
  • the above-prepared conjugates AP2, AP12 and comparison AP31-contrast AP35 were prepared into 0.3 mg/mL solutions using DMEM medium.
  • Administration began 21 days after U118MG cells were inoculated, and the day of administration was recorded as D1.
  • mice inoculated with U118MG subcutaneous tumors were randomly divided into 8 groups, with 2 mice in each group:
  • mice For the two groups of mice, AP2 or AP12 was administered to each mouse in each group, and the single administration volume was 10 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 3 mg/kg, which were recorded in sequence as Test group 15A-15B;
  • each mouse in each group was given contrast AP31, contrast AP32, contrast AP33, contrast AP34, or contrast AP35, with a single administration volume of 10 ⁇ L/g.
  • the single dose was calculated to be 3mg/kg, which were recorded as the control group 15C-15G respectively;
  • each mouse was given DMEM culture medium with a dosage volume of 10 ⁇ L/g mouse body weight, which was recorded as the blank control group 15Y.
  • each mouse was imaged in vivo using the small animal in vivo optical imaging system IVIS Lumina Series III. On D6, mice in each group were sacrificed and tumor tissues and kidneys were harvested for fluorescence imaging.
  • FIGs 14A-14C are respectively diagrams showing the fluorescence imaging results in mice at 1h, 24h and 48h after administration of different conjugates, in which the leftmost one of the three mice in each small picture is It is the blank control group of 15Y mice.
  • the blank control group did not show any fluorescence signal; unlike this, 1 hour after administration, the mice in each test group and the control group all showed fluorescence signals at the subcutaneous tumors; as can be seen from Figures 14B and 14C, At 24h and 48h after administration, only the mice in the test groups 15A and 15B showed strong fluorescence signals at the subcutaneous tumors, while the mice in the blank control group 15Y and the control groups 15C-15G showed no fluorescence signals at all.
  • Figure 14D is a diagram showing the fluorescence signal imaging of tumor tissues and kidneys of mice in each group after the mice were sacrificed on D6, where Blank represents the blank control group 15Y.
  • the tumor tissues of the mice in the blank control group 15Y and the control group 15C-15G showed no fluorescent signal at all; in contrast, the tumors of the mice in the test group 15A or 15B that were administered the conjugate provided by the present disclosure
  • the tissues all showed strong fluorescence signals, while only weak fluorescence signals were displayed at the metabolic organ kidney, indicating that compared with the control conjugate, the conjugate provided by the present disclosure can target tumor tissue stably and efficiently, and does not Various conjugates having the sequence shown in formula (1) have not shown targeting effects on tumor tissue.
  • U118MG human glioma cells purchased from Guangzhou Genio Biotechnology Co., Ltd. were cultured according to the method of Experimental Example 4. Take the U118MG human glioma cells growing in the logarithmic phase, digest them with (0.25% trypsin), collect the cells, centrifuge to remove the supernatant, and resuspend the cells in serum-free DMEM medium to make a concentration of 1 ⁇ 10 8 cells/mL of cell culture medium.
  • mice 8 NOD-SCID mice (purchased from Spefford (Beijing) Biotechnology Co., Ltd.), male, 12 weeks old.
  • the above cell culture medium was inoculated subcutaneously on the right back of NOD-SCID mice.
  • the inoculation volume was 100 ⁇ L per mouse, that is, 1 ⁇ 10 7 cells were inoculated into each mouse. injection The mice were then continued to be fed for 21 days.
  • Conjugates AP2, AP4 and comparison conjugate AP13 were dissolved into a conjugate solution with a concentration of 0.3 mg/mL (based on aptamer) using serum-free DMEM medium.
  • mice 21 days after U118MG cell inoculation, 8 mice were randomly divided into 4 groups, 2 mice in each group. Drugs were administered to mice in each group, and the day of administration was recorded as D1. The experiment used tail vein injection as a single dose.
  • conjugates AP2, AP4 or comparison conjugate AP13 were administered to each mouse in different groups respectively.
  • the single administration volume was 10 ⁇ L/g mouse body weight.
  • the single administration dose was calculated. is 3mg/kg, which are respectively recorded as test groups 16a, 16b and 16c.
  • DMEM was administered to each group of mice respectively.
  • the single administration volume was 10 ⁇ L/g mouse body weight, which was recorded as the blank control group 16Y.
  • mice 24 hours after administration, the mice were euthanized, and the tumor tissues were collected and fixed in 4% paraformaldehyde solution.
  • the tumor tissues were dehydrated in 15% sucrose for 24 hours, and then dehydrated in 30% sucrose for 24 hours.
  • OCT embedding agent infiltrated the tissues, quick-frozen in liquid nitrogen, using a freezing microtome (model POLAR-D-JC, purchased from Sakura Seiki Co., Ltd.) to slice the quick-frozen tissue into 10 ⁇ m thickness to obtain tumor tissue sections.
  • a laser confocal imager (model LSM 900 Basic Operation, purchased from Carl Zeiss (Shanghai) Management Co., Ltd.) was used to perform imaging analysis on the DAPI-stained sections obtained above.
  • the parameter selection was Cy5 laser intensity 20% (em 650nm, ex 670nm) , DAPI laser intensity 1.5% (em 360nm, ex 460nm), shot in Best signal mode. See Figure 15 for the results.
  • Figure 15 is a graph showing laser confocal imaging results of U118MG glioma cells 24 hours after administration of different conjugates to mice subcutaneously inoculated with U118MG glioma.
  • the experimental results show that compared with the blank control group DMEM and the comparison conjugate AP13, the conjugates containing the delivery group of the present disclosure can efficiently and specifically deliver the diagnostic agent group into the interior of U118MG glioma, showing Excellent targeting effect and potential diagnostic ability.
  • U118MG human glioma cells purchased from Guangzhou Genio Biotechnology Co., Ltd. were cultured according to the method of Experimental Example 4. Take the U118MG human glioma cells growing in the logarithmic phase, digest them with (0.25% trypsin), collect the cells, centrifuge to remove the supernatant, and resuspend the cells in serum-free DMEM medium to make a concentration of 1 ⁇ 10 8 cells/mL of cell culture medium.
  • NOD-SCID mice purchased from Spefford (Beijing) Biotechnology Co., Ltd.
  • the above cell culture medium was inoculated subcutaneously on the right back of NOD-SCID mice.
  • the inoculation volume was 100 ⁇ L per mouse, that is, 1 ⁇ 10 7 cells were inoculated into each mouse. Mice were kept for 21 days after injection.
  • Conjugate AP2 conjugate 15 and conjugate 36 were dissolved into a conjugate solution with a concentration of 0.5 mg/mL (based on aptamer) using serum-free DMEM medium.
  • Administration began 21 days after U118MG cells were inoculated, and the day of administration was recorded as D1.
  • conjugate AP2 was administered to each mouse respectively, and the single administration volume was 10 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 5 mg/kg.
  • conjugate 15 was administered to each mouse respectively, and the single administration volume was 10 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 5 mg/kg.
  • conjugate 36 was administered to each mouse respectively, and the single administration volume was 10 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 5 mg/kg.
  • mice were anesthetized with isoflurane (0.41ml/min isoflurane in 4L/min fresh air flow), and the small animal in vivo optical imaging system IVIS Lumina Series was used III Perform in vivo imaging of each mouse.
  • Figures 16A and 16B show fluorescence imaging results in mice after administration of different conjugates.
  • the results show that various conjugates of the present disclosure including siRNA groups and delivery groups formed by the aptamers of the present disclosure can effectively target and enrich tumor tissues at different times after administration. It has excellent drug-making ability.
  • PANC-1 human pancreatic cancer was cultured in DMEM complete medium (MACGENE, Cat. No. CM15019) containing 10% FBS (Gibco, Cat. No. 10099-141) at 37 in an incubator containing 5% CO 2 /95% air.
  • DMEM complete medium MACGENE, Cat. No. CM15019
  • FBS Gibco, Cat. No. 10099-141
  • Conjugate 37, conjugate 38 and conjugate AP2 were respectively dissolved into conjugate solutions with a concentration of 20 ⁇ M using DMEM.
  • DMEM medium containing 10% FBS was added to each well.
  • control group blank control group DMEM (hereinafter referred to as the control group), conjugate 37, conjugate 38 or conjugate AP2 to the corresponding culture medium, and freely ingest at a concentration of 2 ⁇ M.
  • RNA was extracted and recorded as The control group, conjugate group 9, conjugate group 10 and conjugate AP2 group (the latter three groups are collectively referred to as the test group).
  • a magnetic bead tissue RNA extraction kit (purchased from Wuhan Nanoci Biotechnology Co., Ltd., Cat. No. NMR0211-20) was used to extract total RNA from cells in each well according to the instruction manual to obtain solutions containing total RNA.
  • Step 1 Reverse-transcribe the total RNA of the cells in each well. After the reaction is completed, add 80 ⁇ l of DEPC water to the reverse transcription reaction system to obtain 100 ⁇ L of cDNA-containing solution.
  • Figure 17 is a bar graph showing the inhibition rate of hSTAT3 mRNA in PANC-1 human pancreatic cancer cells by Conjugate 37, Conjugate 38 and Conjugate AP2 of the present disclosure, respectively.
  • the inhibition rate of hSTAT3 mRNA in PANC1 human pancreatic cancer cells was 33%; after administration of conjugate 38, the inhibition rate of hSTAT3 mRNA in PANC1 human pancreatic cancer cells reached 44%.
  • the above results show that the conjugates of the present disclosure with different connection methods, different linking groups and different modified nucleotide sequences can be freely taken up into tumor cells and show the effect of inhibiting target mRNA.
  • U118MG human glioma cells purchased from Guangzhou Genio
  • U118MG cells were cultured according to the method of Experimental Example 4. Biological Technology Co., Ltd).
  • U118MG cells were digested with (0.25% trypsin) and cultured in a 12-well plate at 2 ⁇ 10 5 cells/well in a 37°C cell culture incubator with 5% CO2 for 24 hours.
  • Conjugate 15, conjugate 16, conjugate 17, conjugate 18 and conjugate 19 were dissolved in serum-free DMEM medium to a concentration of 200 ⁇ M.
  • Laser confocal imaging The parameter selection is Cy5 laser intensity 2% (em 650nm, ex 670nm), DAPI laser intensity 1.5% (em 360nm, ex 460nm). Shooting in Best signal mode, see Figure 18 for the results.
  • Figure 18 is a graph showing laser confocal imaging results of conjugates of the present disclosure within U118MG glioma.
  • This experimental example evaluates and compares the ability of conjugate 15, conjugate 18 and conjugate AP2 to freely take up into U118-MG glioma cells in vitro. Take U118MG human glioma cells grown in the logarithmic phase as cultured in Experimental Example 4, digest with 0.25wt% trypsin and collect the cells, centrifuge to remove the supernatant, and resuspend the cells in DMEM medium supplemented with 10% FBS. Prepare a cell culture medium with a concentration of 1 ⁇ 10 5 cells/mL, mix thoroughly, and plate on a 24-well plate at 1000 ⁇ L/well, that is, 1 ⁇ 10 5 cells/well.
  • the culture medium was drained and 900 ⁇ L of serum-free DMEM was added to each well.
  • test group 20a, test group 20b and control group 20c take 100 ⁇ L of DMEM culture medium and image in a high-content imaging system.
  • the average fluorescence intensity of DMEM in the blank control group Degree normalization that is, the fluorescence intensity of test group 20a, test group 20b and control group 20c is imaged after subtracting the average fluorescence intensity of the blank control group. The results are shown in Figure 19.
  • Figure 19 is a bar graph showing the fluorescence intensity results of the conjugates of the present disclosure in U118MG glioma, where DMEM represents the blank control group.
  • the results of Figure 6 show that the conjugate provided by the present disclosure can effectively deliver siRNA groups into tumor cells, thereby facilitating the efficient production of RNAi effects in tumor cells.
  • U118MG human glioma cells expressing the Luciferase (Photinus pyralis) reporter gene were cultured according to the method of Experimental Example 4. Take the U118MG-luc human glioma cells growing in the logarithmic phase, digest them with 0.25wt% trypsin and collect the cells. After centrifugation, aspirate the supernatant and resuspend the cells in serum-free DMEM medium to make the cell density: Cell culture medium of 4 ⁇ 10 7 cells/mL.
  • mice 24 Balb/C-nude nude mice (purchased from Spefford (Beijing) Biotechnology Co., Ltd.), male, 12 weeks old.
  • the above cell culture fluid was inoculated into the striatum of Balb/C-nude nude mice, and the mouse striatum injection method was used to inject the cell culture fluid into the right striatum of the mouse at the AP (anteroposterior/posterior/anterior) position. position): 1 mm, ML (medial lateral): 1.5 mm, DV (dorsal ventral): 3.5 mm, injection volume 10 ⁇ L, that is, each mouse is inoculated with 4 ⁇ 10 5 cells. After in situ tumor inoculation, the mice were continued to be fed for 14 days.
  • Conjugate 20 was dissolved into a conjugate solution with a concentration of 1 mg/mL (based on aptamer) using 1 ⁇ DMEM (purchased from Zhongke Maichen (Beijing) Technology Co., Ltd., batch number: K1902200) medium.
  • Conjugate 39 and comparative conjugate 43 were respectively dissolved into conjugate solutions with a concentration of 0.8 mg/mL (based on aptamer).
  • the small animal in vivo optical imaging system IVIS Lumina Series III was used to conduct in vivo imaging of each mouse. According to the brain fluorescence intensity, there were 6 mice in each group. The day of administration was recorded as D1 (i.e. , the first day of the experiment, the following D4, D8, etc. correspond to the fourth day, the eighth day of the experiment, and so on).
  • In vivo imaging method Each mouse was intraperitoneally injected with 10 ⁇ L/g body weight concentration of 15 mg/mL D-luciferin potassium salt working solution (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.), and in vivo imaging was performed 10 min after injection ( Lumina III small animal in vivo imaging system). After imaging, circle the mouse brain fluorescence Optical region (ROI), software measures fluorescence intensity (Radiance). Under these conditions, the Luciferase (Photinus pyralis) reporter gene expressed in U118MG-luc human glioma cells can produce a fluorescent response, so the fluorescence intensity can reflect the proliferation number of glioma cells. The higher the fluorescence intensity, the greater the number of glioma cells.
  • ROI mouse brain fluorescence Optical region
  • mice in each group were administered drugs on D1, D4, D8 and D12 respectively. Weigh before administration and administer according to weight.
  • conjugate 20 was administered to each mouse respectively, and the single administration volume was 5 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 5 mg/kg, and the dose containing MMAE was equivalent to 0.3 mg/kg.
  • conjugate 39 was administered to each mouse respectively, and the single administration volume was 5 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 4 mg/kg, and the dose containing MMAE was equivalent to 0.3 mg/kg.
  • control conjugate 43 was administered to each mouse respectively, and the single administration volume was 5 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 4 mg/kg, and the dose containing MMAE was equivalent to 0.3mg/kg.
  • DMEM culture medium was given to each mouse respectively, and the single administration volume was 5 ⁇ L/g mouse body weight.
  • Figure 20 is a line graph showing changes in tumor fluorescence intensity over time in U118MG orthotopic tumor model mice after administration of the conjugates or control compounds provided by the present disclosure.
  • the tumor fluorescence intensity (Radiance) of the blank control group and the control group increased significantly with the prolongation of observation time, indicating that the number of U118MG human glioma cells increased significantly;
  • the tumor fluorescence intensity of test groups 21a and 21b administered with the conjugate provided by the present disclosure was significantly reduced, and the reduction amplitude was up to 1 order of magnitude, and compared with the control group, it was more than 2 orders of magnitude, indicating that U118MG human colloid
  • the number of tumor cells is significantly reduced, and may be reduced to 1/10 of that at the beginning of the experiment, and may even be reduced to less than 1% of that of the control group.
  • the conjugate containing the tumor therapeutic agent group and the delivery group formed by the targeting aptamer provided by the present disclosure can effectively penetrate the blood-brain barrier and achieve high It effectively targets into brain glioma and has a good effect on inhibiting tumor growth, showing good treatment compliance and high drugability in effectively inhibiting tumors.
  • U118MG human glioma cells purchased from Guangzhou Genio Biotechnology Co., Ltd. were cultured according to the method of Experimental Example 4. Take the U118MG human glioma cells growing in the logarithmic phase, digest them with (0.25% trypsin), collect the cells, centrifuge to remove the supernatant, and resuspend the cells in serum-free DMEM medium to make a concentration of 1 ⁇ 10 8 cells/mL of cell culture medium.
  • mice 36 NOD-SCID mice (purchased from Spefford (Beijing) Biotechnology Co., Ltd.), male, 12 weeks old.
  • the above cell culture medium was inoculated subcutaneously on the right back of NOD-SCID mice, and the inoculation volume was 100 ⁇ L per mouse, that is, 1 ⁇ 10 7 cells were inoculated into each mouse.
  • the mice were kept for 7 days after injection.
  • Conjugate 20 was dissolved in serum-free DMEM medium to a conjugate solution with a concentration of 1 mg/mL (based on aptamer).
  • Conjugate 39, conjugate 40 and conjugate 41 were respectively dissolved into conjugate solutions with a concentration of 0.8 mg/mL (based on aptamer).
  • Administration began 7 days after U118MG cells were inoculated, and the day of administration was recorded as D8.
  • the experiment adopted abdominal subcutaneous administration, with administration once each on D8, D12, D16 and D20, for a total of 4 administrations.
  • DMEM was administered to each group of mice respectively, and the single administration volume was 5 ⁇ L/g mouse body weight;
  • MMAE was administered to each group of mice respectively, and the single administration volume was 5 ⁇ L/g mouse body weight. Calculation showed that the single administration dose was 0.3 mg/kg;
  • conjugate 20 was administered to each mouse respectively, and the single administration volume was 5 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 5 mg/kg, and the dose containing MMAE was equivalent to 0.3 mg/kg;
  • conjugate 39 For the three groups of mice, conjugate 39, conjugate 40 or conjugate 41 were administered to each group of mice respectively.
  • the single administration volume was 5 ⁇ L/g mouse body weight.
  • the calculation showed that the single administration dose was 4mg/kg, which contains the dose of MMAE equivalent to 0.3mg/kg; respectively recorded as test groups 22d, 22e and 22f;
  • FIG. 21 is a line graph showing the change in tumor volume over time in mice at different days after administration of different conjugates. As can be seen from Figure 21, compared with the control group 22b or the blank control group, the tumor volume and tumor weight of the mice in the test groups 22c-22f administered the conjugate of the present disclosure were significantly reduced. The above results indicate that conjugates containing delivery groups formed by these aptamers can effectively reach tumor tissues and display good anti-tumor activity.
  • mice inoculated with U118MG subcutaneous tumors were obtained, and the mice were continued to be raised after injection.
  • MMAE Dissolve MMAE into a solution of 0.03 mg/mL and a concentration of 0.01 mg/mL using 10% DMSO + 90% serum-free DMEM medium (volume ratio); dissolve conjugate 39 to a concentration of 0.5 mg/mL and 0.165 mg/mL. solution at a concentration (based on aptamer); control conjugate 44 was dissolved into a solution at a concentration of 0.5 mg/mL and 0.165 mg/mL (based on aptamer).
  • mice Seven days after inoculation of U118MG cells, all mice were randomly divided into 7 groups, with 6 mice in each group, and the mice in each group were administered drugs. The day of administration was recorded as D8. The experiment adopted abdominal subcutaneous administration, with administration once each on D8, D11, D15, D29, D32 and D36, for a total of 6 administrations. Weigh before administration and calculate administration volume based on weight.
  • DMEM culture medium was given to each mouse respectively, and the single administration volume was 10 ⁇ L/g mouse body weight.
  • each mouse was given MMAE at a concentration of 0.01 mg/mL, and the single administration volume was 10 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 0.1 mg/kg.
  • each mouse was administered conjugate 39 at a concentration of 0.165 mg/mL, and the single administration volume was 10 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 1.65 mg/kg (corresponding to MMAE dose is 0.1 mg/kg).
  • each mouse was given a control conjugate at a concentration of 0.165 mg/mL. 44, the single administration volume is 10 ⁇ L/g mouse body weight, and the calculation shows that the single administration dose is 1.65 mg/kg (corresponding MMAE dose is 0.1 mg/kg).
  • each mouse was given MMAE at a concentration of 0.03 mg/mL.
  • the single administration volume was 10 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 0.3 mg/kg.
  • each mouse was given conjugate 39 at a concentration of 0.5 mg/mL, and the single administration volume was 10 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 5 mg/kg (corresponding to MMAE The dose is 0.3mg/kg).
  • each mouse was given a concentration of 0.5 mg/mL of the comparative conjugate 44, and the single administration volume was 10 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 5 mg/kg (corresponding to MMAE dose is 0.3 mg/kg).
  • D9, D16, D19, D21, D24, D26, D29, D32, D36, D39, D43, D47, D53, D57, D60, D64, D67, D71, D74, D78, D81, D84, D88, D92 , D95 and D99 were used to measure the tumor volume.
  • the blank control group was measured at D53
  • the test groups 7a, 7b and the control group 7c were measured at D60 and the experiment was terminated.
  • the long diameter and short diameter of the tumor were determined by in vitro measurement. Tumor volume was calculated according to the formula 1/2 (long diameter ⁇ short diameter 2 ). After the experiment was terminated, the tumor tissue was taken, weighed and the average value calculated. The results are shown in Figure 22.
  • Figure 22 is a line graph showing changes in tumor volume over time in U118MG subcutaneous tumor model mice after administration of different concentrations of conjugates or control compounds provided by the present disclosure.
  • the tumor volume of test group 23b was significantly smaller than that of test group 23a and control group 23c, which had a similar MMAE content; during the test period, the tumor volume of test group 23e was significantly smaller than that of test group 23d and control group 23f, which had a similar MMAE content. , showing better anti-tumor activity than the administration of MMAE test groups 23a and 23d alone, as well as the administration of comparison conjugate 44.
  • the tumor weight in the mice administered the conjugate of the present disclosure was also significantly lower than that in the MMAE group and the control group.
  • mice inoculated with U118MG subcutaneous tumors were obtained, and the mice were continued to be raised after injection.
  • MMAE Dissolve MMAE into a solution with a concentration of 0.02 mg/mL using 10% DMSO + 90% serum-free DMEM medium (volume ratio); dissolve conjugate 20 and comparison conjugate 44 to a concentration of 0.33 mg/mL (to adapt Conjugate 39 and Conjugate 41 were dissolved into a solution with a concentration of 0.26 mg/mL (based on aptamer); Conjugate 42 was dissolved into a solution with a concentration of 0.23 mg/mL (based on aptamer) (calculated) solution.
  • mice inoculated above were divided into groups, with 6 mice in each group.
  • the mice in each group were administered drugs, and the day of administration was recorded as D8. Weigh before administration, and calculate the administration volume based on the average weight of each mouse of 20g.
  • the experiment adopted abdominal subcutaneous administration, once each on D8, D12, D15 and D19, for a total of 4 administrations.
  • DMEM medium was administered to each mouse respectively, with a single administration volume of 100 ⁇ L/g.
  • MMAE was administered to each mouse respectively, with a single administration volume of 100 ⁇ L, and calculation showed that the single administration dose was 0.1 mg/kg.
  • conjugate 20 was administered to each mouse respectively, with a single administration volume of 100 ⁇ L, and the calculation showed that the single administration dose was 1.65 mg/kg.
  • the comparison conjugate 44 was administered to each mouse respectively, with a single administration volume of 100 ⁇ L, and the calculation showed that the single administration dose was 1.65 mg/kg.
  • conjugate 39 was administered to each mouse respectively, with a single administration volume of 100 ⁇ L, and the calculation showed that the single administration dose was 1.32 mg/kg.
  • conjugate 41 was administered to each mouse respectively, with a single administration volume of 100 ⁇ L, and the calculation showed that the single administration dose was 1.32 mg/kg.
  • conjugate 42 was administered to each mouse respectively, with a single administration volume of 100 ⁇ L, and the calculation showed that the single administration dose was 1.17 mg/kg.
  • the MMAE dose corresponding to a single dose was 0.1 mg/kg.
  • Tumor volume was measured on D1, D9, D16, D19, D22, D26, D30, D36, D40, D43, D47, D50, D54, D57, D61, D64, D68 and D71.
  • the blank control group was measured on D54, and the 8a group (only given MMAE) was measured on D64 and the experiment was terminated.
  • the long diameter and short diameter of the tumor were determined by in vitro measurement. Tumor volume was calculated according to the formula 1/2 (long diameter ⁇ short diameter 2 ). After the experiment was terminated, tumor tissues were collected from each group, weighed, and the average value was calculated. The results are shown in Figure 23.
  • Figure 23 shows a line graph of tumor volume over time and D72 tumor weight in each group of mice. It can be seen from the results of Figure 23 that the tumor volume of the blank control group increased rapidly, and the tumor volume increase rate of the other groups decreased; and compared with the test group 24a and the control group 24c that were only given MMAE, the conjugate of the present disclosure was given In each test group, the tumor volume increase rate was significantly further reduced when the equivalent single dose of MMAE was 0.1 mg/kg. In addition, conjugate 20, conjugate 39, conjugate 41 and conjugate 42 reduced the tumor weight by at least 58% compared with test group 24a at the end point of the experiment D72, showing more excellent anti-tumor effects.
  • the culture conditions of A549 human lung adenocarcinoma cells were DMEM complete medium (MACGENE Company, catalog number CM15019) containing 10% FBS (Gibco, catalog number 10099-141), at 37.5% Culture in a CO 2 /95% air incubator. Digest and collect the cells with 0.25wt% trypsin, aspirate the supernatant and resuspend the cells in serum-free DMEM medium to prepare a cell culture medium with a cell density of 1 ⁇ 10 8 cells/mL.
  • DMEM complete medium (MACGENE Company, catalog number CM15019) containing 10% FBS (Gibco, catalog number 10099-141)
  • FBS Gibco, catalog number 10099-141
  • mice inoculated with A549 subcutaneous tumors were obtained, and the mice were continued to be raised after injection.
  • Conjugate 20 and conjugate 39 were dissolved in serum-free DMEM medium to a concentration of 1 mg/mL. degree (in terms of aptamer) of the conjugate solution.
  • Comparative conjugate 43 was dissolved into a conjugate solution with a concentration of 0.8 mg/mL (based on aptamer).
  • 10% DMSO + 90% serum-free DMEM medium (volume ratio) MMAE was dissolved into a solution with a concentration of 0.06 mg/mL.
  • mice Seven days after inoculation of A549 human lung cancer cells, all mice were divided into groups, with 6 mice in each group, and each mouse was administered a drug. The day of administration was recorded as D8. Weigh before administration, and calculate the administration volume based on mouse body weight.
  • mice in each group were administered once each on D8, D12, D15 and D19, for a total of 4 times.
  • DMEM was administered to each group of mice respectively, with a single administration volume of 5 ⁇ L/g mouse body weight;
  • MMAE was administered to each group of mice respectively, and the single administration volume was 5 ⁇ L/g mouse body weight. Calculation showed that the single administration dose was 0.3 mg/kg;
  • conjugate 20 was administered to each mouse respectively, and the single administration volume was 5 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 5 mg/kg, and the dose containing MMAE was equivalent to 0.3 mg/kg;
  • conjugate 39 was administered to each mouse respectively.
  • the single administration volume was 5 ⁇ L/g mouse body weight.
  • the calculation showed that the single administration dose was 5 mg/kg, and the dose containing MMAE was equivalent to 0.3 mg/kg;
  • control conjugate 43 was administered to each mouse respectively, and the single administration volume was 5 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 5 mg/kg, and the dose containing MMAE was equivalent to 0.3mg/kg;
  • mice in each group were administered once each on D46, D50 and D54.
  • the volume of single administration was 10 ⁇ L/g mouse body weight. Calculation showed that the dose containing MMAE in a single administration was equivalent to 0.6 mg/kg.
  • the tumor volume was measured on D1, D9, D16, D19, D22, D26, D30, D36, D40, D43, D47, D50, D54 and D57.
  • the experiment of the blank control group was stopped after the measurement on D50.
  • the long diameter and short diameter of the tumor were determined by in vitro measurement. Tumor volume was calculated according to the formula 1/2 (long diameter ⁇ short diameter 2 ). The results are shown in Figure 24.
  • Figure 24 is a diagram showing that after administration of different concentrations of conjugates or control compounds provided by the present disclosure, Line graph showing changes in tumor volume over time in A549 subcutaneous tumor model mice. It can be seen from the results in Figure 24 that the tumor volume of mice in the blank control group increased rapidly, and the increase rate of tumor volume in the other groups decreased; at each time period, the tumors of mice given conjugate 20 and conjugate 39 The tumor volume was smaller than that of the control group 25b and 25e. The above results indicate that the conjugate containing the delivery group formed by the aptamer of the present disclosure can effectively target and reach tumor tissue and show good anti-tumor activity.
  • any combination of various embodiments of the present disclosure can also be carried out, and as long as they do not violate the idea of the present disclosure, they should also be regarded as the contents disclosed in the present disclosure.

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Abstract

一种适配体，该适配体包含一段连续的核苷酸序列，连续的核苷酸序列具有式(1)所示的序列。还提供一种缀合物，该缀合物包含适配体形成的递送基团以及功能性基团。提供的适配体可以特异性地靶向并内化进入肿瘤细胞；提供的缀合物可以靶向递送至肿瘤组织，从而对肿瘤及肿瘤相关疾病进行诊断和/或治疗。

Description

适配体、缀合物与组合物及制备方法和用途 技术领域
本公开涉及一种适配体，以及含有基于该适配体的递送基团的缀合物与药物组合物。本公开还涉及适配体、缀合物与药物组合物的制备方法和用途。
背景技术
肿瘤是指机体在各种致瘤因子作用下，局部组织细胞增生所形成的新生物。其中，肿瘤细胞发生转移与侵入周遭组织的情形，称为恶性肿瘤。按照生成肿瘤的来源组织细胞分类，一般分为上皮细胞产生的恶性肿瘤(癌症)、间叶组织细胞产生的恶性肿瘤(肉瘤)、血液干细胞产生的恶性肿瘤(白血病等)、以及神经胶质细胞产生的恶性肿瘤(胶质瘤)等。其中，胶质瘤是最常见的颅内原发性恶性肿瘤，约占脑肿瘤的40％-50％，全球年发病率为每10万人3-8例。按照WHO病理分型标准，胶质瘤属于神经上皮肿瘤，包括多种病理类型，包括但不限于毛细胞性星形细胞瘤、弥漫星形细胞瘤、间变星形细胞瘤、胶质母细胞瘤、少突胶质细胞瘤、间变型少突胶质细胞瘤等。
目前，本领域中对肿瘤、特别是胶质瘤的诊断和治疗的重要问题之一是如何将诊断剂和/或治疗剂特异性地递送至肿瘤组织和细胞内，并在合适的时间、以合适的方式使这些诊断剂和/或治疗剂产生相应的诊断和/或治疗作用。
适配体或称核酸适体，是一种能够与多种的目标分子，像小分子化合物、蛋白质、核酸，甚至细胞、组织与器官等等结合的寡核苷酸分子。适配体能够提供“对特定分子辨识”这一重要特性，因此与抗体类似地常应用于生物技术与治疗等方面。适配体可以在试管中设计、并能够快速的利用化学方法合成、同时具有易于保存、免疫原性较低或无免疫原性的优异性质，因此近年来逐渐得到本领域研究人员的重视。然而，适合用于肿瘤靶向递送的适配体在本领域中仍需要进一步开发与应用。
发明内容
本公开的发明人意外发现了一种能够特异性靶向肿瘤细胞、特别是 胶质瘤细胞的适配体，该适配体对肿瘤细胞、特别是胶质瘤细胞显示出高度特异性，从而可以在肿瘤细胞、特别是胶质瘤细胞中有效富集。进一步地，通过将该适配体与各种功能性基团进行共价连接缀合，所获得的缀合物能够有效将适合用于各类肿瘤的诊断剂和/或治疗剂递送至肿瘤细胞、特别是胶质瘤组织和/或细胞，显示出优异的诊断和/或治疗效果，从而，发明人作出如下发明：
在一方面，本公开提供了一种适配体，包含一段连续的核苷酸序列，连接相邻的两个核苷酸的基团独立地为磷酸酯基或者具有修饰基团的磷酸酯基，每个核苷酸选自修饰或未修饰的A、U、C或G中的一种，所述连续的核苷酸序列具有以下式(1)所示的序列：
5′-T1-S1-Na-S2-Nb-S3-Nc-S4-T2-3′    式(1)
其中，T1是由1-3个核苷酸组成的基序，T2是由0-15个核苷酸组成的基序，并且T2中不包含与T1完全反向互补的基序；
S1和S4各自是由3-7个核苷酸组成的基序，S1与S4长度相同并且完全反向互补；
Na和Nc各自是由1-4个核苷酸组成的基序，Na中的每个核苷酸与Nc中的每个核苷酸均不互补，并且Na和Nc中U的总个数占Na和Nc中全部核苷酸总个数的50％以上；
S2和S3各自是由1-4个核苷酸组成的基序，S2与S3长度相同并且完全反向互补；
Nb是由3-6个核苷酸组成的基序，并且Nb两端的核苷酸之间不形成AU或GC互补。
在另一方面，本公开还提供了一种缀合物，包含一个或多个递送基团和一个或多个功能性基团，所述递送基团由本公开提供的适配体去除一个氢原子或官能团形成，每个所述递送基团独立地与所述功能性基团经共价键连接，或通过连接基团连接，每个所述功能性基团独立地为诊断剂基团、对肿瘤、特别是胶质瘤具有治疗作用的小分子治疗剂基团、对肿瘤、特别是胶质瘤具有治疗作用的功能性寡核苷酸基团和递送助剂基团中的一种。
在又一方面，本公开还提供了一种药物组合物，包含本公开提供的 缀合物以及药学上可接受的载体。
在又一方面，本公开还提供了本公开提供的适配体和/或缀合物和/或药物组合物在制备用于诊断和/或治疗肿瘤及肿瘤相关疾病或症状的药物中的应用。
在又一方面，本公开还提供了一种诊断和/或治疗肿瘤及肿瘤相关疾病或症状的方法，所述方法包括向有需要的受试者给予有效量的本公开提供的缀合物和/或药物组合物。
在又一方面，本公开还提供了一种试剂盒，包括本公开提供的缀合物和/或药物组合物。
以引用的方式并入
本说明书中提及的所有出版物、专利以及专利申请均以引用的方式并入本公开，其程度与每一单独的出版物、专利或专利申请均专门并且单独地以引用的方式并入本公开的程度相同。
有益效果
采用本公开提供的适配体，能够特异性地把功能性基团递送至各种肿瘤细胞，特别是胶质瘤组织和细胞。
本公开提供的缀合物和药物组合物具有优异的靶向肿瘤、特别是胶质瘤组织和细胞的能力，能够显著提高肿瘤相关疾病和/或症状的诊断准确性和/或显著治疗或缓解肿瘤相关的疾病和/或症状。
在一方面，本公开提供的缀合物能够在体外高效、特异性地进入肿瘤细胞。例如，与随机序列的对比缀合物相比，本公开提供的缀合物在U118MG胶质瘤细胞中均显示出显著更高的平均荧光强度，R值基本保持在2以上，表明本公开提供的缀合物具有优异的进入U118MG胶质瘤细胞的能力。又例如，本公开提供的缀合物具有很强的进入U118MG胶质瘤细胞、U251人胶质瘤细胞、A549人非小细胞肺癌细胞、MCF-7人乳腺癌细胞等各种癌细胞的能力，同时基本不进入正常细胞如SVGp12正常星形胶质细胞和293T人肾上皮细胞。又例如，与对比AP10相比，本公开提供的缀合物具有很强的进入U118MG和A549肿瘤球内部的能力。 又例如，不同长度和序列的本公开提供的缀合物均能够有效靶向对U118MG胶质瘤组织。
在另一方面，本公开提供的适配体能够将各种诊断剂基团，如不同荧光基团递送至肿瘤组织。例如，本公开提供的包含荧光基团的不同缀合物在小鼠体内接种肿瘤处均能显示出强烈的荧光信号，表明本公开提供的适配体能够特异性地靶向U118MG胶质瘤；进一步地，本公开提供的缀合物在给药后24h至48h时仍显示出较强的荧光信号，表明在较长时间内均能够稳定靶向胶质瘤组织。又例如，本公开提供的缀合物在尾静脉给药时仍能到达并靶向脑胶质瘤，暗示本公开提供的适配体还具有穿透血脑屏障(Blood-Brain-Barrier，BBB)，并将诊断剂基团跨血脑屏障递送至脑胶质瘤的优异能力。又例如，具有各种不同修饰的本公开提供的缀合物均能够稳定靶向U118MG胶质瘤、A549人非小细胞肺癌肿瘤和PAN02胰腺癌肿瘤细胞，显示出广泛的特异性靶向肿瘤的能力。又例如，通过形成缀合物，本公开提供的适配体能够将不同的诊断剂基团(例如不同荧光基团Cy3或Cy5)高效、特异性地递送至U118MG胶质瘤，并且能在较长时间内保持稳定。又例如，经由激光共聚焦显微镜的观察，结果表明本公开提供的缀合物能够将诊断剂基团高效、特异性地递送进入U118MG胶质瘤的肿瘤细胞内部，显示出优异的靶向效果和潜在的诊断能力。
在又一方面，本公开提供的缀合物能够将各种小分子药物基团，如小分子毒素基团特异性地递送至肿瘤组织，并显示出优异的肿瘤抑制效果。例如，本公开提供的缀合物能够有效地将MMAE递送至肿瘤组织，在显示出肿瘤靶向能力的同时，还降低了MMAE分子在其它组织分布带来的毒性风险，且各种给药方式均能够有效抑制肿瘤体积的增加速度和肿瘤重量，表明本公开的缀合物能够有效抑制肿瘤增殖。此外，进一步提高给药剂量的缀合物可使肿瘤体积在测试期间几乎不增加，显示出更加优异的抗肿瘤效果。
在又一方面，本公开提供的缀合物能够将各种功能性寡核苷酸基团，如siRNA基团特异性地递送至肿瘤组织，并显示出优异的肿瘤细胞活力抑制效果。例如，本公开提供的适配体在缀合siRNA后，不仅能够高效靶向肿瘤细胞并抑制目标mRNA含量，还能不显著影响正常细胞中的相应 mRNA水平，具有优异的靶向递送效率和高的安全性。又例如，在给药后的不同时间时，本公开的各种包含siRNA基团的缀合物均能够有效地靶向并富集至U118MG肿瘤组织。又例如，本公开的缀合物能够在体外实验中经自由摄取进入PANC1人胰腺癌细胞，并且显示出显著抑制hSTAT3 mRNA的效果。又例如，激光共聚焦成像以及高内涵成像系统中成像结果表明，本公开的包含siRNA基团的缀合物能够有效进入U118MG肿瘤细胞内，从而有利于在肿瘤细胞中高效产生RNAi作用。
进一步地，本公开提供的发明人意外地发现，本公开提供的缀合物和/或药物组合物能够高效地通过血脑屏障，在全身给药的情况下就能够靶向至脑内的胶质瘤中，从而进一步提高功能性基团的递送效率、节省成本并降低不期望的副反应。例如，本公开提供的缀合物能够在皮下给药的情况下，穿越血脑屏障，有效靶向进入原位U118MG胶质瘤，并显著抑制肿瘤体积的增加甚至降低至初始体积的1/10以下、甚至相比对对照组可降低至1/100以下，表明本公开的缀合物能够有效穿透血脑屏障并高效靶向进入脑胶质瘤，并具有良好的抑制肿瘤生长效果，显示出良好的治疗依从性和高效抑制肿瘤的能力。
由此说明，本公开提供的适配体具有优异的靶向肿瘤、特别是胶质瘤组织和细胞的能力，包含本公开提供的适配体和诊断剂基团(例如荧光显影剂基团)的缀合物能够显著提高肿瘤的诊断成功率，并且包含本公开提供的适配体和治疗剂基团的缀合物能够显著有效抑制肿瘤生长，或者降低肿瘤细胞中的癌症相关基因表达水平，具有良好的应用前景。
附图说明
图1A和图1B分别是示出了AP1和对比AP10进入U118MG胶质瘤细胞和SVGp12正常星形胶质细胞的高内涵成像图。
图1C-1G依次分别是示出了AP1和对比AP10进入U251人胶质瘤细胞、A549人非小细胞肺癌细胞、MCF-7人乳腺癌细胞和293T人肾上皮细胞的高内涵成像图。
图2A和2B依次分别是示出了AP1和对比AP10进入U118MG胶质瘤肿瘤球和A549人非小细胞肺癌肿瘤球的高内涵成像图。
图3A-3D依次分别是示出了给药后1h、4h、24h和48h时，给予不同 缀合物后的U118MG皮下瘤模型小鼠活体成像及肿瘤组织成像的图。
图4A和4B分别是示出了给药后24h和48h时，给予不同缀合物后建立U118MG原位瘤模型小鼠脑组织的荧光成像图。
图5A-5E分别是示出给予不同序列的本公开提供的缀合物以及对比缀合物后，不同时间点的小鼠体内荧光成像图；图5F-5H分别是不同时间点的肿瘤组织成像图。
图6是示出了在给予缀合物后1h时，各组中1只小鼠的脏器荧光影像的图。
图7是示出了给予本公开提供的缀合物后，建立PAN02皮下瘤模型的小鼠中各器官组织的荧光成像的图。
图8A-8E分别是示出了给药后48h或96h时，给予本公开提供的缀合物的小鼠的脏器荧光影像的图。
图9是示出了给予缀合物后D9时，各组小鼠的肿瘤荧光影像的图。
图10是示出了给予本公开提供的缀合物或对照化合物后，各组小鼠中肿瘤体积随时间变化的折线图。
图11A和图11B分别是示出了不同浓度的缀合物AP1和缀合物19对U118-MG神经胶质瘤细胞和SVGp12人星形胶质细胞的细胞活力的影响的图。
图12是示出了给予包含不同连接基团的本公开提供的缀合物后，小鼠体内的荧光成像结果的图。
图13A-13C依次分别是示出了给予不同缀合物后1h、24h和48h时，小鼠体内的荧光成像结果的图。图13D是示出了D5时处死小鼠后，各组小鼠肿瘤组织和肾脏的荧光信号成像的图。
图14A-14C依次分别是示出了给予不同缀合物后1h、24h和48h时，小鼠体内的荧光成像结果的图。图14D是示出了D6时处死小鼠后，各组小鼠肿瘤组织和肾脏的荧光信号成像的图。
图15是示出了给予不同缀合物24h时，U118MG胶质瘤内的激光共聚焦成像结果的图。
图16A和图16B是示出了给予不同缀合物后，小鼠体内的荧光成像结果的图。
图17是示出了本公开的缀合物在PANC-1人胰腺癌细胞中对 hSTAT3 mRNA的相对抑制率的柱状图。
图18是示出了本公开的缀合物在U118MG胶质瘤细胞内的激光共聚焦成像结果的图。
图19是示出了本公开的缀合物在U118MG胶质瘤内的相对荧光强度结果的柱状图。
图20是示出了给予本公开提供的缀合物或对照化合物后，U118MG原位瘤模型小鼠中肿瘤荧光强度随时间变化的折线图。
图21是示出了给予本公开提供的缀合物或对照化合物后，U118MG皮下瘤模型小鼠肿瘤体积随时间变化的折线图。
图22是示出了给予不同浓度本公开提供的缀合物或对照化合物后，U118MG皮下瘤模型小鼠肿瘤体积随时间变化的折线图。
图23是示出了给予不同浓度本公开提供的缀合物或对照化合物后，U118MG皮下瘤模型小鼠肿瘤体积随时间变化的折线图。
图24是示出了给予不同浓度本公开提供的缀合物或对照化合物后，A549皮下瘤模型小鼠肿瘤体积随时间变化的折线图。
具体实施方式
以下对本公开的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本公开，并不用于限制本公开。
定义
本公开中，如无特别说明，A、U、C、G和T分别指腺嘌呤核苷酸、尿嘧啶核苷酸、胞嘧啶核苷酸、鸟嘌呤核苷酸和胸腺嘧啶核苷酸，2-甲基胞嘧啶核苷酸指胞嘧啶核苷酸中的胞嘧啶碱基上2′位的氢被甲基取代得到的核苷酸。这些核苷酸的结构为本领域技术人员所公知。如本公开所使用的，“核酸基序”或“基序”是指适配体中的核酸序列片段，由1个或多个核苷酸组成。在一些实施方式中，基序是具有生物学功能的核酸序列片段。
如本公开所使用的，“烷基”是指具有指定数量的碳原子的直链和支链饱和烃基，所述数量通常为1至20个碳原子，例如1至10个碳原子， 如1至8个或1至6个碳原子。例如，C1-C6烷基指包含1至6个碳原子的直链和支链烷基。当提及具有特定数量的碳的烷基残基时，旨在涵盖具有该数量的碳的所有支链和直链形式；因此，例如，“丁基”意味着包括正丁基、仲丁基、异丁基和叔丁基；“丙基”包括正丙基和异丙基。亚烷基是烷基的子集，指与烷基相同、但具有两个连接点的残基。
如本公开所使用的，“烯基”是指具有一个或多个碳-碳双键的不饱和支链或直链烷基，所述碳-碳双键是通过从母体烷基的相邻碳原子中除去一分子氢而获得的。该基团可以处于双键的顺式或反式构型。典型的烯基基团包括但不限于：乙烯基；丙烯基，如丙-1-烯-1-基、丙-1-烯-2-基、丙-2-烯-1-基(烯丙基)、丙-2-烯-2-基；丁烯基，例如丁-1-烯-1-基、丁-1-烯-2-基、2-甲基丙-1-烯-1-基、丁-2-烯-1-基、丁-2-烯-2-基、丁-1，3-二烯-1-基、丁-1，3-二烯-2-基等等。在某些实施方式中，烯基基团具有2到20个碳原子，而在其他实施方式中，具有2至10个、2至8个或2至6个碳原子。亚烯基是烯基的一个子集，指与烯基相同、但具有两个连接点的残基。
如本公开所使用的，“炔基”是指具有一个或多个碳-碳三键的不饱和支链或直链烷基，所述碳-碳三键是通过从母体烷基的相邻碳原子中除去两分子氢而获得的。典型的炔基基团包括但不限于：乙炔基；丙炔基，如丙-1-炔-1-基，丙-2-炔-1-基；丁炔基，例如丁-1-炔-1-基，丁-1-炔-3-基，丁-3-炔-1-基等。在某些实施方式中，炔基具有2到20个碳原子，而在其他实施方式中，具有2至10、2至8或2至6个碳原子。亚炔基是炔基的一个子集，指的是与炔基相同、但有两个连接点的残基。
如本公开所使用的，“杂环基”是指稳定的3-至18-元非芳香族环基，包含2-12个碳原子和1-6个杂原子，所述杂原子选自氮、氧和硫。除非说明书中另有说明，杂环基是单环、双环、三环或四环系统，可包括稠环或桥环系统。杂环基中的杂原子可以是被氧化的杂原子。一个或多个氮原子(如果存在的话)可以是季铵化的氮原子。杂环基是部分饱和或完全饱和的。杂环基可以通过任何环原子连接至分子的其余部分。此类杂环基的实例包括但不限于：二噁烷基、噻吩基[1，3]二硫酰基(thienyl[1，3]dithianyl)、十氢异喹啉基、咪唑啉基、咪唑烷基、异噻唑烷 基、异噁唑烷基、吗啉基、八氢吲哚基、八氢异吲哚基、2-氧杂哌嗪基、2-氧杂哌啶基、2-氧杂吡咯烷基、噁唑烷基、哌啶基、哌嗪基、4-哌啶酮基、吡咯烷基、吡唑烷基、奎宁环基、噻唑烷基、四氢呋喃基、三硫酰基(trithianyl)、四氢吡喃基、硫代吗啉基(thiomorpholinyl)、硫杂吗啉基(thiamorpholinyl)、1-氧代硫吗啉基(1-oxo-thiomorpholinyl)和1，1-二氧代硫吗啉基(1，1-dioxo-thiomorpholinyl)。亚杂环基是杂环基的一个子集，指与杂环基相同、但具有两个附着点的残基。
如本公开所使用的，“芳基”是指通过从环碳原子中除去氢原子而衍生自芳香族单环或多环烃环系统形成的基团。所述芳香族单环或多环烃环系统仅含有氢和6至18个碳原子的碳，其中所述环系统中的一个或多个环是完全不饱和的，即，包含根据Hückel理论的环状、离域的(4n+2)π-电子体系。芳基包括但不限于苯基、芴基和萘基等基团。亚芳基是芳基的子集，指与芳基相同、但具有两个连接点的残基。
“杂芳基”指由3-至18-元芳香环自由基衍生而成的基团，包含2个至17个碳原子和选自氮、氧和硫的1至6个杂原子。如本公开所使用的，杂芳基可以是单环、双环、三环或四环系统，其中环系统中的一个或多个环是完全不饱和的，即，包含根据Hückel理论的环状离域(4n+2)π-电子体系。杂芳基包括稠环或桥环系统。杂芳基中的杂原子可以是被氧化的杂原子。一个或多个氮原子(如果存在的话)可以是季铵化的氮原子。杂芳基通过任何环原子附着至分子的其余部分。杂芳基的实例包括但不限于：氮杂环庚三烯基、吖啶基、苯并咪唑基、苯并吲哚基、1，3-苯并二噁唑基、苯并呋喃基、苯并噁唑基、苯并[d]噻唑基、苯并噻二唑基、苯并[b][1，4]二噁庚英基(benzo[b][1，4]dioxepinyl)、苯并[b][1，4]噁嗪基(benzo[b][1，4]oxazinyl)、1，4-苯并二噁烷基(1，4-benzodioxanyl)、苯并萘并呋喃基、苯并噁唑基、苯并间二氧杂环戊烯基(benzodioxolyl)、苯并二噁英基(benzodioxinyl)、苯并吡喃基、苯并吡喃酮基、苯并呋喃基、苯并呋喃酮基、苯并噻吩基、苯并噻吩并[3，2-d]嘧啶基、苯并三唑基、苯并[4，6]咪唑并[1，2-a]吡啶基、咔唑基、噌啉基(cinnolinyl)、环戊烷并[d]嘧啶基、6，7-二氢-5H-环戊烷并[4，5]噻吩并[2，3-d]嘧啶基、5，6-二氢苯并[h]喹唑啉基(5，6-dihydrobenzo[h]quinazolinyl)、5，6-二氢苯并[h]噌啉基 (5，6dihydrobenzo[h]cinnolinyl)、6，7-二氢-5H-苯并[6，7]环庚烷并[1，2-c]哒嗪基、二苯并呋喃基、二苯并噻吩基、呋喃基、呋喃酮基、呋喃并[3，2-c]吡啶基、5，6，7，8，9，10-六氢环辛烷并[d]嘧啶基、5，6，7，8，9，10-六氢环辛烷并[d]哒嗪基、5，6，7，8，9，10-六氢环辛烷并[d]吡啶基、异噻唑基、咪唑基、吲唑基(indazolyl)、吲哚基、异吲哚基、二氢吲哚基、异二氢吲哚基、异喹啉基、吲哚嗪基(indolizinyl)、异噁唑基、5，8-甲醇-5，6，7，8-四氢喹唑啉基(5，8-methano-5，6，7，8-tetrahydroquinazolinyl)、萘啶基(naphthyridinyl)、1，6-萘啶酮基(1，6-naphthyridinonyl)、噁二唑基、2-氧杂吖庚因基(2-oxoazepinyl)、噁唑基、氧杂环丙烷基(oxiranyl)、5，6，6a，7，8，9，10，10a-八氢苯并[H]喹唑啉基、1-苯基-1H-吡咯基、吩嗪基、吩噻嗪基、吩噁嗪基、酞嗪基(phthalazinyl)、蝶啶基(pteridinyl)、嘌呤基、吡咯基、吡唑基、吡唑并[3，4-d]嘧啶基、吡啶基、吡啶并[3，2-d]嘧啶基、吡啶并[3，4-d]嘧啶基、吡嗪基、嘧啶基、哒嗪基、吡咯基、喹唑啉基、喹喔啉基(quinoxalinyl)、喹啉基、四氢喹啉基、5，6，7，8-四氢喹唑啉基、5，6，7，8-四氢苯并[4，5]噻吩并[2，3-d]嘧啶基、6，7，8，9-四氢-5H-环庚烷并[4，5]噻吩并[2，3-d]嘧啶基、5，6，7，8-四氢吡啶并[4，5-c]哒嗪基、噻唑基、噻二唑基、三唑基、四唑基、三嗪基、噻吩并[2，3-d]嘧啶基、噻吩并[3，2-d]嘧啶基、噻吩并[2，3-c]吡啶基(thieno[2，3-c]pridinyl)和噻吩基(thiophenyl/thienyl)。亚杂芳基是杂芳基的子集，指与杂芳基相同、但具有两个连接点的残基。
本公开提供的适配体
在一方面，本公开提供了一种适配体，包含一段连续的核苷酸序列，连接相邻的两个核苷酸的基团独立地为磷酸酯基或者具有修饰基团的磷酸酯基，每个核苷酸选自修饰或未修饰A、U、C或G中的一种，所述连续的核苷酸序列具有式(1)所示的序列：
5′-T1-S1-Na-S2-Nb-S3-Nc-S4-T2-3′   式(1)
其中，T1是由1-3个核苷酸组成的基序。发明人发现，T1的存在有利于本公开提供的适配体显示出高的肿瘤靶向作用。在一些实施方式中，T1由2个核苷酸组成，此时，本公开提供的适配体具有更优异的肿瘤靶向能力。在一些实施方式中，T1由2个核苷酸组成并且含有至少一个C。 在一些实施方式中，按照5′-3′方向，T1为CU、UC或AC。
T2是由0-15个核苷酸组成的基序。发明人发现，具有这些核苷酸数量和各种核苷酸序列的T2均不会显著影响适配体的肿瘤靶向能力。在一些实施方式中，T2由0-10个核苷酸组成。在一些实施方式中，按照5′-3′方向，T2由U起始的1-9个核苷酸组成，此时，适配体可能具有更优异的稳定性。
T2中不包含与T1完全反向互补的基序。在本公开的上下文中，“反向互补”是指两段核苷酸序列或基序之间，按照核酸碱基配对的规律形成氢键连接，并且一段核苷酸序列或基序按照5′-3′方向的每一个核苷酸依次与另一端核苷酸序列或基序按照3′-5′方向的每一个核苷酸能够形成碱基配对。在一些实施方式中，“反向互补”包括AU、GC和UG互补中的一种或多种。
本公开提供的适配体中，S1和S4各自是由3-7个核苷酸组成的基序，且S1与S4长度相同并且完全反向互补。具有上述S1和S4基序的适配体具有较优的稳定性，能够较长时间地靶向肿瘤组织和细胞。在一些实施方式中，S1和S4各自由3-5个核苷酸组成且长度相同。在一些实施方式中，S1和S4形成的反向互补中，GC互补占全部互补数量的40％以上，此时，本公开提供的适配体具有进一步更优的稳定性和肿瘤靶向能力。在一些实施方式中，按照5′-3′方向，S1为GCU且S4为AGC，或S1为GAGU且S4为GCUC，或S1为GGAGU且S4为GCUCU，或S1为UAUGG且S4为CCAUG。
本公开提供的适配体中，Na和Nc各自是由1-4个核苷酸组成的基序，Na中的每个核苷酸与Nc中的每个核苷酸均不互补，并且Na和Nc中U的总个数占Na和Nc中全部核苷酸总个数的50％以上。具有上述Na和Nc基序的适配体显示出优异的肿瘤组织靶向能力，当Na和/或Nc基序缺失、或Na和Nc中U的总个数不足时，适配体基本不显示肿瘤组织靶向效果。在一些实施方式中，Na和Nc中核苷酸的数量之和为2-4的整数。在一些实施方式中，Na和Nc中核苷酸的数量之和为3或4，并且所述Na和Nc中U的数量之和为2或3。在一些实施方式中，按照5′-3′方向，Na和/或Nc为U、UU、UC或CU。
本公开提供的适配体中，S2和S3各自是由1-4个核苷酸组成的基序，S2与S3长度相同并且完全反向互补。通过包含S2和S3基序，本公开提供的适配体显示出好的稳定性和优异的肿瘤靶向能力。在一些实施方式中，S2和S3各自由2-3个核苷酸组成且长度相同。在一些实施方式中，S2和S3形成的反向互补中至少包含一个GC互补，此时该反向互补具有更好的稳定性。在一些实施方式中，按照5′-3′方向，S2为CA且S3为UG，或S2为AC且S3为GU，或S2为GCC且S3为GGU。
Nb是由3-6个核苷酸组成的基序，并且Nb两端的核苷酸不是AU或GC互补。不受理论限制地，具有上述Nb基序的适配体能够在空间方面保持特定构型，从而使本公开提供的适配体稳定、高效地靶向肿瘤组织和细胞。在一些实施方式中，Nb由4-5个核苷酸组成。在一些实施方式中，按照5′-3′方向，Nb为GACG、GACGU、GACCG、UACU、GUUG或GAUCU。
本公开的发明人意外地发现，具有上述式(1)所示序列的本公开提供的适配体能够有效地靶向肿瘤、特别是胶质瘤组织，从而允许本公开提供的适配体作为递送载体，将对肿瘤具有诊断/治疗效果的功能性基团递送至肿瘤。进一步地，本公开提供的适配体能能够特异性地进入肿瘤细胞，从而从细胞水平、甚至基因水平上更有效地递送诊断剂/治疗剂基团。
在一些实施方式中，本公开提供的适配体中，所述连续的核苷酸序列的长度为18-50个核苷酸，或者20-40个核苷酸，或者21-36个核苷酸，或者24-32个核苷酸。具有这些连续的核苷酸序列长度的适配体能够更容易地靶向至肿瘤，并且在合成成本和靶向效果方面具有良好的平衡。
在一些实施方式中，本公开提供的适配体中，连续的核苷酸序列具有以下SEQ ID NO：1、SEQ ID NO：2或SEQ ID NO：3所示的序列：
5′-CUGCUUCAGACGUGUUAGCUU-3′(SEQ ID NO：1)
其中，按照5′-3′方向，T1为CU，S1为GCU，Na为U，S2为CA，Nb为GACG，S3为UG，Nc为UU，S4为AGC，T2为UU；
5′-CUGAGUUCAGACGUGUUGCUCU-3′(SEQ ID NO：2)
其中，按照5′-3′方向，T1为CU，S1为GAGU，Na为U，S2为CA， Nb为GACG，S3为UG，Nc为UU，S4为GCUC，T2为U；
5′-UCUAUGGCUGCCGAUCUGGUCUCCAUGUACGU-3′(SEQ ID NO：3)，
其中，按照5′-3′方向，T1为CU，S1为GAGU，Na为U，S2为CA，Nb为GACG，S3为UG，Nc为UU，S4为GCUC，T2为U。
在一些实施方式中，所述连续的核苷酸序列具有SEQ ID NO：4所示的核苷酸序列：
5′-N6GGAGUUCAN1N2N3N4UGN5GCUCN7-3′(SEQ ID NO：4)，
其中，N1、N2和N3各自独立地为A、U、C和G中的一种，N4为U、C或G或者由U、C或G中的两个组成的基序；N5为U、CU或UU；N6为CU、UC或AC；N7为U、UU或UUN8，N8为由1-15个核苷酸组成的基序。
上述核苷酸序列中，按照5′-3′方向，T1为N6所示的基序，S1为GGAGU表示的基序，Na为U，S2为CA，Nb为N1、N2、N3和N4组成的基序N1N2N3N4，S3为UG，Nc为N5表示的基序，S4为GCUC以及N7中的第一个核苷酸组成的基序，T2为N7中的其余核苷酸组成的基序。
包含上述SEQ ID NO：4所示的核苷酸序列的适配体能够更有效地靶向肿瘤、特别是胶质瘤并在肿瘤组织中富集。
实验验证表明，SEQ ID NO：4所示的核苷酸序列中，N1、N2、N3和N4的上述选择不会显著影响本公开提供的适配体的肿瘤靶向能力。在一些实施方式中，N1、N2、N3和N4组成的基序N1N2N3N4为GACG、GACGU、GACCG、UACU、GUUG或GAUCU中的一种，包含这些基序的适配体具有更高的肿瘤特异性靶向效果。
在一些实施方式中，SEQ ID NO：4所示的核苷酸序列中，N5为U或UU。此时，本公开提供的适配体对肿瘤均具有优异的靶向作用。
在一些实施方式中，所述连续的核苷酸序列具有SEQ ID NO：5-11中任意一项所示的核苷酸序列：
5′-CUGGAGUUCAGACGUGUUGCUCUU-3′(SEQ ID NO：5)
5′-CUGGAGUUCAGACGUUGUGCUCUU-3′(SEQ ID NO：6)
5′-CUGGAGUUCAGACCGUGUGCUCUU-3′(SEQ ID NO：7)
5′-CUGGAGUUCAGACGUGUUGCUCU-3′(SEQ ID NO：8)
5′-ACGGAGUUCAGACGUGUUGCUCUU-3′(SEQ ID NO：9)
5′-CUGGAGUUCACUACUGUUGCUCUU-3′(SEQ ID NO：10)
5′-CUGGAGUUCAGUUGUGUUGCUCUU-3′(SEQ ID NO：11)。
具有上述核苷酸序列的本公开提供的适配体显示出对肿瘤的高度靶向作用。
在一些实施方式中，基序N8由1-15个核苷酸组成。在一些实施方式中，N8由1-8个核苷酸组成。
在一些实施方式中，基序N8的存在使得本公开提供的适配体对体内的核酸外切酶更加稳定，从而在体内能够更长时间地发挥肿瘤的靶向作用。在一些实施方式中，N8能够增加或维持本公开提供的适配体对肿瘤的靶向作用。从稳定性、靶向性和合成效率的平衡出发考虑，在一些实施方式中，基序N8由8个核苷酸组成。在一些实施方式中，按照5′-3′方向，基序N8的核苷酸序列为CCGAUCUC。在一些实施方式中，连续的核苷酸序列具有SEQ ID NO：12-14中任意一项所示的核苷酸序列：
5′-CUGGAGUUCAGACGUGUUGCUCUUCCGAUCUC-3′                             (SEQ ID NO：12)
5′-CUGGAGUUCAGACGUUGUGCUCUUCCGAUCUC-3′                             (SEQ ID NO：13)
5′-CUGGAGUUCAGACCGUGUGCUCUUCCGAUCUC-3′                             (SEQ ID NO：14)
本公开提供的适配体中，5′末端核苷酸的核糖5′端以及3′末端核苷酸的核糖3′端的末端基团独立地是羟基或磷酸基，这些末端基团的选择不会改变本公开提供的适配体的靶向能力。在一些实施方式中，本公开提供的适配体中，5′末端核苷酸的核糖5′端以及3′末端核苷酸的核糖3′端的末端基团均为羟基。
本公开提供的适配体中，每个核苷酸均可以是修饰或未修饰的核苷酸。一般而言，核苷酸的修饰可能会改变本公开提供的适配体的稳定性 和/或对肿瘤的靶向能力。在一些实施方式中，本公开提供的适配体中的至少一个核苷酸为修饰的核苷酸。在一些实施方式中，本公开提供的适配体中的至少一个连接相邻的两个核苷酸的基团具有修饰基团的磷酸酯基。
核苷酸的修饰包括但不限于对糖的修饰、对碱基的修饰和/或以核苷酸类似物代替核苷酸。在一些实施方式中，本公开提供的适配体中，每个所述修饰的核苷酸独立地为为2′-卤素修饰的核苷酸、2′-烷氧基修饰的核苷酸、2′-烷基修饰的核苷酸、2′-经取代的烷基修饰的核苷酸、2′-氨基修饰的核苷酸、2′-经取代的氨基修饰的核苷酸、2′-脱氧核苷酸、碱基经修饰的核苷酸和核苷酸类似物中的一种。
在本公开的上下文中，“氟代修饰的核苷酸”指核苷酸的核糖基2′位的羟基被氟取代形成的核苷酸，其具有以下式(7)所示的结构。“非氟代修饰的核苷酸”指核苷酸的核糖基2′位的羟基被非氟基团取代形成的核苷酸、或核苷酸类似物。在一些实施方式中，每一个非氟代修饰的核苷酸独立地选自核苷酸的核糖基2′位的羟基被非氟基团取代形成的核苷酸或核苷酸类似物中的一种。
这些核糖基2′位的羟基被非氟基团取代形成的核苷酸是本领域技术人员所公知的，这些核苷酸可以选自2′-烷氧基修饰的核苷酸、2′-烷基修饰的核苷酸、2′-经取代的烷基修饰的核苷酸、2′-氨基修饰的核苷酸、2′-经取代的氨基修饰的核苷酸、2′-脱氧核苷酸中的一种。
在一些实施方式中，2′-烷氧基修饰的核苷酸为甲氧基修饰的核苷酸(2′-OMe)，如式(8)所示。在一些实施方式中，2′-氨基修饰的核苷酸(2′-NH2)如式(9)所示。在一些实施方式中，2′-脱氧核苷酸(DNA)如式(10)所示：
本领域技术人员知晓各种对核苷酸的碱基进行修饰的方式。在一些 实施方式中，碱基修饰包括但不限于在碱基上增加一个或多个甲基。在一些实施方式中，将胸腺嘧啶(T)视为碱基经修饰的尿嘧啶(U)的一种。在一些实施方式中，将2-甲基胞嘧啶视为碱基经修饰的胞嘧啶(C)的一种。
核苷酸类似物指能够在核酸中代替核苷酸，但结构不同于腺嘌呤核糖核苷酸、鸟嘌呤核糖核苷酸、胞嘧啶核糖核苷酸、尿嘧啶核糖核苷酸或胸腺嘧啶脱氧核糖核苷酸的基团。在一些实施方式中，核苷酸类似物可以是异核苷酸、桥联的核苷酸(bridged nucleic acid，简称BNA)或无环核苷酸。
BNA是指受约束的或不能接近的核苷酸。BNA可以含有五元环、六元环、或七元环的具有“固定的”C3′-内切糖缩拢的桥联结构。通常将该桥掺入到该核糖的2′-、4′-位处以提供一个2′，4′-BNA核苷酸。在一些实施方式中，BNA可以是LNA、ENA、cET BNA等，其中，LNA如式(12)所示，ENA如式(13)所示，cET BNA如式(14)所示：
无环核苷酸是核苷酸的糖环被打开形成的一类核苷酸。在一些实施方式中，无环核苷酸可以是解锁核酸(UNA)、甘油核酸(GNA)或肽核酸(PNA)，其中，UNA如式(15)所示，GNA如式(16)所示：
上述式(15)和式(16)中，R选自H、OH或烷氧基(O-烷基)。
肽核酸是多肽骨架取代糖苷-磷酸主链形成的一类核苷酸类似物。在 一些实施方式中，肽核酸可以是例如以2-氨基乙基甘氨酸键取代糖苷-磷酸单元形成的核苷酸类似物。
异核苷酸是指核苷酸中碱基在核糖环上的位置发生改变而形成的化合物。在一些实施方式中，异核苷酸可以是碱基从核糖环的1′-位移动至2′-位或3′-位而形成的化合物，如式(17)或(18)所示。
上述式(17)-式(18)化合物中，Base表示核酸碱基，例如A、U、G、C或T；R选自H、OH、F或者如上所述的非氟基团。
在一些实施方式中，核苷酸类似物选自异核苷酸、LNA、ENA、cET、UNA和GNA中的一种。
在一些实施方式中，每一个非氟代修饰的核苷酸均为甲氧基修饰的核苷酸，在上文和下文中，所述甲氧基修饰的核苷酸指核糖基的2′-羟基被甲氧基取代而形成的核苷酸。
在上文及下文中，“氟代修饰的核苷酸”、“2′-氟修饰的核苷酸”、“核糖基团的2′-羟基被氟取代的核苷酸”和“具有2′-氟代核糖基的核苷酸”意义相同，均指核苷酸的2′-羟基被氟取代，而形成的具有如式(7)所示结构的化合物；“甲氧基修饰的核苷酸”、“2′-甲氧基修饰的核苷酸”、“核糖基团的2′-羟基被甲氧基取代的核苷酸”和“具有2′-甲氧基核糖基的核苷酸”意义相同，均指核苷酸核糖基团的2′-羟基被甲氧基取代而形成的具有如式(8)所示结构的化合物。
在一些实施方式中，本公开提供的适配体中的所述连续的核苷酸序列中的每个胞嘧啶核苷酸为氟代修饰的胞嘧啶核苷酸，和/或所述所述连续的核苷酸序列中的每个尿嘧啶核苷酸为氟代修饰的尿嘧啶核苷酸。在一些实施方式中，本公开提供的适配体中的所述连续的核苷酸序列中的每个核苷酸均为2′-甲氧基修饰的核苷酸。在一些实施方式中，本公开提供的适配体中的一个或多个尿嘧啶核苷酸具有修饰的碱基。。
连接相邻的两个核苷酸的基团可以是磷酸酯基或修饰的磷酸酯基。磷酸酯基的修饰例如将磷酸酯基中的至少一个非桥接氧原子替换为硫原子，形成硫代磷酸酯基或二硫代磷酸酯基。在一些实施方式中，本公开提供的适配体中的至少一个连接相邻的两个核苷酸的基团为硫代磷酸酯基。在一些实施方式中，本公开提供的适配体中的5′末端前四个核苷酸之间的3个连接相邻的两个核苷酸的基团中至少1个为硫代磷酸酯基。在一些实施方式中，本公开提供的适配体中的5′末端前四个核苷酸之间的3个连接相邻的两个核苷酸的基团中至少2个为硫代磷酸酯基。在一些实施方式中，本公开提供的适配体中的3′末端前四个核苷酸之间连接相邻的两个核苷酸的基团中至少1个为硫代磷酸酯基。在一些实施方式中，本公开提供的适配体中的5′末端前四个核苷酸之间的3个连接相邻的两个核苷酸的基团中至少2个为硫代磷酸酯基。在一些实施方式中，本公开提供的适配体中的每个连接相邻的两个核苷酸的基团均为硫代磷酸酯基。
具有上述修饰的适配体不仅成本低，而且可使体内的核糖核酸酶不易切割适配体，由此增加适配体的稳定性，使其具有更强的抵抗核酸酶水解的性能。同时，上述修饰的适配体具有较高的靶向肿瘤组织和/或细胞的活性。
在一些实施方式中，连续的核苷酸序列具有SEQ ID NO：15-39中的一种所示的核苷酸序列：
5′-CfUfGGAGUfUfCfAGACfGUfGUfUfGCfUfCfUfUfCfCfGAUfCfUfCf-3′(SEQ ID NO：15)
5′-CfUfGGAGUfUfCfAGACfGUfGUfUfGCfUfCfUfUf-3′(SEQ ID NO：16)
5′-CfUfGGAGUfUfCfAGACfGUfUfGUfGCfUfCfUfUfCfCfGAUfCfUfCf-3′(SEQ ID NO：17)
5′-CfUfGGAGUfUfCfAGACfGUfUfGUfGCfUfCfUfUf-3′(SEQ ID NO：18)
5′-CfUfGGAGUfUfCfAGACfCfGUfGUfGCfUfCfUfUfCfCfGAUfCfUfCf-3′(SEQ ID NO：19)
5′-CfUfGGAGUfUfCfAGACfCfGUfGUfGCfUfCfUfUf-3′(SEQ ID NO：20)
5′-CmUmGmGmAmGmUmUmCmAmGmAmCmGmUmGmUmUmGmCmUmCmUmUmCmCmGmAmUmCmUmCm-3′(SEQ ID NO：21)
5′-CmUmGmGmAmGmUmUmCmAmGmAmCmGmUmGmUmUmGmCmUmCmUmUm-3′(SEQ ID NO： 22)
5′-CmUmGmGmAmGmUmUmCmAmGmAmCmGmUmUmGmUmGmCmUmCmUmUmCmCmGmAmUmCmUmCm-3′(SEQ ID NO： 23)
5′-CmUmGmGmAmGmUmUmCmAmGmAmCmGmUmUmGmUmGmCmUmCmUmUm-3′(SEQ ID NO： 24)
5′-CmUmGmGmAmGmUmUmCmAmGmAmCmCmGmUmGmUmGmCmUmCmUmUmCmCmGmAmUmCmUmCm-3′(SEQ ID NO： 25)
5′-CmUmGmGmAmGmUmUmCmAmGmAmCmCmGmUmGmUmGmCmUmCmUmUm-3′(SEQ ID NO： 26)
5′-CmUmGmGmAmGmUmUmCmAmGmAmCmGmUmGmUmUmGmCmUmCmUm-3′(SEQ ID NO： 27)
5′-CmUmGmAmGmUmUmCmAmGmAmCmGmUmGmUmUmGmCmUmCmUm-3′(SEQ ID NO： 28)
5′-CmUmGmCmUmUmCmAmGmAmCmGmUmGmUmUmAmGmCmUmUm-3′(SEQ ID NO： 29)
5′-AmCmGmGmAmGmUmUmCmAmGmAmCmGmUmGmUmUmGmCmUmCmUmUm-3′(SEQ ID NO： 30)
5′-CmUmGmGmAmGmUmUmCmAmCmUmAmCmUmGmUmUmGmCmUmCmUmUm-3′(SEQ ID NO： 31)
5′-CmUmGmGmAmGmUmUmCmAmGmUmUmGmUmGmUmUmGmCmUmCmUmUm-3′(SEQ ID NO： 32)
5′-UmCmUmAmUmGmGmCmUmGmCmCmGmAmUmCmUmGmGmUmCmUmCmCmAmUmGmUmAmCmGmUm-3′(SEQ ID NO： 33)
5′-CmsUmsGmGmAmGmUmUmCmAmGmAmCmGmUmGmUmUmGmCmUmCmUmUmCmCmGmAmUmCmsUmsCm-3′(SEQ ID NO： 34)
5′-CmsUmsGmGmAmGmUmUmCmAmGmAmCmGmUmGmUmUmGmCmUmCmsUmsUm-3′(SEQ ID NO： 35)
5′-CmsUmsGmGmAmGmUmUmCmAmGmAmCmGmUmUmGmUmGmCmUmCmUmUmCmCmGmAmUmCmsUmsCm-3′(SEQ ID NO： 36)
5′-CmsUmsGmGmAmGmUmUmCmAmGmAmCmGmUmUmGmUmGmCmUmC msUmsUm-3′(SEQ ID NO：37)
5′-CmsUmsGmGmAmGmUmUmCmAmGmAmCmCmGmUmGmUmGmCmUmCmUmUmCmCmGmAmUmCmsUmsCm-3′(SEQ ID NO：38)
5′-CmsUmsGmGmAmGmUmUmCmAmGmAmCmCmGmUmGmUmGmCmUmCmsUmsUm-3′(SEQ ID NO：39)
其中，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；小写字母s表示该字母s左右两个核苷酸之间为硫代磷酸酯基连接。
本公开提供的适配体可以通过本领域常规的寡核苷酸制备方法(例如固相合成和液相合成的方法)得到。其中，固相合成已经有商业化订制服务。可以通过使用具有相应修饰的核苷单体来将修饰的核苷酸基团引入本公开提供的适配体中，制备具有相应修饰的核苷单体的方法及将修饰的核苷酸基团引入适配体的方法也是本领域技术人员所熟知的。所有修饰的核苷单体均可以商购得到或者采用已知方法制备得到。
缀合物
在另一方面，本公开还提供了一种缀合物，包含一个或多个递送基团和一个或多个功能性基团，所述递送基团由本公开提供的适配体去除一个氢原子或官能团形成，每个所述递送基团独立地与所述功能性基团经共价键连接，或通过连接基团连接，每个所述功能性基团独立地为诊断剂基团、对肿瘤具有治疗作用的小分子治疗剂基团、对肿瘤具有治疗作用的功能性寡核苷酸基团和递送助剂基团中的一种。通过经共价键或连接基团连接功能性基团来形成缀合物，本公开提供的适配体能够将功能性基团缀合递送至肿瘤。
递送基团由本公开提供的适配体去除一个或多个氢原子或官能团形成。在一些实施方式中，本公开提供的适配体的5′末端核苷酸的核糖5′基团以及3′末端核苷酸的核糖3′基团均为羟基，递送基团由本公开提供的适配体在5′末端核苷酸的5′羟基中去除一个氢原子形成；在一些实施方式中，递送基团由本公开提供的适配体在3′末端核苷酸的3′羟基中去除一个氢原 子形成；在一些实施方式中，递送基团由本公开提供的适配体在5′末端核苷酸中去除5′羟基形成；在一些实施方式中，递送基团由本公开提供的适配体在3′末端核苷酸中去除3′羟基形成。在一些实施方式中，递送基团由本公开提供的适配体在其包含的核苷酸中去除核糖2′-羟基形成。
在一些实施方式中，本公开提供的缀合物具有式(101)所示的结构：
其中，每个RAP基团独立地为具有如式(102)所示的结构的基团：
式中，每个AP基团相同或不同，独立地表示一个所述递送基团；每个A0基团相同或不同，独立地表示一个所述功能性基团；Rj、每个Rk或每个Ri相同或不同，分别独立地表示共价键或者连接基团，且Ri和Rk二者不同时为共价键；m0为1-6的整数；n0为1-6的整数，每个n1各自独立地表示0-4的整数；表示基团共价连接的位点。
在一些实施方式中，m0为1-6的整数，即，式(101)所示的缀合物中包含1-6个所述功能性基团A0。从递送效率和成本的角度考虑，在一些实施方式中，m0为1-4的整数，即，式(101)所示的缀合物中包含1-4个所述功能性基团A0。在一些实施方式中，m0为1，即，式(101)所示的缀合物中包含1个所述功能性基团A0。
在一些实施方式中，n0为1-6的整数，即，式(101)所示的缀合物中包含1-6个RAP基团。从递送效率和成本的角度考虑，在一些实施方式中，n0为1-3的整数，即，式(101)所示的缀合物中包含1-3个RAP基团。在一些实施方式中，n0为1，即，式(101)所示的缀合物中包含1个RAP基团。
在一些实施方式中，每个n1各自独立地表示0-4的整数，且Ri和Rk二者不同时为共价键，从而，每个RAP基团中包含1-5个由本公开提供的 适配体得到的递送基团AP。在一些实施方式中，每个n1各自独立地表示0-1的整数，从而，每个RAP基团中包含1-2个递送基团AP。在一些实施方式中，n0为1、且n1为0，此时，式(101)所示的缀合物中包含1个递送基团AP。
RAP基团中，Rk和Ri的作用是将递送基团AP共价连接至Rj基团，并经由Rj基团连接至功能性基团A0。因此，只要是能够实现上述连接、并且不对递送基团AP和功能性基团A0的作用产生负面影响的任何Rk或Ri均可用于本发明。在一些实施方式中，每个所述Rk或每个所述Ri独立地为长度为1-70个碳原子的直链亚烷基，或者，所述直链亚烷基中的一个或多个碳原子被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、OP(O)2、OP(O)(S)、C5-C8亚糖苷基、C2-C10亚烯基、C2-C10亚炔基、C6-C10亚芳基、C3-C18亚杂环基和C5-C10亚杂芳基；并且其中，所述直链亚烷基可具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C10烷基、C6-C10芳基、C5-C10杂芳基、C1-C10卤代烷基、-OC1-C10烷基、-OC1-C10烷基苯基、-C1-C10烷基-OH、-OC1-C10卤代烷基、-SC1-C10烷基、-SC1-C10烷基苯基、-C1-C10烷基-SH、-SC1-C10卤代烷基、卤素取代基、-OH、-SH、-NH2、-C1-C10烷基-NH2、-N(C1-C10烷基)(C1-C10烷基)、-NH(C1-C10烷基)、-N(C1-C10烷基)(C1-C10烷基苯基)、-NH(C1-C10烷基苯基)、氰基、硝基、-CO2H、-C(O)O(C1-C10烷基)、-CON(C1-C10烷基)(C1-C10烷基)、-CONH(C1-C10烷基)、-CONH2，-NHC(O)(C1-C10烷基)、-NHC(O)(苯基)、-N(C1-C10烷基)C(O)(C1-C10烷基)、-N(C1-C10烷基)C(O)(苯基)、-C(O)C1-C10烷基、-C(O)C1-C10烷基苯基、-C(O)C1-C10卤代烷基、-OC(O)C1-C10烷基、-SO2(C1-C10烷基)、-SO2(苯基)、-SO2(C1-C10卤代烷基)、-SO2NH2、-SO2NH(C1-C10烷基)、-SO2NH(苯基)、-NHSO2(C1-C10烷基)、-NHSO2(苯基)和-NHSO2(C1-C10卤代烷基)。从合成成本和难度以及缀合物的肿瘤靶向效果出发考虑，在一些实施方式中，每个n1均为0，每个Ri独立地为共价键，或者为以下连接基团的一种或多种的连接组合：C1-C20亚烷基、磷酸酯键、硫代磷酸酯键、酰胺键、酯键、醚键、硫醚键、二硫键、1，2，3-三唑亚基、聚乙二醇亚基、吡咯烷亚基、2-氧代吡咯烷亚基、亚苯基、亚环己基、2-丁二酰亚胺亚基、2-硫代丁二酰亚胺亚基、氨基酸亚基、 核苷酸亚基。
在一些实施方式中，连接基团Rj包含本领域技术人员知晓能够用于抗体偶联药物的连接基团。连接基团Rj可以是可裂解的或不可裂解的。在一些实施方式中，连接基团Rj可以是可裂解的。在上下文中，“可裂解”是指在本公开的缀合物靶向至肿瘤后，连接基团Rj在肿瘤内环境下和/或肿瘤细胞内发生共价键断裂，释放出单独的功能性基团产生治疗或诊断效果。在一些实施方式中，连接基团Rj包含活化酶连接基团、硫酸酯酶-可切割连接基团、半乳糖可切割连接基团、对溶酶体蛋白酶敏感的连接基团、肽基连接基团、葡糖苷酸连接基团、酸敏可切割连接基团、或对谷胱甘肽敏感的二硫化物连接基团的一种或多种。在一些实施方式中，连接基团Rj包含肽基连接基团。在一些实施方式中，肽基连接基团选自缬氨酸-瓜氨酸二肽连接子(Val-Cit)、丙氨酸-丙氨酸二肽连接子(Ala-Ala)、缬氨酸-丙氨酸二肽连接子(Val-Ala)、甘氨酸-甘氨酸-苯丙氨酸-甘氨酸的四肽连接子(Gly-Gly-Phc-Gly)中的一种或多种。在一些实施方式中，连接基团Rj选自N-琥珀酰亚基4-(2-二硫吡啶)丁酸盐(SPDB)、N-琥珀酰亚胺-4-(2-硫代吡啶亚基)戊酸盐(SPP)、(S)-2-((S)-2-氨基-3-甲基丁酰胺)-5-脲基戊酸(Val-Cit-PAB-OH)、N-琥珀酰亚胺基-4-(N-马来酰亚胺甲基)环己烷-1-羧酸盐(SMCC)或者2-(磷酸酯基-(CH2)6-S-)-马来酰亚胺己酰基-缬氨酸-瓜氨酸-对氨基苯甲基亚基中的一种。在一些实施方式中，连接基团Rj包含Mckertish CM，Kayser V.Advances and Limitations of Antibody Drug Conjugates for Cancer.Biomedicines.2021 Jul 23；9(8)：872.中列举的连接基团，以引用的方式将该文献的全部内容整体并入本文。
在一些实施方式中，连接基团Rj包含缬氨酸-瓜氨酸二肽连接子(Val-Cit)、聚乙二醇亚基、亚氨基己基亚基、N-琥珀酰亚胺基、GAU三核苷酸连接基团中的一种或多种。在一些实施方式中，每个Ri独立地为共价键、二硫键、亚十二烷基、缬氨酸-瓜氨酸二肽连接子(Val-Cit)、聚乙二醇亚基、亚氨基己基亚基、N-琥珀酰亚胺基或者GAU三核苷酸亚基中的一种或2种的连接组合。
Rj基团的作用是将RAP基团与功能性基团A0相连接，从而通过RAP 基团中递送基团AP的肿瘤靶向作用将功能性基团A0特异性地递送至肿瘤组织和/或细胞。因此，任何能够实现上述连接、且不会影响递送基团AP的肿瘤靶向作用和功能性基团A0的效果的Rj基团都能够实现本发明的目的、解决本发明所要解决的技术问题。在一些实施方式中，在式(101)所示的缀合物到达肿瘤组织和/或进入肿瘤细胞后，所述Rj发生裂解，释放出单独的功能性基团A0对应的药物活性分子。在一些实施方式中，所述Rj在体内不发生裂解，此时缀合物中Rj基团和RAP基团的存在不会影响功能性基团A0发挥诊断和/或治疗作用。
在一些实施方式中，Rj为共价键，m0为1，此时，式(101)所示的缀合物中包含1个功能性基团A0和1个RAP基团，每个RAP基团直接连接至该功能性基团A0。在一些实施方式中，每个RAP基团连接至功能性基团A0的同一原子。在一些实施方式中，每个RAP基团连接至功能性基团A0的不同原子。
在一些实施方式中，Rj为连接基团，所述连接基团Rj包含主链部分、侧链部分和缀合连接部。
主链部分分别与缀合连接部和侧链部分连接。在一些实施方式中，主链部分为长度为1-70个碳原子的直链亚烷基，或者，所述直链亚烷基中的一个或多个碳原子被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、OP(O)2、C5-C8亚糖苷基、C2-C10亚烯基、C2-C10亚炔基、C6-C10亚芳基、C3-C18亚杂环基和C5-C10亚杂芳基；并且其中，所述直链亚烷基可具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C10烷基、C6-C10芳基、C5-C10杂芳基、C1-C10卤代烷基、-OC1-C10烷基、-OC1-C10烷基苯基、-C1-C10烷基-OH、-OC1-C10卤代烷基、-SC1-C10烷基、-SC1-C10烷基苯基、-C1-C10烷基-SH、-SC1-C10卤代烷基、卤素取代基、-OH、-SH、-NH2、-C1-C10烷基-NH2、-N(C1-C10烷基)(C1-C10烷基)、-NH(C1-C10烷基)、-N(C1-C10烷基)(C1-C10烷基苯基)、-NH(C1-C10烷基苯基)、氰基、硝基、-CO2H、-C(O)O(C1-C10烷基)、-CON(C1-C10烷基)(C1-C10烷基)、-CONH(C1-C10烷基)、-CONH2，-NHC(O)(C1-C10烷基)、-NHC(O)(苯基)、-N(C1-C10烷基)C(O)(C1-C10烷基)、-N(C1-C10烷基)C(O)(苯基)、-C(O)C1-C10烷基、 -C(O)C1-C10烷基苯基、-C(O)C1-C10卤代烷基、-OC(O)C1-C10烷基、-SO2(C1-C10烷基)、-SO2(苯基)、-SO2(C1-C10卤代烷基)、-SO2NH2、-SO2NH(C1-C10烷基)、-SO2NH(苯基)、-NHSO2(C1-C10烷基)、-NHSO2(苯基)和-NHSO2(C1-C10卤代烷基)。
侧链部分分别与主链部分和RAP基团连接。在一些实施方式中，每个侧链部分独立地是共价键，或者是长度为1-70个碳原子的直链亚烷基，或者，所述直链亚烷基中的一个或多个碳原子被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、OP(O)2、C5-C8亚糖苷基、C2-C10亚烯基、C2-C10亚炔基、C6-C10亚芳基、C3-C18亚杂环基和C5-C10亚杂芳基；并且，所述直链亚烷基可具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C10烷基、C6-C10芳基、C5-C10杂芳基、C1-C10卤代烷基、-OC1-C10烷基、-OC1-C10烷基苯基、-C1-C10烷基-OH、-OC1-C10卤代烷基、-SC1-C10烷基、-SC1-C10烷基苯基、-C1-C10烷基-SH、-SC1-C10卤代烷基、卤素取代基、-OH、-SH、-NH2、-C1-C10烷基-NH2、-N(C1-C10烷基)(C1-C10烷基)、-NH(C1-C10烷基)、-N(C1-C10烷基)(C1-C10烷基苯基)、-NH(C1-C10烷基苯基)、氰基、硝基、-CO2H、-C(O)O(C1-C10烷基)、-CON(C1-C10烷基)(C1-C10烷基)、-CONH(C1-C10烷基)、-CONH2，-NHC(O)(C1-C10烷基)、-NHC(O)(苯基)、-N(C1-C10烷基)C(O)(C1-C10烷基)、-N(C1-C10烷基)C(O)(苯基)、-C(O)C1-C10烷基、-C(O)C1-C10烷基苯基、-C(O)C1-C10卤代烷基、-OC(O)C1-C10烷基、-SO2(C1-C10烷基)、-SO2(苯基)、-SO2(C1-C10卤代烷基)、-SO2NH2、-SO2NH(C1-C10烷基)、-SO2NH(苯基)、-NHSO2(C1-C10烷基)、-NHSO2(苯基)和-NHSO2(C1-C10卤代烷基)；
缀合连接部分别与主链部分和功能性基团A0连接。在一些实施方式中，每个缀合连接部独立地为共价键或者以下连接结构的一种或多种的连接组合：C1-C10直链亚烷基、磷酸酯键、硫代磷酸酯键、酰胺键、酯键、醚键、二硫键、1，2，3-三唑亚基、聚乙二醇亚基、吡咯烷亚基、2-氧代吡咯烷亚基、亚苯基、亚环己基、2-丁二酰亚胺亚基、2-硫代丁二酰亚胺亚基、氨基酸亚基、核苷酸亚基。
在一些实施方式中，连接基团Rj中的每个所述缀合连接部分别与所 述主链部分和一个所述功能性基团A0连接；所述侧链部分为n0个，每个侧链部分分别与所述主链部分和一个所述RAP基团连接。从而，每个功能性基团A0和RAP基团各自独立地连接至连接基团Rj。在一些实施方式中，全部侧链部分连接至主链部分中的同一原子；或者，每个侧链部分连接至主链部分中的不同原子。
在一些实施方式中，m0为1，所述连接基团Rj包含如式(301)所示的结构：
其中，k为1-3的整数；LC为所述主链部分，LA为所述侧链部分，LB为所述缀合连接部，表示基团共价连接的位点。
所述主链部分LC为共价键或2-4价、直链或支链的C1-C25饱和烃基，或者，所述饱和烃基中的一个或多个碳原子被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、OP(O)2、C5-C8亚糖苷基、C2-C5亚烯基、C2-C5亚炔基、C6-C10亚芳基、C3-C8亚杂环基和C5-C10亚杂芳基；其中，所述饱和烃基可具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C5烷基、C6-C10芳基、C5-C10杂芳基、-O-C1-C5烷基、-OC1-C5烷基苯基、-C1-C5烷基-OH、-SC1-C5烷基、硝基、-C(O)O(C1-C5烷基)、-CON(C1-C5烷基)(C1-C5烷基)、-CONH(C1-C5烷基)、-CONH2，-NHC(O)(C1-C5烷基)、-NHC(O)(苯基)、-N(C1-C5烷基)C(O)(C1-C5烷基)、-N(C1-C5烷基)C(O)(苯基)、-C(O)C1-C5烷基、-C(O)C1-C5烷基苯基、-OC(O)C1-C5烷基、-SO2(C1-C5烷基)、-SO2(苯基)、-SO2NH2、-SO2NH(C1-C5烷基)、-SO2NH(苯基)、-NHSO2(C1-C5烷基)和-NHSO2(苯基)。在一些实施方式中，LC为2-4价的C5-C20饱和烃基，或者，所述饱和烃基中的一个或多个碳原子被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、OP(O)2、C5-C8亚糖苷基、C2-C5亚烯基、C2-C5亚炔基、C6-C10亚芳基、C3-C8亚杂环基和C5-C10亚杂芳基；其中，所述饱和烃基可具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C5烷基、C6-C10芳基、C5-C10杂芳基、-O-C1-C5烷基、-OC1-C5烷基苯基、-C1-C5烷基-OH、-SC1-C5烷基、硝基、 -CONH2。在一些实施方式中，LC的长度为5-30个原子，其中所述LC的长度指LC中与LA直接连接的原子到与LB直接连接的原子形成的最长的原子链上的成链原子的个数。为了简化结构，在一些实施方式中，LC的长度为8-25个原子。
所述侧链部分LA为共价键，或者C1-C20亚烷基，或者，所述亚烷基中的一个或多个碳原子被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、OP(O)2、C5-C8亚糖苷基、C2-C5亚烯基、C2-C5亚炔基、C6-C10亚芳基、C3-C8亚杂环基和C5-C10亚杂芳基；其中，所述亚烷基可具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C5烷基、C6-C10芳基、C5-C10杂芳基、-O-C1-C5烷基、-OC1-C5烷基苯基、-C1-C5烷基-OH-SC1-C5烷基、-SC1-C5烷基苯基、-C1-C5烷基-SH、-OH、-SH、-NH2、-C1-C5烷基-NH2、-N(C1-C5烷基)(C1-C5烷基)、-NH(C1-C5烷基)、-N(C1-C5烷基)(C1-C5烷基苯基)、-NH(C1-C5烷基苯基)、硝基、-C(O)O(C1-C5烷基)、-CON(C1-C5烷基)(C1-C5烷基)、-CONH(C1-C5烷基)、-CONH2，-NHC(O)(C1-C5烷基)、-NHC(O)(苯基)、-N(C1-C5烷基)C(O)(C1-C5烷基)、-N(C1-C5烷基)C(O)(苯基)、-C(O)C1-C5烷基、-C(O)C1-C5烷基苯基、-OC(O)C1-C5烷基、-SO2(C1-C5烷基)、-SO2(苯基)、-SO2NH2、-SO2NH(C1-C5烷基)、-SO2NH(苯基)、-NHSO2(C1-C5烷基)和-NHSO2(苯基)。
所述缀合连接部LB为1-5个以下连接键中的一种或多种的连接组合：磷酸酯键、硫代磷酸酯键、酰胺键、酯键、醚键、二硫键。
在一些实施方式中，k为1-3的整数；LC含有如式(L1)-(L3)所示的基团中的任意一个，通过如式(L1)-(L3)所示的基团中的醚键与LA部分连接：
表示基团连接至分子其余部分的位点；
在一些实施方式中，k＝1，LC含有如式(L1)所示的基团，基团(L1)中的O原子和LA直接相连。在一些实施方式中，k＝2，LC含有如式(L2)所示的基团，基团(L1)中的2个O原子各自和1个LA直接相连。在一些实施方式中，k＝4，LC含有如式(L3)所示的基团，基团(L3)中的3个O原子各自和1个LA直接相连。
LB为磷酸酯键或二硫键；
每个LA为共价键，或者每个LA选自于由基团(L4)-(L23)及其连接组合所组成的组：
式中，每个j1为1-10的整数；
每个R′为C1-C10烷基；
每个Ra为氢原子，C1-C10烷基，或者选自由基团(L24)-(L37)组成的组：
在一些实施方式中，LA的长度为3-35个原子，其中所述LA的长度指LA中与LC直接连接的原子到与LA中与RAP直接连接的原子形成的最长的原子链上的成链原子的个数。在一些实施方式中，每个LA为基团(L4)-(L9)、(L13)、(L14)、(L18)中至少2个的连接组合。在一些实施方式中，每个LA为基团(L4)、(L5)、(L7)、(L9)、(L13)、(L14)、(L18)中至少2个的连接组合。
在一些实施方式中，LA具有如式(302)所示的包含酰胺键的结构， LB具有如式(303)所示的包含N-酰基吡咯烷的结构，含有羰基和氧原子，LC为基于羟甲基氨基甲烷、二羟甲基氨基甲烷或三羟甲基氨基甲烷的连接基团：
其中，n302、q302和p302各自独立地为2-6的整数，可选地，n302、q302和p302各自独立地为2或3；n303为4-16的整数，可选地，n303为8-12的整数，表示基团共价连接的位点。
在一些实施方式中，每个所述侧链部分LA分别与一个RAP基团通过磷酸酯键、醚键或酯键而连接，并通过所述主链部分LC中羟基的氧原子与所述主链部分LC形成醚键而连接；所述缀合连接部LB通过式(303)中的羰基与所述主链部分LC中氨基的氮原子形成酰胺键而连接，并通过式(303)中的氧原子与所述功能性基团A0形成磷酸酯键、醚键或酯键而连接。在一些实施方式中，主链部分LC是基于羟甲基氨基甲烷、二羟甲基氨基甲烷或三羟甲基氨基甲烷的连接基团，所述主链部分LC经由羟基的氧原子与各个所述侧链部分LA通过醚键相连接，并且经由氨基的氮原子与所述缀合连接部LB通过酰胺键相连接。从而，该连接基团Rj中1-3个侧链连接至同一氨甲基的碳原子上，并通过缀合连接部LB连接至包含递送基团的RAP基团。
在一些实施方式中，所述缀合物具有如式(305)所示的结构：
在一些实施方式中，所述连接基团Rj包含如式(306)所示的结构：
其中，n306为0-3的整数，每个p306独立地为1-6的整数，表示基团共价连接的位点；由全部吡咯烷亚基和任何可能的磷酸二酯基团形成的连接组合构成主链部分，由连接至吡咯烷亚基上氮原子的羰基与由*标出的氧原子之间的原子链构成每个侧链部分，所述侧链部分通过由*标出的氧原子与RAP基团形成醚键连接；由#标出的氧原子中的至少一个为缀合链接部并与功能性基团A0形成醚键、酯键或磷酸酯键而连接，其余由#标出的氧原子与氢原子连接形成羟基，或者与C1-C3烷基连接形成C1-C3烷氧基。从而，该连接基团Rj中1-3个侧链部分连接至主链部分中不同的碳原子上，并通过氧原子连接至包含递送基团的RAP基团。
在一些实施方式中，本公开提供的缀合物具有如式(307a)、(307b)或(307c)所示的结构：
在一些实施方式中，本公开提供的缀合物具有如式(308)所示的结构：
其中，n308可为1-10的整数；在一些实施方式中，从合成容易程度、结构/工艺成本和肿瘤细胞特异性等多方面综合考虑，n308为2-6的整数。在一些实施方式中，n308为3或4。
每个R3独立地为功能性基团A0，或者包含递送基团AP的RAP基团；。在一些实施方式中，至少一个R3为功能性基团A0，且至少一个R3为RAP。在一些实施方式中，一个R3为功能性基团A0，其余R3为RAP基团。
在一些实施方式中，当每个m308独立地选自2-10的整数时，认为可能使得该缀合物中，多个递送基团AP之间的空间位置更适合于与肿瘤细胞表面的相应受体发生相互作用，为了使式(308)表示的化合物更为简单，更容易合成和/或降低成本，根据本公开的一些实施方式，每个m308独立地为2-5的整数，在一些实施方式中，每个m308均相等。
本领域技术人员可以理解，当每个R308独立地选自H、C1-C10烷基、C1-C10卤代烷基和C1-C10烷氧基时，不改变式(308)表示的缀合物的性 质，均可实现本公开的目的。在一些实施方式中，每个R308独立地选自H、甲基或乙基。在一些实施方式中，每个R308均为H。
连接至所述功能性基团A0的每个L1表示所述缀合连接部，并且连接至所述RAP的每个L1表示所述侧链部分。在一些实施方式中，一个R3为所述功能性基团A0，其余R3为所述RAP基团。在一些实施方式中，一个或多个L1作为所述侧链部分，将RAP基团与含氮骨架上的N原子连接；并且另外的一个或多个L1作为所述缀合连接部，将功能性基团A0与含氮骨架上的N原子连接。所述含氮骨架共同组成连接基团Rj的主链部分。在本公开的上下文中，“含氮骨架”是指式(308)所示结构中的链结构，其中连接有R308的碳原子与N原子互相连接。
从递送效率和合成成本的角度出发考虑，在一些实施方式中，每个L1的长度独立地为3-25个原子。在一些实施方式中，每个L1的长度独立地为4-15个原子。本领域技术人员会理解，尽管为了方便起见，L1被定义为线性亚烷基，但是它可能不是线性基团或者名称不同，例如由于上述替换和/或取代而产生的胺或烯基。为了本公开的目的，L1的长度是连接两个连接点的链中的原子数。为此目的，将替换所述直链亚烷基的碳原子而得到的环(如亚杂环基或亚杂芳基)按照环上连接点之间的最小原子数计算链中与该环相应部分的长度。
在一些实施方式中，L1选自上述式L4-L23所示基团及其任意连接组合所组成的组。在一些实施方式中，每个L1独立地选自于由基团L4-L9、L13、L14、L18中至少2个的连接组合所组成的组。在一些实施方式中，每个L1独立地为基团L4、L5、L7、L9、L13、L14、L18中至少2个的连接组合。
在一些实施方式中，式(308)所示的缀合物中，每个L1上同时含有与含氮骨架上的N原子连接的连接位点和与功能性基团A0或所述RAP基团连接的连接位点，与含氮骨架上的N原子连接的位点与该N原子形成酰胺键。在一些实施方式中，一个或多个L1选自B5、B6、B5′或B6′：
其中，表示基团共价连接的位点，q2为1-10的整数。在一些实施方式中，q2为1-5的整数。
在一些实施方式中，每个RAP基团中包含一个或多个递送基团。在一些实施方式中，式(308)所示的化合物中包含多个功能性基团。在一些实施方式中，式(308)化合物中每个功能性基团是相同的功能性基团。在一些实施方式中，式(308)化合物中每个功能性基团是用于同一目的和功能的功能性基团。在一些实施方式中，式(308)化合物中含有不同种类的用于不同目的和功能的功能性基团。
在一些实施方式中，所述式(308)所示的化合物具有式(403)、(404)、(405)、(406)、(407)、(408)、(409)、(410)、(411)、(412)、(413)、(414)、(415)、(416)、(417)、(418)、(419)、(420)、(421)、(422)、(423)、(424)、(425)、(426)或(427)所示的结构：








在一些实施方式中，连接基团Rj包含核苷酸序列I和核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II各自包含5-25个修饰或未修饰的核苷酸，所述苷酸序列I和所述核苷酸序列II至少部分地反向互补，所述递送基团连接至所述核苷酸序列I，所述功能性基团连接至所述核苷酸序列II，并且所述核苷酸序列I和所述核苷酸序列II在受试者体内不引发免疫反应或毒性反应。在一些实施方式中，所述核苷酸序列I和所述核苷酸序列II实质上反向互补或者完全反向互补；或者，所述核苷酸序列I和所述核苷酸序列II的长度相等并且均为10-20个修饰或未修饰的核苷酸；或者，所述核苷酸序列I和所述核苷酸序列II均由17个核苷酸组成并且完全反向互补。在一些实施方式中，所述递送基团3′末端经磷酸酯键连接至所述核苷酸序列I的5′末端核苷酸的核糖5′位，所述功能性基团连接至所述核苷酸序列II的5′末端核苷酸的核糖5′位；或者，所述功能性基团包含一段核苷酸序列，所述核苷酸序列的3′末端经磷酸酯键连接至所述核苷酸序列I的5′末端核苷酸的核糖5′位。在一些实施方式中，所述核苷酸序列I和所述核苷酸序列II分别具有SEQ ID NO：40和SEQ ID NO：41所示的序列：
5′-GUACAUUCUAGAUAGCC-3′(SEQ ID NO：40)
5′-GGCUAUCUAGAAUGUAC-3′(SEQ ID NO：41)。
在一些实施方式中，所述核苷酸序列I和所述核苷酸序列II分别具有 SEQ ID NO：42和SEQ ID NO：43所示的序列：
5′-GmUfAmCfAmUfUfCfUfAmGmAmUfAmGmCfCf-3′(SEQ ID NO：42)
5′-GmGmCfUfAmUfCfUfAmGmAmAmUfGmUfAmCf-3′(SEQ ID NO：43)。
本公开提供的缀合物可以包含一个或多个功能性基团。在本公开的上下文中，功能性基团是指，由具有相应功能性的活性剂分子(例如，诊断剂化合物、对肿瘤、特别是胶质瘤具有治疗作用的小分子治疗剂基团化合物和/或调控肿瘤细胞中癌症相关基因表达水平的功能性寡核苷酸化合物)去除一个或多个原子、官能团或部分结构基团(例如，去除一个氢原子)而形成的包含自由基的基团，并且所述功能性基团通过该自由基与本公开提供的缀合物的分子其它部分形成共价连接。
在一些实施方式中，本公开提供的缀合物中的至少一个功能性基团为诊断剂基团，每个所述诊断剂基团独立地选自造影剂基团或荧光成像基团(荧光基团)。在一些实施方式中，本公开提供的缀合物中的全部功能性基团为上述诊断剂基团。通过包含诊断剂基团，本公开提供的缀合物能够将该诊断剂基团递送至肿瘤，从而特异性地、高效、准确地对肿瘤的存在以及疾病进展状态等相关疾病和/或症状信息进行诊断。在一些实施方式中，所述诊断剂基团可用于在体外实验中对待测样品中的肿瘤细胞/组织进行诊断。在一些实施方式中，所述诊断剂基团可用于在受试者体内对肿瘤细胞/组织的存在和/或性质进行诊断。
在一些实施方式中，本公开提供的缀合物中的至少一个功能性基团为对肿瘤、特别是胶质瘤具有治疗作用的小分子治疗剂基团，每个所述小分子治疗剂基团独立地选自细胞毒素基团、抗生素基团、血管生成抑制剂或抗体药物基团。在一些实施方式中，本公开提供的缀合物中的全部功能性基团为上述小分子治疗剂基团。通过包含上述小分子治疗剂基团，本公开提供的缀合物能够特异性地将该小分子治疗剂基团递送至肿瘤，从而通过该小分子治疗剂基团的作用，对肿瘤的疾病进程或症状进行治疗和/或缓解，例如，通过本公开提供的缀合物将细胞毒素基团特异性地递送至肿瘤，使肿瘤中的癌细胞特异性地消亡，从而在减少细胞毒 素本身低靶向性带来的副作用的同时，显著减少肿瘤中的癌细胞数量，从而对肿瘤进行治疗。
在一些实施方式中，本公开提供的缀合物中的至少一个功能性基团为功能性寡核苷酸基团，所述功能性寡核苷酸基团能够调节肿瘤细胞中癌症相关基因的表达水平。在一些实施方式中，本公开提供的缀合物中的全部功能性基团为上述功能性寡核苷酸基团。通过包含该功能性寡核苷酸基团，本公开提供的缀合物能够特异性地将该功能性寡核苷酸基团递送至肿瘤，从而通过该功能性寡核苷酸基团的作用，例如RNAi效应，对肿瘤细胞内癌症相关基因的表达水平进行调控，例如抑制致癌基因的表达，从而对肿瘤相关的疾病进程或症状进行治疗和/或缓解。在一些实施方式中，所述功能性寡核苷酸是siRNA。在一些实施方式中，所述功能性寡核苷酸是靶向STAT3 mRNA、RRM2 mRNA或PLK1的siRNA。在一些实施方式中，所述功能性寡核苷酸是正义链如SEQ ID NO：44所示、反义链如SEQ ID NO：45所示的siRNA，或者正义链如SEQ ID NO：67所示、反义链如SEQ ID NO：68所示的siRNA，或者正义链如SEQ ID NO：69所示、反义链如SEQ ID NO：70所示的siRNA中的一种或多种：
5′-CmsUmsAmGmAmAmAfAfCfUmGmGmAmUmAmAmCmGmUm-3′(SEQ ID NO：44)
5′-AmsCfsGmUmUmAfUmCmCmAmGmUmUmUfUmCfUmAmGmsCmsCm-3′(SEQ ID NO：45)
5′-CUUCUUAUUGACACUUACAdT-S-dT-3′(SEQ ID NO：67)
5′-UGUAAGUGUCAAUAAGAAGdT-S-dT-3′(SEQ ID NO：68)
5′-CAAGAAGAAUGAAUACAGUdT-S-dT-3′(SEQ ID NO：69)
5′-ACUGUAUUCAUUCUUCUUGdT-S-dT-3′(SEQ ID NO：70)
所述功能性基团可通过任何合适的方式包含于本公开提供的缀合物中。例如，可以通过前述的缀合连接部将功能性基团A0与所述主链部分相连接。
在一些实施方式中，本公开提供的缀合物中的至少一个功能性基团为递送助剂基团，所述递送助剂基团选自于C10-C30烃基、胆固醇基、磷脂基团中的一种或多种。通过包含该递送助剂基团，本公开提供的缀合 物能够更好地与中枢神经系统中的体内环境相容，可能具有更好的生物利用度，和/或使本公开提供的缀合物更有效地递送至肿瘤。
本领域技术人员可以采用任意合理的合成路线制备本公开提供的缀合物。
在一些实施方式中，本公开提供的缀合物的合成方法包括在脱保护反应条件下，在溶剂中，将经保护的缀合物与脱保护试剂接触，分离获得本公开提供的缀合物。经保护的缀合物是本公开提供的缀合物中任意活性官能团均被保护基团保护而形成的化合物。在一些实施方式中，所述活性官能团包括但不限于羟基、氨基和/或磷酸基，所述保护基团相应地为羟基保护基、氨基保护基和/或磷酸羟基保护基(例如，氰乙基保护基)。根据保护基团的不同，对所使用的溶剂、脱保护反应条件和脱保护试剂进行选择和确定。在一些实施方式中，所述脱保护反应条件、溶剂和脱保护试剂是核酸固相合成中使用的脱保护反应条件、溶剂和试剂。在一些实施方式中，所述方法包括将经保护的缀合物加入甲胺水溶液和氨水的混合溶液中，所述脱保护反应条件包括在常温常压下反应1-5h。在一些实施方式中，以等体积混合所述甲胺水溶液与饱和浓氨水得到所述混合溶液，所述溶液相对于经保护的缀合物的用量为0.1-10ml/μmol。在一些实施方式中，所述分离包括通过柱色谱分离进行纯化，收集产品洗脱液并除去溶剂。纯化条件可以是例如使用制备型离子色谱纯化柱，以氯化钠水溶液与磷酸钠水溶液的梯度洗脱剂进行洗脱。在一些实施方式中，以20mM磷酸钠(pH 8.1)，溶剂为水/乙腈＝9∶1(体积比)；洗脱剂B：1.5M氯化钠，20mM磷酸钠(pH 8.1)，溶剂为水/乙腈＝9∶1(体积比)；洗脱梯度：洗脱剂A∶洗脱剂B＝100∶0-50∶50梯度洗脱。
在一些实施方式中，本公开提供的缀合物具有式(101)所示的结构，所述经保护的缀合物的合成方法包括在有机溶剂中，在偶联反应条件下，将包含活性基团Rx1和递送基团的化合物与包含活性基团Rx2和功能性基团的化合物接触，反应获得所述经保护的缀合物，其中，所述递送基团由本公开提供的适配体去除一个或多个氢原子或一个或多个官能团形成，每个所述功能性基团独立地为诊断剂基团、对肿瘤具有治疗作用的小分子治疗剂基团和对肿瘤具有治疗作用的功能性寡核苷酸基团中的一种， 其中所述递送基团和功能性基团中的任何活性基团均被保护基团保护，所述活性基团Rx1和所述活性基团Rx2是能够经反应形成共价键或者连接基团Rj的基团。在一些实施方式中，连接有活性基团Rx1的递送基团和连接有活性基团Rx2的功能性基团的摩尔比为m0∶n0。在一些实施方式中，所述递送基团和功能性基团中的活性基团包括但不限于羟基、氨基、磷酸基中的一种或多种。
本领域的技术人员可以通过各种方法获得包含活性基团Rx1和递送基团的化合物。在一些实施方式中，所述包含活性基团Rx1和递送基团的化合物可以通过本领域技术人员熟知的核酸合成方法，例如亚磷酰胺固相合成或者磷酸二酯法/磷酸三酯法液相合成获得。在一些实施方式中，所述包含活性基团Rx1和递送基团的化合物采用亚磷酰胺固相合成法得到，该方法包括，在亚磷酰胺固相合成条件下，按照寡核苷酸单链中核苷酸的顺序，将核苷单体依次连接，其中，至少一个核苷单体为具有活性基团Rx1的核苷单体，或者，在连接全部核苷单体后，再按照亚磷酰胺固相合成法连接一个具有活性基团Rx1的亚磷酰胺单体或经保护的亚磷酰胺单体，随后再脱除该保护基团形成活性基团Rx1。亚磷酰胺固相合成法为本领域技术人员所公知，其过程和条件在Methods in Molecular Biology，vol.288：Oligonucleotide Synthesis：Methods and Applications，P17-P31中详细公开，以引用的方式将其全部内容整体并入本文。
在一些实施方式中，所述偶联反应条件是缩合反应条件或者巯基-二硫键交换反应的条件。
在一些实施方式中，所述偶联反应条件是缩合反应条件，所述缩合反应条件是酰基化缩合反应条件、脱水缩合反应条件或者点击化学反应的条件，活性基团Rx1与活性基团Rx2是能够发生前述缩合反应的基团。在一些实施方式中，所述缩合反应条件是酰基化缩合反应的条件，所述活性基团Rx1和Rx2是能够发生酰基化缩合反应形成RI的基团。在一些实施方式中，所述缩合反应条件是脱水缩合反应的条件，所述活性基团Rx1和Rx2中的一个是包含酰卤基团或羧基的基团，另一个是包含氨基或羟基的基团。在一些实施方式中，所述缩合反应条件是点击化学的条件，所述活性基团Rx1和Rx2中的一个是包含炔基的基团，另一个是包含叠氮基 团的基团。在一些实施方式中，所述缩合反应条件是Michael加成反应的条件，所述活性基团Rx1和Rx2中的一个是包含巯基的基团，另一个是包含琥珀酰亚胺基的基团。在一些实施方式中，所述缩合反应条件是N-羟基琥珀酰亚胺-碳二亚胺(NHS-EDC)联用偶联反应的条件，所述活性基团Rx1和Rx2中的一个是包含N-羟基琥珀酰亚胺(NHS)的基团，另一个是包含碳二亚胺基(EDC)的基团。
在一些实施方式中，所述包含活性基团Rx1和递送基团的化合物是通过在偶联反应的条件下，将具有活性基团Rx0的本公开提供的适配体和交联剂接触制备得到的，所述交联剂含有点击化学活性基团和酰基化基团。所述活性基团RX0与所述酰基化基团通过发生偶联反应而形成共价连接，使所述点击化学活性基团连接至本公开的适配体。
在一些实施方式中，活性基团Rx1是末端含有1-3个点击化学活性基团的活性基团，所述点击化学活性基团包含末端炔基。在一些实施方式中，所述酰基化基团是活性酯基团，例如可以是NHS酯基、亚氨酸酯基以及五氟苯酯基中的一种。本领域的技术人员可以通过各种方法获得所述交联剂，例如，当所述酰基化基团是五氟苯酯基，所述点击化学基团包含末端炔基时，所述交联剂可以按照如Michael E.，et al.″Efficient synthesis and biological evaluation of 5′-GalNAc conjugated antisense oligonucleotides.″Bioconjugate chemistry 26.8(2015)：1451-1455中Scheme 1a(A)中描述的方法制备获得，以引用的方式将其全部内容整体并入本文。在一些实施方式中，所述活性基团Rx0是氨基。在一些实施方式中，所述偶联条件是碱性条件。在一些实施方式中，所述碱性条件是有弱碱水溶液存在的条件，例如有碳酸氢钠水溶液存在的条件。
本领域的技术人员可以通过各种方式获得所述具有活性基团Rx0的适配体，在一些实施方式中，所述具有活性基团Rx0的适配体是通过在合成适配体的过程中在相应位置使用含有活性基团的亚磷酰胺单体制备得到的。本领域的技术人员可以通过各种方式获得含有活性基团的亚磷酰胺单体。在一些实施方式中，所述活性基团Rx0是氨基，含有Rx0的亚磷酰胺单体可以通过本领域技术人员熟知的方法商购获得或制备获得，例如，含有Rx0的亚磷酰胺单体可以是容易商购获得的6-(三氟乙酰氨基)-己基- (2-氰乙基)-(N，N-二异丙基)-亚磷酰胺单体，其中，活性基团Rx0为氨基，该活性基团Rx0可以是通过亚磷酰胺固相合成法将所述亚磷酰胺单体连接至寡核苷酸单链后，经本领域技术人员容易实现的脱保护反应(如浓氨水氨解)脱除三氟乙酰基保护基而获得。
在一些实施方式中，所述偶联反应条件是巯基-二硫键交换反应中的一种，所述活性基团Rx1和Rx2中的一个是包含巯基的基团，另一个包含经二硫键连接的离去基团。为了避免副反应发生，在一些实施方式中，上述含有活性基团Rx1的亚磷酰胺单体中的Rx1以被保护的Rx1’形式存在，所述制备方法还包含在脱保护反应条件下，通过将制备得到的包含被保护的活性基团Rx1’和递送基团的化合物与脱保护试剂接触，分离得到包含活性基团Rx1和递送基团的化合物的步骤。在一些实施方式中，所述Rx1’含有二硫键离去基团，所述脱保护反应条件是巯基-二硫键交换反应条件，所述脱保护试剂是二硫键活化剂。在一些实施方式中，所述二硫键活化剂是二硫二吡啶。本领域的技术人员可以通过各种方法获得上述含有活性基团Rx1或Rx1’的亚磷酰胺单体，在一些实施方式中，所述含有活性基团Rx1或Rx1’的亚磷酰胺单体是商购得到的，例如可以通过商购得到如式(105)所示的亚磷酰胺单体。
其中n105和m105各自独立地是1-10的整数。
本领域的技术人员可以通过各种方式获得所述包含活性基团Rx2和功能性基团的化合物。在一些实施方式中，所述偶联反应条件是巯基-二硫键交换反应条件，所述活性基团Rx2包含巯基，所述包含活性基团Rx2和功能性基团的化合物可以由本领域技术人员按照各种已知的方式获得，例如通过使用包含巯基的亚磷酰胺单体通过亚磷酰胺固相合成方法制备获得，或者商购获得。在一些实施方式中，所述功能性基团是功能性寡核苷酸基团，所述偶联反应条件是亚磷酰胺固相合成反应条件，活性基 团Rx1是羟基，所述活性基团Rx2是亚磷酰胺基团，所述制备方法包括将连接有递送基团和活性基团Rx1的固相载体按照功能性寡核苷酸的核酸序列，按照亚磷酰胺固相合成反应的方式依次连接核苷单体。在一些实施方式中，所述功能性基团是诊断剂基团或小分子治疗剂基团，所述偶联反应条件是亚磷酰胺固相合成反应条件，所述活性基团Rx2是亚磷酰胺基团，所述包含活性基团Rx2和功能性基团的化合物可以是例如容易商购获得的包含亚磷酰胺基团和荧光基团或小分子治疗剂基团的化合物。在一些实施方式中，在一些实施方式中，所述偶联反应条件是Michael加成反应条件，所述活性基团Rx2是N-琥珀酰亚胺基团，所述包含活性基团Rx2和功能性基团的化合物可以是例如容易商购获得的包含N-琥珀酰亚胺基团和小分子治疗剂基团的化合物。在一些实施方式中，所述功能性基团是功能性寡核苷酸基团，所述活性基团Rx1与活性基团Rx2分别是核苷酸序列I和核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II各自包含5-25个修饰或未修饰的核苷酸，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补，所述递送基团连接至所述核苷酸序列I，所述功能性基团连接至所述核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II在受试者体内不引发免疫反应或毒性反应，所述偶联反应条件是退火形成核酸双链的反应条件。在一些实施方式中，所述功能性寡核苷酸基团是siRNA，所述核苷酸序列I的5′末端与所述递送基团通过磷酸二酯键连接，所述所述核苷酸序列II的3′末端与所述siRNA的5′末端通过磷酸二酯键连接。在一些实施方式中，所述核苷酸序列I和所述核苷酸序列II均由17个核苷酸组成并且完全反向互补。在一些实施方式中，所述核苷酸序列I和所述核苷酸序列II分别具有SEQ ID NO：40和SEQ ID NO：41所示的序列。在一些实施方式中，所述核苷酸序列I和所述核苷酸序列II分别具有SEQ ID NO：42和SEQ ID NO：43所示的序列。
在一些实施方式中，所述缀合物还可以其药学上可接受的盐或前体化合物的形式用于本公开。在本公开的上下文中，“药学上可接受的盐”是指为了增加药物的稳定性、溶解性和/或生物利用度而将药物形成相应的在药学上不对人体产生额外副作用的盐，例如钾盐、钠盐、羧酸盐等。“前体化合物”是指尽管其本身与所述缀合物结构和功能上都不完全相同，但在进入体内之后或在体液环境下能够发生反应、形成所述包含本 公开提供的缀合物、从而发挥效果、实现本公开提供的目的的化合物。某些情况下，这些前体化合物能够增加药物的稳定性、延长缓释时间、增加生物利用度等作用。在一些实施方式中，所述前体化合物包括可在人体内反应形成缀合物中全部功能性基团A0的前体基团。在一些实施方式中，所述前体化合物包括缀合物中全部活性羟基被乙酰氧基取代形成的化合物。在一些实施方式中，所述前体化合物包括药物前体基团，所述药物前体基团是由缀合物中所述功能性基团对应的诊断剂、治疗剂和/或功能性寡核苷酸的前体化合物形成的残基。在一些实施方式中，所述药物前体基团可以是例如所述功能性基团中羟基或氨基官能团中的活性氢被酰基、烷基、或者磷酰基取代而形成的基团。本领域技术人员可以理解，这些药学上可接受的盐以及前体化合物的应用同样处于本公开的范围之内。
药物组合物
在一方面，本公开还提供一种药物组合物，所述药物组合物包含本公开提供的缀合物以及药学上可接受的载体。
所述药学上可接受的载体可以是本领域常规使用的载体，例如但不限于水、生理盐水、磁性纳米粒(magnetic nanoparticles，如基于Fe3O4或Fe2O3的纳米粒)、碳纳米管(carbon nanotubes)、介孔硅(mesoporous silicon)、磷酸钙纳米粒(calcium phosphate nanoparticles)、聚乙烯亚胺(polyethylenimine，PEI)、聚酰胺型树形高分子(polyamidoamine(PAMAM)dendrimer)、聚赖氨酸(poly(L-lysine)，PLL)、壳聚糖(chitosan)、1，2-二油酰基-3-三甲铵丙烷(1，2-dioleoyl-3-trimethylammonium-propane，DOTAP)、聚D型或L型乳酸/羟基乙酸共聚物(poly(D&L-lactic/glycolic acid)copolymer，PLGA)、聚(氨乙基乙撑磷酸酯)(poly(2-aminoethyl ethylene phosphate)，PPEEA)和聚(甲基丙烯酸-N，N-二甲氨基乙酯)(poly(2-dimethylaminoethyl methacrylate)，PDMAEMA)以及它们的衍生物中的一种或多种。
在一些实施方式中，所述药学上可接受的载体含有生理学可接受的化合物，所述化合物起到例如稳定药物组合物或增加或减少缀合物和/或药物组合物的吸收的作用。所述生理学可接受的化合物选自如下化合物 中的一种或多种：碳水化合物，例如葡萄糖、蔗糖和/或葡聚糖；抗氧化剂，例如抗坏血酸和/或谷胱甘肽；螯合剂；低分子量蛋白；降低任何共同给药的物质的清除或水解的组合物；赋形剂；稳定剂和缓冲剂。也可以将清洁剂用于稳定组合物或增加或减少药物组合物的吸收。所述生理学可接受的化合物还可以包括湿润剂、乳化剂、分散剂或特别用于防止微生物生长或作用的防腐剂中的一种或多种。所述生理学可接受的化合物是本领域技术人员已知的，本公开不再赘述。本领域技术人员容易理解的是，药学上可接受的载体以及生理学可接受的化合物的选择取决于例如给药途径和任何共同给药的物质的特定生理化学特性。
在一些实施方式中，药学上可接受的载体是无菌的并且通常不含不合需要的物质。本公开提供的药物组合物可根据需要进一步包含可药用的辅助物质以接近生理学条件，例如pH调节剂和缓冲剂，毒性调节剂等，例如乙酸钠、氯化钠、氯化钾、氯化钙、乳酸钠等，药物组合物中的本公开提供的缀合物的浓度可以在大的区间内变化，并且主要依据流体体积、粘度、体重等按照特定的给药方式加以选择。
在一些实施方式中，所述药物组合物中，对缀合物和药学上可接受的载体的含量没有特别要求，在一些实施方式中，缀合物与药学上可接受的载体的重量比可以为1∶(1-500)，在一些的实施方式中，上述重量比为1∶(1-50)。
在一些实施方式中，所述药物组合物中，还可以包含药学上可接受的其它辅料，该辅料可以为本领域常规采用的各种制剂或化合物的一种或多种。例如，所述药学上可接受的其它辅料可以包括pH缓冲液、保护剂和渗透压调节剂中的至少一种。
所述pH缓冲液可以为pH值7.5-8.5的三羟甲基胺基甲烷盐酸盐缓冲液和/或pH值5.5-8.5的磷酸盐缓冲液，例如可以为pH值5.5-8.5的磷酸盐缓冲液。
所述保护剂可以为肌醇、山梨醇、蔗糖、海藻糖、甘露糖、麦芽糖、乳糖和葡萄糖中的至少一种。以所述药物组合物的总重量为基准，所述保护剂的含量可以为0.01-30重量％。
所述渗透压调节剂可以为氯化钠和/或氯化钾。所述渗透压调节剂的含量使所述药物组合物的渗透压为200-700毫渗摩尔/千克(mOsm/kg)。根据所需渗透压，本领域技术人员可以容易地确定所述渗透压调节剂的含量。在一些实施方式中，所述药物组合物所制成的制剂在给药过程中的剂量会因给药方式的不同而发生调整。
在一些实施方式中，所述药物组合物可以为液体制剂，例如注射液；也可以为冻干粉针剂，实施给药时与液体辅料混合，配制成液体制剂。所述液体制剂可以但不限于用于皮下、肌肉或静脉注射给药，也可以但不限于通过穿刺注射、或通过口咽吸入、或鼻腔给药等方式递送所述药物组合物。在一些实施方式中，所述药物组合物用于皮下、肌肉、静脉或鞘内注射给药。
在一些实施方式中，所述药物组合物可以为脂质体制剂的形式。在一些实施方式中，所述脂质体制剂中使用的药学上可接受的载体包含含胺的转染化合物(下文也可将其称为有机胺)、辅助脂质和/或聚乙二醇化脂质。其中，所述有机胺、辅助脂质和聚乙二醇化脂质可分别选自于中国专利申请CN103380113A(通过引用的方式将其整体并入本公开)中所描述的含胺的转染化合物或其药学上可接受的盐或衍生物、辅助脂质和聚乙二醇化脂质中的一种或多种。
在一些实施方式中，所述有机胺可为中国专利申请CN103380113A中描述的如式(201)所示的化合物或其药学上可接受的盐：
其中：
X101和X102各自独立地是O、S、N-A或C-A，其中A是氢或C1- C20烃链；
Y101和Z101各自独立地是C＝O、C＝S、S＝O、CH-OH或SO2；
R101、R102、R103、R104、R105、R106和R107各自独立地是氢，环状或无环的、被取代的或未被取代的、支链或直链脂族基团，环状或无环的、被取代的或未被取代的、支链或直链杂脂族基团，被取代的或未被取代的、支链或直链酰基，被取代的或未被取代的、支链或直链芳基，被取代的或未被取代的、支链或直链杂芳基；
x是1-10的整数；
n是1-3的整数，m是0-20的整数，p是0或1；其中，如果m＝p＝0，则R102是氢；
并且，如果n或m中的至少一个是2，那么R103和在式(201)中的氮形成如式(202)或式(203)所示的结构：
其中，g、e和f各自独立地是1-6的整数，“HCC”代表烃链，且每个*N代表式(201)中的氮原子。
在一些实施方式中，R103是多胺。在其它实施方式中，R103是缩酮。在一些实施方式中，在式(201)中的R101和R102中的每一个独立地是任意的被取代的或未被取代的、支链或直链烷基或烯基，所述烷基或烯基具有3至约20个碳原子，诸如8至约18个碳原子，和0至4个双键，诸如0至2个双键。
在一些实施方式中，如果n和m中的每一个独立地具有1或3的值，那么R103可以是下述式(204)-式(213)中的任一个：
其中，式(204)-式(213)中，g、e和f各自独立地是1-6的整数，每个“HCC”代表烃链，且每个*显示R103与在式(201)中的氮原子的可能连接点，其中在任意*位置上的每个H可以被替换以实现与在式(201)中的氮原子的连接。
本领域技术人员可以通过任何合理的方法获得式(201)所示的化合物。在一些实施方式中，式(201)所示化合物可以根据中国专利申请CN103380113A中的描述制备。
在一些实施方式中，所述有机胺为如式(214)所示的有机胺和/或如式(215)所示的有机胺：
所述辅助脂质为胆固醇、胆固醇的类似物和/或胆固醇的衍生物；
所述聚乙二醇化脂质为1，2-二棕榈酰-sn-甘油-3-磷脂酰乙醇胺-N-[甲氧基(聚乙二醇)]-2000。
在一些实施方式中，所述药物组合物中，所述有机胺、所述辅助脂质和所述聚乙二醇化脂质三者之间的摩尔比为(19.7-80)∶(19.7-80)∶(0.3-50)，例如可以为(50-70)∶(20-40)∶(3-20)。
在一些实施方式中，由本公开提供的缀合物与上述含胺的转染试剂形成的药物组合物颗粒具有约30nm至约200nm的平均直径，通常为约40nm至约135nm，更通常地，该脂质体颗粒的平均直径是约50nm至约120nm、约50nm至约100nm、约60nm至约90nm或约70nm至约90nm，例如，该脂质体颗粒的平均直径是约30、40、50、60、70、75、80、85、90、100、110、120、130、140、150或160nm。
在一些实施方式中，由本公开提供的缀合物与上述含胺的转染试剂形成的药物组合物中，缀合物与全部脂质(例如有机胺、辅助脂质和/或聚乙二醇化脂质)的重量比(重量/重量比)在从约1∶1至约1∶50、从约 1∶1至约1∶30、从约1∶3至约1∶20、从约1∶4至约1∶18、从约1∶5至约1∶17、从约1∶5至约1∶15、从约1∶5至约1∶12、从约1∶6至约1∶12或从约1∶6至约1∶10的范围内，例如，本公开提供的缀合物与全部脂质的重量比为约1∶5、1∶6、1∶7、1∶8、1∶9、1∶10、1∶11、1∶12、1∶13、1∶14、1∶15、1∶16、1∶17或1∶18。
在一些实施方式中，所述药物组合物在销售时各组分可以独立存在，在使用时可以液体制剂的形式存在。在一些实施方式中，本公开提供的缀合物与上述药学上可接受的载体形成的药物组合物可以按照已知的各种方法制备，只是用本公开提供的缀合物替代现有适配体或缀合物即可；在一些实施方式中，可以按照如下方法制备：
将有机胺、辅助脂质和聚乙二醇化脂质按照上述摩尔比悬浮于醇中并混匀得到脂质溶液；醇的用量使得到的脂质溶液的总质量浓度为2-25mg/mL，例如可以为8-18mg/mL。所述醇选自药学上可接受的醇，诸如在室温附近为液体的醇，例如，乙醇、丙二醇、苯甲醇、甘油、聚乙二醇200，聚乙二醇300，聚乙二醇400中的一种或多种，例如可以为乙醇。
将本公开提供的缀合物溶解于缓冲盐溶液中，得到缀合物水溶液。缓冲盐溶液的浓度为0.05-0.5M，例如可以为0.1-0.2M，调节缓冲盐溶液的pH至4.0-5.5，例如可以为5.0-5.2，缓冲盐溶液的用量使缀合物的浓度不超过0.6mg/mL，例如可以为0.2-0.4mg/mL。所述缓冲盐选自可溶性醋酸盐、可溶性柠檬酸盐中的一种或多种，例如可以为醋酸钠和/或醋酸钾。
将脂质溶液和缀合物水溶液混合，将混合后得到的产物在40-60孵育至少2分钟，例如可以为5-30分钟，得到孵育后的脂质体制剂。脂质溶液和缀合物水溶液的体积比为1∶(2-5)，例如可以为1∶4。
将孵育后的脂质体制剂浓缩或稀释，去除杂质，除菌，得到本公开提供的药物组合物，其理化参数为pH值为6.5-8，包封率不低于80％，粒径为40-200nm，多分散指数不高于0.30，渗透压为250-400mOsm/kg；例如理化参数可以为pH值为7.2-7.6，包封率不低于90％，粒径为60- 100nm，多分散指数不高于0.20，渗透压为300-400mOsm/kg。
其中，浓缩或稀释可以在去除杂质之前、之后或同时进行。去除杂质的方法可以采用现有各种方法，例如可以使用切相流系统、中空纤维柱，在100K Da条件下超滤，超滤交换溶液为pH7.4的磷酸盐缓冲液(PBS)。除菌的方法可以采用现有各种方法，例如可以在0.22μm滤器上过滤除菌。
本公开提供的适配体和/或缀合物的应用
在又一方面，本公开还提供了本公开提供的适配体和/或缀合物和/或药物组合物在制备用于对肿瘤及肿瘤相关疾病或症状进行诊断和/或治疗的药物中的应用。
在又一方面，本公开还提供了一种对肿瘤及肿瘤相关疾病或症状进行诊断和/或治疗的方法，所述方法包括向有需要的受试者给予本公开提供的缀合物和/或药物组合物。
通过给予本公开提供的缀合物和/或药物组合物，本公开提供的方法能够有效诊断和/或治疗肿瘤及肿瘤相关疾病或症状；并且，在本公开提供的缀合物的高特异性靶向作用下，可以减少诊断剂和/或治疗剂在不期望的身体其它器官/组织处的分布，降低潜在的副反应。特别是对于肿瘤治疗领域中常用的、已知副反应明显的放疗和/或化疗药物而言，具有重要意义和显著价值。在一些实施方式中，所述药物可用于在体外实验中对待测样品中的肿瘤细胞/组织进行诊断。在一些实施方式中，所述药物可用于在受试者体内对肿瘤细胞/组织进行诊断。在一些实施方式中，所述药物可用于对受试者体内的肿瘤或肿瘤相关疾病以及症状进行治疗。
本公开所使用的术语“给药/给予”是指通过使得至少部分地将缀合物和/或药物组合物定位于期望的位点以产生期望效果的方法或途径，将缀合物和/或药物组合物放置入受试者体内。适于本公开方法的给药途径包括局部给药和全身给药。一般而言，局部给药导致与受试者整个身体相比将更多缀合物和/或药物组合物递送至特定位点；而全身给药导致将所述缀合物和/或药物组合物递送至受试者的基本整个身体。
进一步地，本公开提供的发明人意外地发现，本公开提供的缀合物 和/或药物组合物能够高效地通过血脑屏障，在全身给药的情况下就能够靶向至脑内的肿瘤中，从而进一步提高功能性基团的递送效率、节省成本并降低不期望的副反应。
可通过本领域已知的任何合适途径向受试者给药，所述途径包括但不仅限于：口服或胃肠外途径，如静脉内给药、肌肉内给药、皮下给药、经皮给药、气道给药(气雾剂)、肺部给药、鼻部给药、直肠给药和局部给药(包括口腔含化给药和舌下给药)。给药频率可以是每天、每周、每两周、每三周、每个月或每年1次或多次。
本公开提供的缀合物和/或药物组合物的使用剂量可为本领域常规的剂量，所述剂量可以根据各种参数、尤其是受试者的年龄、体重和性别来确定。可在细胞培养或实验动物中通过标准药学程序测定毒性和疗效，例如测定LD50(使50％的群体致死的剂量)和ED50(在量反应中指能引起50％最大反应强度的剂量，在质反应中，指引起50％实验对象出现阳性反应时的剂量)。可基于由细胞培养分析和动物研究得到的数据得出人用剂量的范围。
在给予本公开提供的缀合物和/或药物组合物时，例如，对于雄性或雌性、6-12周龄、体重18-25g的C57BL/6J或C3H/HeNCrlVr小鼠，以所述缀合物和/或药物组合物中的缀合物的量计：对于缀合物与药学上可接受的缀合分子形成的缀合物，其缀合物用量可以为0.001-100mg/kg体重，在一些实施方式中为0.01-50mg/kg体重，在进一步的实施方式中为0.05-20mg/kg体重，在更进一步的实施方式中为0.1-15mg/kg体重，在又进一步的实施方式中为0.1-10mg/kg体重。在给予本公开提供的缀合物和/或药物组合物时，可优选上述用量。
试剂盒
本公开提供了一种试剂盒，所述试剂盒包含本公开提供的缀合物和/或药物组合物。
在一些实施方式中，本公开提供的试剂盒可在一个容器中提供缀合物和/或药物组合物。在一些实施方式中，本公开提供的试剂盒可包含一个提供药学上可接受的赋形剂的容器。在一些实施方式中，所述试剂盒 中还可包含其它成分，如稳定剂或防腐剂等。在一些实施方式中，本公开提供的试剂盒可在不同于提供本公开提供的缀合物和/或药物组合物的容器以外的其它容器中包含至少一种其它治疗剂。在一些实施方式中，所述试剂盒可包含用于将缀合物和/或药物组合物与药学上可接受的载体和/或辅料或其它成分(若有的话)进行混合的说明书。
在本公开提供的试剂盒中，所述缀合物和药学上可接受的载体和/或辅料以及所述药物组合物，和/或药学上可接受的辅料可以任何形式提供，例如液体形式、干燥形式或冻干形式。在一些实施方式中，所述缀合物和药学上可接受的载体和/或辅料以及所述药物组合物和任选的药学上可接受的辅料基本上纯净和/或无菌。在一些实施方式中，可在本公开提供的试剂盒中提供无菌水。
下面将通过实施例来进一步说明本公开，但是本公开并不因此而受到任何限制。
实施例
除非特别说明，以下实施例中所用到的试剂、培养基均为市售商品，所用到的核酸电泳、real-time PCR等操作均参照Molecular Cloning(Cold Spring Harbor LBboratory Press(1989))所记载的方法进行。
制备例1-9和12本公开提供的缀合物的合成
通过固相合成方法分别合成了表1A中编号为AP1-AP8和AP12的缀合物，并且，在分别按照表1A中AP1-AP8和AP12对应的核苷酸序列从3′-5′方向依次连接全部核苷单体后，再按照固相合成方法连接核苷亚磷酰胺单体的方式连接Cy5亚磷酰胺单体(购自苏州吉玛公司，批号CY5P21H1B)。随后，将核苷酸序列加入甲胺水溶液与氨水等体积混合溶液中，溶液相对于缀合物的用量为0.5ml/μmol，在25℃下反应2h，过滤除去固体，将上清液真空浓缩至干。
利用制备型离子色谱纯化柱(Source 15Q)，通过NaCl水溶液的梯度洗脱，完成制备的缀合物的纯化。具体而言为：洗脱剂A：20mM磷酸钠(pH 8.1)，溶剂为水/乙腈＝9∶1(体积比)；洗脱剂B：1.5M氯化钠，20mM磷酸钠(pH 8.1)，溶剂为水/乙腈＝9∶1(体积比)；洗脱梯度：洗脱 剂A∶洗脱剂B＝100∶0-50∶50梯度洗脱。收集产品洗脱液后合并，采用反相色谱纯化柱进行脱盐，具体条件包括采用葡聚糖凝胶柱进行脱盐，填料为葡聚糖凝胶G25，以去离子水洗脱。将获得的洗脱液浓缩除去溶剂并冻干，分别获得了5′末端核苷酸的核糖5′位通过磷酸酯基连接基团连接荧光基团Cy5的缀合物AP1-AP8。
按照同样的方法，制备获得5′末端核苷酸的核糖5′位通过磷酸酯基连接基团连接荧光基团Cy3的缀合物AP9，区别仅在于，按照表1A中AP9对应的核酸序列依次连接核苷单体，并使用Cy3亚磷酰胺单体(购自上海兆维公司，货号OP-038)代替Cy5亚磷酰胺单体进行制备。
在上述缀合物AP1-AP9和AP12合成完成后，使用超纯水(Milli-Q超纯水仪自制，电阻率18.2MΩ*cm(25))将制备获得的缀合物稀释至浓度为0.2mg/mL后，利用液质联用仪(LC-MS，Liquid Chromatography-Mass Spectrometry，购于Waters公司，型号：LCT Premier)进行分子量检测。其结果，分子量实测值与理论值一致，表明获得了目标缀合物。
对比制备例10、11、13和14对比缀合物的合成
按照制备例1-9和12的方法，分别合成了表1A中编号为对比AP10、对比AP11、对比AP13和对比AP14的缀合物，区别仅在于，分别按照表1A中对应于对比AP10、对比AP11、对比AP13和对比AP14的序列，依次连接核苷单体。从而，分别获得了5′末端核苷酸的核糖5′位通过磷酸酯基连接基团连接荧光基团Cy5的对比缀合物对比AP10、对比AP11、对比AP13和对比AP14。其中，对比AP10和对比AP11分别是具有不同序列或修饰方案的阴性对照缀合物，其适配体序列具有一定随机性并且与本公开提供的适配体几乎不具有序列同源性。对比AP13和对比AP14分别是序列分别与缀合物AP2和AP4仅存在少量核苷酸差异的对比缀合物。具体来说，对比AP13与AP2相比，5′末端多了两个核苷酸G和A、3′末端少一个核苷酸C，并且链内在对应于AP2的5′端起算第17-18个核苷酸的位置处缺失了一个核苷酸U；对比AP14与AP4相比，5′末端多了两个核苷酸G和A、3′末端少一个核苷酸U，并且链内在对应于AP2的5′端起算第17-18个核苷酸的位置处缺失了一个核苷酸U。
表1A缀合物的核苷酸序列
表1A中，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；小写字母s表示该字母s左右两个核苷酸之间为硫代磷酸酯基连接；CY3和CY5分别表示荧光染料基团Cy3(Cyanine 3)和Cy5(Cyanine 5)基团在适配体上的连接位点。
制备例15-16缀合物15-16的合成
本制备例15中，按照以下步骤制备获得了缀合物15。
(15-1)寡核苷酸C1的制备
通过制备例1-8和12的方法合成表1B中缀合物15中的寡核苷酸序列，区别仅在于，该正义链序列中包含siRNA的正义链，该siRNA的正 义链3′末端经作为连接基团的Spacer18-GfAfUf连接至缀合物AP2中包含的递送基团，其中，Spacer18是指包含依次连接的6个乙氧基亚基、包含长度为18个原子的六聚乙二醇亚基连接基团。连接基团从3′-5′方向依次连接核苷单体GfAfUf后，再按照固相合成方法连接六聚乙二醇(HEG)-亚磷酰胺单体。HEG-亚磷酰胺单体具有式(15-a)所示结构。
在固相合成的过程中，在连接完5′端最后一个核苷单体之后，按照连接核苷亚磷酰胺单体的方法，另外连接含有-NH-(CH2)6-基团的亚磷酰胺单体(购自Hongene Biotech公司)，从固相载体上切割下寡核苷酸单链，经过氨解浓缩后粗品经超滤脱盐并浓缩干得到如式(15-b)所示的寡核苷酸单链C1(40.00mg，5.49μmol)：
式中，代表缀合物15对应的寡核苷酸序列。
(15-2)寡核苷酸C2的合成：
用5.4ml溶剂DMF溶解Cy5-NHS 346mg。取浓缩干的寡核苷酸单链C1，用100mM碳酸氢钠-水溶液1.34mL溶解备用。将Cy5-NHS的DMF溶液加入到寡核苷酸单链C1溶液中，加入三乙胺，室温下避光反应过夜。向反应液中加入甲基叔丁基醚/乙醇溶液(v∶v＝4∶1)，加入量是反应液的3倍体积，室温下涡旋1min，离心机上3200g离心15min，结束后移除上清液，沉淀即是所需寡核苷酸单链C1粗品。用纯水将核酸样品溶解，安捷伦半制备反相柱纯化，使用以下流动相梯度洗脱：洗脱液A：100mM TEAA-水(pH＝7.2)，洗脱液B：乙腈(ACN)，梯度：15-75％(B  v/v％)。浓缩除去溶剂，获得通过-NH-(CH2)6-基团和磷酸酯基连接基团连接有Cy5荧光基团的寡核苷酸单链C2 43.4mg。
按照同样方法，合成了缀合物16的正义链，区别仅在于，按照表1B中缀合物16的正义链核苷酸序列，依次连接核苷酸。与缀合物3中，递送基团连接至siRNA正义链3′末端不同，该缀合物4的siRNA正义链在5′末端经作为连接基团的Spacer18-GfAfUf连接连接至缀合物AP2包含的递送基团的3′末端，并且该递送基团的5′末端核苷酸的核糖5′位通过-NH-(CH2)6-基团和磷酸酯基连接基团连接有荧光基团Cy5。
按照核酸固相合成方法，合成表1B中编号缀合物15和16的缀合物的反义链序列。
分别将每一缀合物的正义链序列与反义链序列溶于注射用水中，得到40mg/mL的溶液，以等摩尔比混合，50℃加热15min，室温冷却后，使它们通过氢键形成双链结构。随后按照制备例1-2记载的方法对所获得的缀合物进行纯化、脱盐、除去溶剂和冻干，分别获得缀合物15和缀合物16。合成完成后，对于所获得的缀合物3和4的正义链序列和反义链序列，分别采用离子交换色谱(IEX-HPLC)检测纯度，并采用液质联用色谱(LC-MS)分析分子量，缀合物3和缀合物4的正义链MS理论值均为：18986.82；缀合物3实测值为18984.81，缀合物4实测值为18984.84。缀合物3和缀合物4的反义链相同，理论值为6881.59，MS分子离子峰实测值为：1719.2(4价阴离子)，测得的实测值与理论值进行一致。缀合物15和缀合物16分别是包含双链siRNA和缀合物AP2包含的递送基团的缀合物，其中递送基团经Spacer18-GfAfUf连接基团连接至siRNA正义链的5′末端或3′末端，并且在整条包含siRNA正义链的核苷酸序列的5′末端核苷酸的核糖5′位通过NH-(CH2)6-基团和磷酸酯基连接基团缀合连接有Cy5荧光基团。
表1B缀合物中的核苷酸序列

表1B中，大写字母C、G、U、A、T表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母d表示该字母d右侧相邻的一个核苷酸为脱氧核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；小写字母s表示该字母s左右两个核苷酸之间为硫代磷酸酯基连接；CY3和CY5分别表示荧光染料基团Cy3(Cyanine 3)和Cy5(Cyanine 5)基团在适配体 上的连接位点；CY5-(NH-C6H12)表示siRNA中经由正己基氨基连接基团连接至荧光染料基团Cy5的位点；大写字母X表示亚丙基-磷酸酯基，小写字母组合hd表示该字母组合hd左侧相邻的一个核苷酸为2′-O-十六烷基修饰的核苷酸；Spacer18表示上述六聚乙二醇亚基连接基团；MMAE表示小分子药物基团MMAE(一甲基澳瑞他汀E)基团在适配体上的连接位点。
制备例17-18缀合物17和缀合物18的合成
按照制备例15-16中相同的方法合成了表1B中编号为缀合物17和缀合物18的缀合物，区别仅在于，分别按照表1B中缀合物17和缀合物18对应的核酸序列依次连接核苷单体；并且，在制备正义链时，C3-spacer亚磷酰胺单体(购自上海兆维公司，货号OP-005)代替Spacer18-GfAfUf连接基团进行连接，该连接重复四次，随后继续按照表1B中的核酸序列继续连接核苷单体。即，所获得的缀合物17是包含双链siRNA和本公开提供的适配体的缀合物，其中适配体经四个依次连接的亚丙基磷酸酯基作为连接基团连接至siRNA正义链的5′末端，并且在适配体的5′末端通过NH-(CH2)6-基团和磷酸酯基连接有Cy5荧光基团；缀合物18是缀合物17中适配体每一末端的前两个核苷酸均具有2’-O-十六烷基修饰的缀合物。
制备例19缀合物19的合成
按照制备例15-16中相同的方法合成了表1B中编号为缀合物19的缀合物，区别仅在于，分别按照表1B中缀合物19对应的核酸序列依次连接核苷单体，获得三条核苷酸序列；并且，在退火时，将所获得的三条核苷酸序列共同溶于注射用水中，并以等摩尔比混合。所获得的缀合物19是包含双链siRNA和本公开提供的缀合物AP2的缀合物，其中适配体经连接基团连接至siRNA，所述连接基团是分别具有SEQ ID NO：28和SEQ ID NO：29所示的序列的互补核苷酸序列形成的双链，适配体连接至SEQ ID NO：28所示序列的5′末端、siRNA连接至SEQ ID NO：29所示序列的3′末端，并且在适配体的5′末端NH-(CH2)6-基团和磷酸酯基连接有Cy5荧光基团。制备完成后，制备12％聚丙烯酰胺凝胶，将所制备的缀合物19溶于核酸上样缓冲液混匀后上样加入所述聚丙烯酰胺凝胶，在 80V电压下垂直电泳1h。电泳结束后，通过Gelstain泡染法，使用3×泡染液浸没凝胶，在室温下缓慢震荡1h对凝胶染色，在凝胶成像仪上成像。成像结果显示所制备的缀合物19显示出单一条带，表明成功获得了双链缀合物，即缀合物19，其中各核苷酸序列间形成双链互补区。
制备例20缀合物20的合成
本制备例中，按照以下步骤制备获得了缀合物20，该缀合物20包含本公开提供的适配体和小分子药物基团MMAE，所述MMAE基团经连接基团连接至适配体的5′末端，所述连接基团为2-(磷酸酯基-(CH2)6-S-)-马来酰亚胺己酰基-缬氨酸-瓜氨酸-对氨基苯甲基亚基。
(20-1)适配体S1的制备
通过固相合成方法合成表1C中缀合物20中的适配体序列，区别仅在于，按照表1C中缀合物20对应的适配体序列，依次连接核苷单体；在固相合成的过程中，在连接完5′端最后一个核苷单体之后，按照连接核苷亚磷酰胺单体的方法，另外连接含有HO-(CH2)6-S-S-(CH2)6-基团的亚磷酰胺单体(购自Hongene Biotech公司)，从固相载体上切割下适配体单链，得到如式(20-a)所示的适配体单链S1(70.00mg，6.42μmol)：
式中，代表缀合物20对应的适配体序列。
(20-2)适配体S2的合成：
用10.0ml纯化水溶解70.0mg步骤(20-1)中制备得到的S1(6.42μmol)后，向所得溶液中加入105mg TCEP(三(2-氯乙基)磷酸酯，0.37mmol，购自毕得医药，批号：BD155793)溶于10.0ml纯化水得到的TCEP水溶液。混匀，在室温下反应2小时，将反应液用10mL纯化水稀释并过滤，得到反应液28mL，将反应液转移至3K规格的超滤管中，在 3900rpm的条件下离心30min。再重复超滤和离心的步骤2次，收集滤膜内产品，获得适配体S2(67.0mg，产率：95.7％)。
(20-3)适配体20的合成：
将24mg Vc MMAE(18.56μmol，5eq，购自CSN公司，批号CSN16143-005)溶于6.0ml DMF中，并加入60μl三乙胺，获得Vc MMAE溶液。用6.0ml纯化水溶解40.0mg步骤(20-2)中制备得到的适配体S2(3.71μmol，1eq)后，向所得溶液中加入上述Vc MMAE溶液，在室温下反应2小时后，获得缀合物20粗品(工艺图中表示为S3)。
将所得到的缀合物20粗品加入0.5ml纯化水稀释，并以0.45μm滤膜过滤，滤液使用安捷伦半制备反相柱色谱纯化，所使用的色谱柱为Kromasil 100-10-C18，10um，21.2*250mm；以100mM三乙胺乙酸盐缓冲液(TEAA，pH＝7.0-7.3)∶乙腈＝95∶5-35∶65作为流动相进行梯度洗脱。收集产物峰洗脱液，蒸发除去溶剂，获得缀合物20(55mg，收率56.7％)。经LC-MS检测分子量，理论值：12092.67，实测值：12091.68，实测值与理论值一致，表明该缀合物20具有S3所示的结构，并且包含本公开提供的适配体和小分子药物基团MMAE，所述MMAE基团经连接基团连接至适配体的5′末端，所述连接基团为2-(磷酸酯基-(CH2)6-S-)-马来酰亚胺己酰基-缬氨酸-瓜氨酸-对氨基苯甲基亚基(2-(磷酸酯基-(CH2)6-S-)-MC-Val-Cit-PAB)。
表1C适配体单链的核苷酸序列

表1C中，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；MMAE表示小分子药物基团MMAE(一甲基澳瑞他汀E)基团在适配体上的连接位点。
制备例21-26本公开提供的缀合物AP21-AP26的合成
按照制备例1-8和12的方法，分别合成了表2中编号为AP21-AP26的缀合物并检测分子量以对合成的缀合物进行确认，区别仅在于，分别按照表2中对应于AP21-AP26的序列，依次连接核苷单体。从而，分别获得了5′末端连接荧光基团Cy5的缀合物AP21-AP26。其中，AP21与前述AP4相比，在3′末端减少1个核苷酸；AP22与前述AP4相比，是对应于式(1)所示序列中S1和S4基序各减少1个核苷酸所示的结构；AP23是AP22对应于式(1)所示序列中S1基序再减少1个核苷酸所示的结构；AP24是AP4改变5′末端前2个核苷酸获得的缀合物；AP25和AP26与前述AP4相比，是对应于式(1)所示序列中Nb基序发生改变获得的缀合物。
在上述缀合物AP11-AP26合成完成后，使用超纯水(Milli-Q超纯水仪自制，电阻率18.2MΩ*cm(25))将制备获得的缀合物稀释至浓度为0.2mg/mL后，利用液质联用仪(LC-MS，Liquid Chromatography-Mass Spectrometry，购于Waters公司，型号：LCT Premier)进行分子量检测。其结果，分子量实测值与理论值一致，表明获得了目标缀合物。
缀合物AP11-AP26的MS理论值和MS实测值值如下表2-a：
对比制备例27-35对照缀合物对比AP27-对比AP35的合成
按照制备例1-9和12的方法，分别合成了表2中编号为对比AP27-对比AP35的缀合物并检测分子量以对合成的缀合物进行确认，区别仅在于，分别按照表2中对应于对比AP27-对比AP35的序列，依次连接核苷单体。从而，分别获得了5′末端连接荧光基团Cy5的对照适配缀合物体对比AP27-对比AP35。其中，对比AP27和对比AP28是序列碱基组成与AP4相同，但核苷酸排列顺序不同的对照缀合物；对比AP29和对比AP30是序列碱基组成与AP2相同，但核苷酸排列顺序不同的对照缀合物；对比AP31与对比AP4相比，在5′末端少2个核苷酸、在3′末端少1个核苷酸；对比AP32与对比AP4相比，对应于式(1)所示序列中缺失了Nb基序；对比AP33与AP4相比，对应于式(1)所示序列中缺失了Na和Nc基序；对比AP34和对比AP35相比，是在S2和S3基序部分各增加了多个核苷酸且改变部分序列的对照缀合物。
表2缀合物的核苷酸序列

表2中，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；CY5表示荧光染料基团Cy5(Cyanine 5)基团在适配体上的连接位点。
在上述对比AP27-对比AP35合成完成后，使用超纯水(Milli-Q超纯水仪自制，电阻率18.2MΩ*cm(25))将制备获得的缀合物稀释至浓度为0.2mg/mL后，利用液质联用仪(LC-MS，Liquid Chromatography-Mass Spectrometry，购于Waters公司，型号：LCT Premier)进行分子量检测。其结果，分子量实测值与理论值一致，表明获得了目标缀合物。
对比AP27-对比AP35的MS理论值和MS实测值值如下表2-b：
表2b：对比缀合物的质谱表征结果
制备例36缀合物36的合成
按照制备例1-8和12中相同的方法合成了表1B中编号为缀合物36的缀合物，区别仅在于，分别按照表1B中缀合物36对应的核酸序列依次连接核苷单体；并且，在制备正义链时，该siRNA正义链3′末端经作为连接基团的6个dT磷酸酯基连接至缀合物AP2中包含的递送基团；该siRNA正义链5′位通过磷酸酯基连接基团连接有Cy5荧光基团。
合成完成后，对于所获得的缀合物36正义链序列和反义链序列，分别采用离子交换色谱(IEX-HPLC)检测纯度，并采用液质联用色谱(LC-MS)分析分子量，缀合物36正义链理论值：19660.11，实测值：19658.23，理论值与实测值一致。缀合物36的反义链理论值：6881.59，实测值：6880.83，理论值与实测值一致。
制备例37-38缀合物37和缀合物38的合成
按照制备例1-8和12中相同的方法合成了表1B中编号为缀合物37和缀合物38的缀合物，区别仅在于，分别按照表1B中缀合物37和缀合物38的正义链或者反义链的核苷酸序列从3′-5′方向依次连接全部核苷单体，得到缀合物正义链和反义链序列。其中：
对于缀合物37，该正义链序列中包含siRNA的正义链，该siRNA正义链3′末端经作为连接基团的Spacer18-GfAfUf连接至缀合物AP2包含的递送基团的5′端，该反义链的5′末端核苷酸的核糖5′位通过磷酸酯基连接基团连接有荧光基团Cy5。
对于缀合物38，该正义链序列中包含siRNA的正义链，该siRNA正义链5′末端经4个依次连接的亚丙基-磷酸酯基连接至缀合物AP2包含的递送基团3′端，该反义链的5′末端核苷酸的核糖5′位通过磷酸酯基连接基团连接有荧光基团Cy5。
合成完成后，对于所获得的缀合物37和缀合物38正义链序列和反义链序列，分别采用离子交换色谱(IEX-HPLC)检测纯度，并采用液质联用色谱(LC-MS)分析分子量，缀合物37正义链分子量：18327.97，实测值：18325.87，理论值与实测值一致。缀合物38正义链理论：19011.37，实测值：19003.59，理论值与实测值一致。缀合物37和缀合 物38的反义链相同，理论值均为：7415.22，实测值均为：7413.32，理论值与实测值一致。结果表明，所获得的缀合物37和缀合物38具有上述表1B中所示的序列和结构。
制备例39-44缀合物39-42和对比缀合物43-44的合成
按照制备20同样方法制备缀合物39，经LC-MS检测分子量，缀合物39理论值：9473.03，实测值9472.06：实测值与理论值一致，表明该缀合物39与表1C序列结构一致。缀合物40、缀合物41、缀合物42、对比缀合物43和对比缀合物44均经LC-MS检测分子量，实测值与理论值一致。按照制备例20的方法，分别合成了表1C中编号为缀合物39、缀合物40、缀合物41、缀合物42、对比缀合物43和对比缀合物44并检测分子量以对合成的缀合物进行确认，区别仅在于，分别按照表1C中对应于的缀合物39、缀合物40、缀合物41、缀合物42、对比缀合物43和对比缀合物44序列，依次连接核苷单体。
缀合物的MS理论值和MS实测值值如下表3：
表3缀合物的质谱表征结果
实验例1本公开提供的缀合物及对照缀合物在体外进入U118-MG胶质瘤细胞能力的评价比较
本实验例中，对缀合物AP1和对照缀合物对比AP10在体外进入U118-MG胶质瘤细胞的能力进行了评价和比较。
在添加有10％的胎牛血清(FBS，Thermo Fisher公司)的DMEM培养基(Thermo Fisher公司)中，于37℃在含5％CO2/95％空气的培养箱中培养U118MG神经胶质瘤细胞(购自中科院上海细胞库)。
将U118MG细胞以5×105细胞/孔接种于96孔板中。用DMEM培养基将前述制备的缀合物AP1和酵母tRNA分别配制成缀合物溶液1A、1B、1C、1D、1E、1F、1G、1H和1I，该缀合物溶液1A-1I中含有终浓度为100μg/mL的酵母tRNA，并且分别含有终浓度依次为5nM、10nM、20nM、40nM、60nM、80nM、100nM、150nM、200nM和500nM的缀合物AP1。
用DMEM培养基将前述制备的对照缀合物对比AP10和酵母tRNA分别配制成缀合物溶液1A′、1B′、1C′、1D′、1E′、1F′、1G′、1H′和1I′，该缀合物溶液1A′-1I′中含有终浓度为100μg/mL的酵母tRNA，并且分别含有终浓度依次为5nM、10nM、20nM、40nM、60nM、80nM、100nM、150nM、200nM和500nM的对照缀合物对比AP10。
向前述含有U118MG细胞的培养孔中，分别加入上述缀合物溶液1A-1I，加入量为200μL/孔，依次记为测试组1A-测试组1I。向前述含有U118MG细胞的培养孔中，分别加入上述缀合物溶液1A′-1I′，加入量为200μL/孔，依次记为对照组1A′-对照组1I′。在另一含有U118MG细胞的培养孔中加入含有终浓度为100μg/mL的酵母tRNA的DMEM培养基200μL，记为空白对照组1J。将含有上述各测试组和空白对照组的96孔板在37℃下避光孵育1h，孵育结束后，对每一测试组和空白对照组中的反应混合物，分别吸去上清液并加入DPBS缓冲液(购自Thermo Fisher公司)洗涤3次，每次100μL。吸去洗涤液后，每一培养孔加入100μL DMEM培养基获得细胞悬液。离心除去上清液，随后加入500μL DPBS缓冲液重悬细胞，使用流式细胞仪(BD Biosciences公司)进行流式细胞分析，每一测试组或空白对照组分析10000个细胞，测定荧光强度分布并计算平均荧光强度(MFI，a.u.)。进一步地，将各缀合物终浓度对应的平均荧光强度列于以下表4中进行对比，并按照以下公式计算相对荧光差值R：R＝(MFIAP1-MFICAP10)/MFICAP10
其中MFIAP1表示缀合物AP1在U118-MG胶质瘤细胞中的平均荧光强度，MFICAP10表示对比AP10在U118-MG胶质瘤细胞中的平均荧光强度。R值反映缀合物AP1进入U118-MG胶质瘤细胞的相对能力，当R＞2时，提示对应缀合物进入细胞的能力较强(参见Engineering of  Targeted Nanoparticles for Cancer Therapy Using Internalizing Aptamers Isolated by Cell-Uptake Selection.ACS Nano.2012 January 24；6(1)在其表2中的记载，以引用的方式将该文献的全部公开内容并入本文)。
表4缀合物平均荧光强度及R值
从表4中可见，在不同浓度下，与随机序列的对比AP10相比，本公开提供的缀合物AP1在U118MG胶质瘤细胞中均显示出显著更高的平均荧光强度，表明本公开提供的缀合物具有优异的进入U118MG胶质瘤细胞的能力。
实验例2荧光成像系统观察缀合物进入不同细胞情况
在本实验中，使用PerkinElmer高内涵成像系统(Operatta公司)对本公开提供的缀合物AP1与对比缀合物对比AP10进入不同细胞的情况进行了观察。
U118MG胶质瘤细胞、SVGp12正常星形胶质细胞、T98G人脑胶质瘤细胞、U251人胶质瘤细胞、A549人非小细胞肺癌细胞、MCF-7人乳腺癌细胞和293T人肾上皮细胞均购自中科院上海细胞库。在实验前，对每一细胞系，选取对数生长的细胞，以每孔5000个细胞接种于96孔板中，每一细胞系接种2个培养孔，于37℃在含5％CO2/95％空气的培养箱中培养24h。吸出上清液，加入DPBS缓冲液(购自Thermo Fisher公司)洗涤2次，每次100μL。
用DMEM培养基将前述制备的缀合物AP1和酵母tRNA分别配制成缀合物溶液2A，该缀合物溶液2A中含有终浓度为100μg/mL的酵母 tRNA和终浓度为100nM的缀合物AP1；
用DMEM培养基将前述制备的对照缀合物对比AP10和酵母tRNA分别配制成对照缀合物溶液2B，该对照缀合物溶液2B中含有终浓度为100μg/mL的酵母tRNA和终浓度为100nM的对比AP10；
对于每一细胞系，在1个培养孔中加入200μL缀合物溶液2A，在37℃下避光孵育30分钟，吸出上清液，加入DPBS缓冲液洗涤2次，每次100μL，得到测试组2X；
对于每一细胞系，在另1个培养孔中加入200μL对照缀合物溶液2B，在37℃下避光孵育30分钟，吸出上清液，加入DPBS缓冲液洗涤2次，每次100μL，得到对照组2Y。
对上述各细胞系的测试组2X和对照组2Y，分别加入100μL 37℃DMEM培养基，在高内涵成像系统中成像。按照对照组2Y的平均荧光强度归一化，即测试组的2X荧光强度减去对照组2Y的平均荧光强度后进行成像。结果分别示于图1A-图1G中。
图1A和图1B分别是示出了AP1和对比AP10进入U118MG胶质瘤细胞和SVGp12正常星形胶质细胞的高内涵成像图。由图1A和1B中可见，仅在经AP1处理的U118MG细胞中显示出了强烈的Cy5荧光信号，而在SVGp12细胞中测试组和对照组均无任何荧光信号，表明与对比AP10相比，本公开提供的缀合物具有很强的进入U118MG胶质瘤细胞的能力，同时基本不进入SVGp12正常星形胶质细胞。上述结果也表明，本发明提供的缀合物能够将功能性基团选择性地靶向递送至U118MG胶质瘤细胞。
进一步地，图1C-1G依次分别是示出了AP1和对比AP10进入U251人胶质瘤细胞、A549人非小细胞肺癌细胞、MCF-7人乳腺癌细胞和293T人肾上皮细胞高内涵成像图。由图1C-1G中可见，在经AP1处理的U251人胶质瘤细胞、A549人非小细胞肺癌细胞、MCF-7人乳腺癌细胞中均显示出强烈的Cy5荧光信号，而上述各组经对比AP10处理的肿瘤细胞以及293T人肾上皮细胞中测试组和对照组均无任何荧光信号，表明与对比AP10相比，本公开提供的缀合物具有很强的进入各种肿瘤细胞的能 力，同时不进入正常细胞，本发明提供的缀合物能够将功能性基团选择性地靶向递送至各种肿瘤细胞。。
实验例3荧光成像系统观察缀合物进入肿瘤球的情况
在本实验中，使用PerkinElmer高内涵成像系统(Operatta公司)对本公开提供的缀合物AP1与对比缀合物对比AP10进入U118MG胶质瘤肿瘤球和A549人非小细胞肺癌肿瘤球的情况进行了观察。
在96孔板的培养孔中，加入80μL新鲜灭菌且未凝固的2％琼脂糖，待完全凝固后，对每一细胞系，选取对数生长的细胞，以每孔2000个细胞接种于96孔板中，每一细胞系接种2个培养孔，每孔200μL细胞悬液，以添加有10％FBS和1％青霉素双抗的DMEM培养基培养，于37℃在含5％CO2/95％空气的培养箱中培养。每隔48h吸出一半上清液，并贴培养孔壁加入新的DMEM培养基。培养7天后，于镜下观察确认获得完整的细胞球，表明肿瘤球培养成功。
按照实验例2的方法向各培养孔中加入AP1或对比AP10，并在高内涵成像系统中成像，区别仅在于，在培养后不进行洗涤，直接进行成像；并且按照Cy5荧光实测强度进行成像。结果参见图2A和2B所示。
图2A和2B依次分别是示出了AP1和对比AP10进入U118MG胶质瘤肿瘤球和A549人非小细胞肺癌肿瘤球的高内涵成像图。由图2A和2B中可见，在经AP1处理的U118MG肿瘤球和A549肿瘤球中内部中显示出了强烈的Cy5荧光信号，而在对比AP10处理的肿瘤球中，仅在肿瘤球边缘显示出明显较弱的荧光信号，表明与对比AP10相比，本公开提供的缀合物具有很强的进入U118MG和A549肿瘤球内部的能力。上述结果说明，本公开提供的适配体能够将功能性基团有效递送至肿瘤内部，具有较高的成药能力。
实验例4缀合物在U118MG细胞皮下瘤模型小鼠体内分布
在添加有10％的胎牛血清(FBS，RMBIO公司)的DMEM完全培养基(MACGENE公司，货号CM15019)中，于37在含5％CO2/95％空气的培养箱中培养U118MG人胶质瘤细胞(购自广州吉妮欧生物科技有限公司)。
取对数期生长的U118MG人胶质瘤细胞，以(0.25％的胰酶)消化，收集细胞，离心去上清，将细胞重悬于添加10％FBS的DMEM培养基中制成浓度为1×108cells/mL的细胞培养液。
实验动物NOD-SCID小鼠10只(购自斯贝福(北京)生物技术有限公司)，雄性，12周龄。将上述细胞培养液接种于NOD-SCID小鼠右前肢皮下位置，接种体积为每只100μL，即，每只小鼠接种1×107个细胞。注射后继续饲养小鼠20天。
用1×DMEM(购自中科迈晨(北京)科技有限公司，批号为K1902200)培养基将AP1、AP2和对比AP11分别溶解成0.3mg/mL浓度(以适配体计)的溶液。
将上述接种U118MG的小鼠随机分为3组，每组4只；对于每组的4只小鼠，分别按10μL/g体重计算给药体积，采取尾静脉注射的方式，向不同组小鼠分别给予AP1、AP2和对比AP11，分别记为测试组4A1、测试组4A2和对照组4A3。从而，每只小鼠的给药剂量均为3mg/kg(按适配体计)。向另外1只上述接种U118MG的小鼠尾静脉注射1×DMEM培养基，给药体积为10μL/g体重，记为空白对照组4Y。
给药后1h时，依次将各组小鼠放置于小动物活体光学成像系统IVIS Lumina Series III(PerkinElmer)中，采用异氟烷气体麻醉小鼠，将麻醉后的小鼠腹部朝上放置于小动物活体光学成像系统中进行活体成像。给药后1h、4h、24h和48h时，分别将各组中的1只小鼠处死，取肿瘤组织进行荧光成像(24h和48h结果未做空白对照组)，结果分别参见图3A、3B、3C和3D所示。
图3A-3D依次分别是示出了给药后1h、4h、24h和48h时的活体成像及肿瘤组织成像图，其中blank表示空白对照组4Y。从图3A-3D可以看出，空白对照组4Y和给予具有随机序列的对比AP11的对照组4A3在小鼠体内基本不显示或仅显示较弱的荧光信号；与此不同，本公开提供的缀合物AP1和AP2在小鼠体内接种肿瘤处显示出强烈的荧光信号，表明本公开提供的适配体能够特异性地将荧光基团靶向递送至U118MG胶质瘤；进一步地，缀合物AP2在给药后24h至48h时仍显示出较强的荧光信号，表明本 公开提供的缀合物在较长时间内均能够稳定靶向胶质瘤组织。
实验例5缀合物在U118MG细胞原位瘤模型小鼠体内分布
取对数期生长的如实验例4中培养的U118MG人胶质瘤细胞，以0.25wt％胰酶消化并收集细胞，吸去上清并将细胞重悬于添加10％FBS的DMEM培养基中制成细胞密度为4×107cells/mL的细胞培养液。
实验动物NOD-SCID小鼠16只(购自斯贝福(北京)生物技术有限公司)，雄性，12周龄。将上述细胞培养液接种于NOD-SCID小鼠中，采用小鼠侧脑室注射方式，将细胞培养液注射于小鼠右侧纹状体，位置为AP(anteroposterior前后/正位)：1mm，ML(medial lateral内侧)：1.5mm，DV(dorsal ventral背腹侧)：3.5mm，注射体积10μL，即，每只小鼠接种4×105个细胞。注射后继续饲养小鼠14天。
用1×DMEM(购自中科迈晨(北京)科技有限公司，批号为K1902200)培养基将AP1、AP2和对比AP11分别溶解成2mg/mL浓度(以适配体计)的缀合物溶液。取10只前述小鼠，通过鞘内注射给药方式分别注射50μL的AP1和AP2溶液，以适配体的量计，每只动物给药量为100μg，每组给予3只小鼠，分别记为测试组5A、5B；向2只小鼠中的每一只分别注射50μL的对比AP11溶液，以适配体的量计，每只动物给药量为100μg，记为对照组5C；向2只小鼠中的每一只分别注射50μL的DMEM培养基，记为空白对照组5Y。
用1×DMEM培养基将AP1、AP2和对比AP11分别溶解成0.3mg/mL浓度(以适配体计)的缀合物溶液。取另外6只前述小鼠，通过尾静脉注射给药，分别注射AP1、AP2和对比AP11溶液，所有动物根据体重计算给药剂量，给药体积均为10μL/g，以适配体的量计，每只动物给药量为3mg/kg，每组给予2只小鼠，分别记为测试组5D、5E和对照组5F。
在给药后24h时，分别杀死各组小鼠中的一只并取脑组织，给药后48h时杀死剩余小鼠并取脑组织，在IVIS Lumina Series III中对小鼠脑组织进行荧光成像。结果分别参见图4A和4B所示。
图4A和4B分别是示出了给药后24h和48h时，给予空白对照组5Y、 测试组5A、5B、5D和5E以及对照组5C和5F后建立U118MG原位瘤模型小鼠脑组织的荧光成像图。从图4A和图4B的结果可以看出，空白对照组和对比AP11的对照组均未显示出肿瘤位置处的明显荧光信号，表明对原位脑胶质瘤没有显著靶向效果；与此不同，给予本公开提供的缀合物的测试组5A和5B在接种肿瘤位置显示出强的荧光信号，表明能够有效靶向肿瘤组织，并且表明本公开提供的适配体能够将诊断剂基团如荧光基团有效递送至肿瘤组织；进一步地，测试组5E在24h和48h时均显示出明显荧光信号，表明缀合物AP2在尾静脉给药时仍能到达并靶向脑胶质瘤，说明本公开提供的缀合物还可以穿透血脑屏障(Blood-Brain-Barrier，BBB)进入脑胶质瘤。
实施例6不同修饰缀合物在U118MG皮下瘤模型小鼠体内分布
按照实验例4描述的方法制备获得U118MG皮下瘤模型小鼠。
用1×DMEM培养基将AP2、AP3和AP12分别溶解成0.3mg/mL浓度(以适配体计)的缀合物溶液。将9只小鼠分为3组，每组3只，向各组的每只小鼠分别尾静脉注射上述AP2、AP3和AP12溶液，所有动物根据体重计算给药剂量，给药体积均为10μL/g，以适配体的量计，每只动物给药量为3mg/kg，3组小鼠依次分别记为测试组6A、测试组6B以及测试组6C。向另外2只小鼠给予DMEM培养基，给药体积为为10μL/g，记为空白对照组6Y。分别于给药后30min、1h、24h、48h将各组小鼠中的1只放置于小动物活体光学成像系统IVIS Lumina Series III(PerkinElmer)中，采用异氟烷气体麻醉小鼠，将麻醉后的小鼠腹部朝上放置于小动物活体光学成像系统中进行活体成像。结果见图5A-5D所示。在给药后第10天，将空白对照组6Y和测试组6A的1只小鼠进行活体成像，结果如图5E所示；在给药后24h和48h时，分别将各组中的1只小鼠处死，取肿瘤组织进行荧光成像，结果分别如图5F和图5G所示。在给药后第10天处死空白对照组6Y和测试组6A的1只小鼠，取肿瘤组织进行荧光成像，结果如图5H所示。图5A-5H中，Blank表示空白对照组6Y。
图5A-5E分别是示出给予不同序列的本公开提供的缀合物以及对比缀合物后，不同时间点的小鼠体内荧光成像图；图5F-5H分别是不同时间点的肿瘤组织成像图。图5A-5H的结果表明：在给药后30min，各测试组和 对照组皮下瘤均可见荧光信号；给药后4h-24h，给予本公开提供的AP2、AP3和AP12的测试组6A、6B和6C小鼠中仍显示出较高的荧光强度；给药后48h-第10天时，给予AP2的测试组6A小鼠仍显示出强的荧光信号。上述结果表明，与对照缀合物相比，本公开提供的缀合物能够在长时间内稳定地靶向肿瘤组织，并且说明本公开提供的适配体能够在长时间内稳定地将诊断剂基团靶向递送至肿瘤。
实验例7缀合物在A549皮下瘤模型小鼠中的体内分布
按照实验例4描述的方法制备获得A549人非小细胞肺癌细胞皮下瘤模型小鼠，区别仅在于，使用A549人非小细胞肺癌细胞代替U118MG胶质瘤细胞制备细胞培养液。
选取11只6-8周龄的NOD-SCID雄性小鼠(购自斯贝福(北京)生物技术公司)，随机分组，其中，空白对照组2只小鼠，其余每组3只小鼠。取上述含有A549肿瘤细胞的培养基分别接种于小鼠右前肢皮下位置，每只小鼠接种A549肿瘤细胞的体积为100μL，从而计算可知，每只小鼠接种细胞数为1×107个。
用1×DMEM(购自中科迈晨(北京)科技有限公司，批号为K1902200)培养基将AP2、AP3和AP12分别溶解成0.3mg/mL浓度(以适配体计)的缀合物溶液。在上述A549肿瘤细胞接种21天时，分别向每只小鼠尾静脉注射1×DMEM以及上述配制的AP2、AP3和AP12缀合物溶液，且分别将注射1×DMEM、AP2、AP3和AP12缀合物溶液的小鼠各自命名为空白对照组、AP2组、AP3组和AP12组。所有动物根据体重计算给药剂量，每只小鼠的给药体积为10μL/g。即，除空白对照组外，其余组中每只小鼠的给药剂量均为3mg/kg(以适配体的量计)；空白对照组仅给予10μL/g DMEM培养基。
在给药后1h和24h时，分别将每组中的1只小鼠处死，并分别取出各只被处死的小鼠的肿瘤组织，在IVIS Lumina Series III中进行荧光成像。将各只小鼠的2个脏器各自依次横向排布，并各自放在同一视野下拍照，结果见图6所示。
图6示出了在给药后1h时，各组中1只小鼠的脏器荧光影像。从图7 可以看出，除空白对照组外，在AP2组、AP3组和AP12组的A549肿瘤中均可检测到较强的荧光信号强度，且AP3组的荧光信号强度较其他组的荧光信号强度更强。
由上述结果可知，本公开具有不同修饰的缀合物均能够特异性地靶向至A549肿瘤。
实验例8本公开提供的缀合物进入PAN02肿瘤细胞的能力
本实验例中采用PAN02肿瘤细胞模型，用来验证本公开提供的缀合物进入PAN02肿瘤细胞的能力。
取对数期生长的PAN02肿瘤细胞(购自中科院上海细胞库)，消化重悬于添加有10％胎牛血清的DMEM培养基中，直至PAN02肿瘤细胞的细胞密度达到1×108cells/mL。
选取1只6周龄的C57/BL小鼠(雄性)，取上述含有PAN02肿瘤细胞的培养基接种于小鼠右前肢皮下位置，小鼠接种PAN02肿瘤细胞的体积为100μL，计算可知，小鼠接种细胞数为1×107个。
用1×DMEM(购自中科迈晨(北京)科技有限公司，批号为K1902200)培养基将AP2溶解成0.3mg/mL浓度(以适配体计)的缀合物溶液。在上述PAN02肿瘤细胞接种21天时，向小鼠尾静脉注射AP2缀合物溶液。根据小鼠体重计算给药剂量，以适配体的量计，给药体积为10μL/g，给药剂量为3mg/kg。
在给药后135min时，将小鼠处死，并取出被处死小鼠的心、肝、脾、肺和肿瘤组织，在IVIS Lumina Series III中进行荧光成像，并放在同一视野下拍照，结果见图7所示。
图7是示出了给予本公开提供的缀合物后，建立PAN02皮下瘤模型的小鼠中各器官组织的荧光成像的图。从图7可以看出，PAN02肿瘤细胞中的荧光信号明显强于其他脏器中的荧光信号强度，表明本公开提供的缀合物能够特异性地靶向PAN02胰腺癌肿瘤细胞。
实验例9缀合不同荧光基团的缀合物进入U118MG胶质瘤细胞的能力
本实验例中采用U118MG胶质瘤模型，验证了本公开提供的分别包含 CY5-荧光基团和CY3-荧光基团的缀合物对U118MG胶质瘤的进入能力。
取对数期生长的U118MG胶质瘤细胞(购自广州吉妮欧生物科技有限公司)，消化重悬于添加有10％胎牛血清的DMEM培养基中，直至U118MG胶质瘤细胞的细胞密度达到1×108cells/mL。
选取11只6-8周龄的NOD-SCID雄性小鼠(购自斯贝福(北京)生物技术公司)，随机分组，其中，第一组3只小鼠，第二组2只小鼠，第三组3只小鼠，第四组3只小鼠。取上述含有U118MG胶质瘤细胞的培养基分别接种于小鼠右前肢皮下位置，每只小鼠接种细胞的体积为100μL，从而计算可知，每只小鼠接种细胞数为1×107个。
用1×DMEM(购自中科迈晨(北京)科技有限公司，批号为K1902200)培养基将AP2、AP9和AP12分别溶解成0.3mg/mL浓度(以适配体计)的缀合物溶液。在上述U118MG胶质瘤接种21天时，分别向每只小鼠尾静脉注射1×DMEM以及上述配制的AP2、AP9和AP12缀合物溶液。其中，第一组的3只小鼠注射AP2缀合物溶液，第二组的2只小鼠注射AP9缀合物溶液，第三组的3只小鼠注射AP12缀合物溶液，第四组的3只小鼠注射1×DMEM作为空白对照组。所有动物根据体重计算给药剂量，每只小鼠的给药体积为10μL/g。即，第一组-第三组中每只小鼠的给药剂量均为3mg/kg(以适配体的量计)。
将给药当天记为D1，在给药后48h、96h和11天(D12)时，分别将每组中的1只小鼠处死，并分别取出各只被处死的小鼠的肿瘤、肺、肝和肾组织，在IVIS Lumina Series III中进行荧光成像。将各只小鼠的4个脏器依次纵向排布，并放在同一视野下拍照，结果分别见图8A-8E所示。其中，组一表示给予AP2的测试组，组二表示给予AP9的测试组，组三表示给予AP12的测试组，组四表示空白对照组。
图8A示出了给药后48h时，第一组中的1只小鼠、第三组中的1只小鼠和第四组中的1只小鼠的脏器荧光影像。从图8A可以看出，第三组和第四组中均未检测到荧光信号。同时，在注射AP2的小鼠肿瘤组织中能够检测出显著的荧光信号，且除代谢器官肾脏以外未在其他组织中检测到荧光信号，表明与对比缀合物相比，AP2可有效地靶向递送至肿瘤组织，并且 在其他组织中无显著的靶向递送。
图8B示出了给药后96h时，第一组中的1只小鼠、第三组中的1只小鼠和第四组中的1只小鼠的脏器荧光影像。从图8B可以看出，在96h后，AP2在肿瘤组织中仍显示出较显著的荧光信号。
图8C示出了给药后48h时，第二组中的1只小鼠和第四组中的1只小鼠的脏器荧光影像。从图8C可以看出，与对照组相比，在注射Cy3标记的AP9的小鼠肿瘤组织中能够检测到显著的荧光信号，表明本公开提供的适配体能高效地将不同的诊断剂基团(如荧光基团)靶向递送至肿瘤组织，本公开提供的包含诊断剂基团的缀合物能够稳定地靶向至肿瘤组织。
图8D示出了给药后96h时，第二组中的1只小鼠和第四组中的1只小鼠的脏器荧光影像。从图8D可以看出，在注射Cy3标记的AP9的小鼠肿瘤组织中能够检测到显著的荧光信号，且除代谢器官肾脏以外未在其他组织中检测到荧光信号。可见，本公开提供的适配体能够将不同的诊断剂基团(例如不同荧光基团)高效递送至U118MG胶质瘤，并且能在较长时间内保持稳定。
图8E示出了给药后11天时，第一组中的1只小鼠、第三组中的1只小鼠和第四组中的1只小鼠的脏器荧光影像。从图8E可以看出，在给药后第11天时，AP2在肿瘤组织中仍能检测到显著的荧光信号，且且除代谢器官肾脏以外未在其他组织中检测到荧光信号。可见，本公开提供的缀合物在长时间内仍然能够稳定地、高特异性地靶向U118MG胶质瘤组织。
从上述结果可知，本公开提供的适配体不仅能够特异性靶向胶质瘤组织，还能够将不同的诊断剂基团递送至胶质瘤。从而，本公开提供的适配体显示出优异的递送能力；同时，本公开提供的包含诊断剂基团的缀合物能够高效、稳定地靶向胶质瘤，从而能够迅速、有效地对胶质瘤的存在进行诊断，且诊断效果可在较长时间内保持稳定。
实验例10不同序列和长度的缀合物对U118MG胶质瘤的靶向效果
本实验例中采用U118MG胶质瘤模型，考察了不同序列和不同长度的缀合物对U118MG胶质瘤的靶向效果。
取对数期生长的U118MG胶质瘤细胞(购自广州吉妮欧生物科技有限 公司)，消化重悬于添加有10％胎牛血清的DMEM培养基中，直至U118MG胶质瘤细胞的细胞密度达到1×108cells/mL。
选取14只6-8周龄的NOD-SCID雄性小鼠(购自斯贝福(北京)生物技术公司)，随机分组，每组2只小鼠。取上述含有U118MG胶质瘤细胞的培养基分别接种于小鼠右前肢皮下位置，每只小鼠接种U118MG胶质瘤的体积为100μL，从而计算可知，每只小鼠接种细胞数为1×107个。
用1×DMEM(购自中科迈晨(北京)科技有限公司，批号为K1902200)培养基将AP2、AP4、AP5、AP6、AP7、AP8和AP12分别溶解成0.3mg/mL浓度(以适配体计)的缀合物溶液。在上述U118MG胶质瘤接种21天时，分别向每只小鼠尾静脉注射上述配制的AP2、AP4、AP5、AP6、AP7、AP8和AP12缀合物溶液。其中，分别将注射AP2、AP4、AP5、AP6、AP7、AP8和AP12的各组小鼠各自命名为AP2组、AP4组、AP5组、AP6组、AP7组、AP8组和AP12组。所有动物根据体重计算给药剂量，以适配体的量计，每只小鼠的给药体积为10μL/g。即，每只小鼠的给药剂量均为3mg/kg。
将给药当天记为D1，给药1h、24h(D2)、48h(D3)和72h(D4)后，依次将各组小鼠放置于小动物活体光学成像系统IVIS Lumina Series III中，采用异氟烷气体麻醉小鼠，将麻醉后的小鼠腹部朝上放置于小动物活体光学成像系统中进行活体成像，动态检测Cy5的荧光信号，追踪各Cy5标记的不同缀合物在动物活体的分布情况。分析观察结果可知，在给药1h后，各个小鼠体内的皮下瘤中均可见荧光信号，且各组小鼠之间的荧光信号强度无显著性差异。在给药24h和48h后，各组小鼠体内仍可检测到荧光信号，但是注射AP6和AP8的小鼠体内的皮下瘤中的荧光信号均要强于注射AP2、AP4、AP5、AP7和AP12的小鼠体内的皮下瘤中的荧光信号。
在D9时，分别将每组中的1只小鼠处死，并分别取出各只被处死的小鼠的肿瘤、肺、肝和肾组织，在IVIS Lumina Series III中进行荧光成像。将各只小鼠的肿瘤组织纵向排布，并放在同一视野下拍照，结果见图9所示。
图9是示出了给药后D9时，各组小鼠的肿瘤荧光影像的图。从图9可以看出，各组小鼠的肿瘤组织中均可检测到Cy5荧光信号，表明本公开不 同长度和序列的缀合物均能够在长时间内稳定有效地靶向至肿瘤组织。
实验例11本公开提供的缀合物在小鼠体内的活性
本实验例考察了制备得到的缀合物20在小鼠体内的抗肿瘤活性。
本实验的小鼠购自斯贝福公司，种系为NOD-SCID，等级为SPF，性别均为雌性，周龄为6-8周龄；U118MG神经胶质瘤细胞购自吉尼欧。
取对数期生长的U118MG细胞，消化重悬于添加有10％的胎牛血清(FBS，GIBCO公司)的DMEM完全培养基(MACGENE公司，货号CM15019)中进行培养，直至细胞密度为1×108细胞/mL，获得含有U118MG细胞的培养液。将上述含有U118MG细胞的培养液接种于各小鼠右前肢皮下位置，注射体积100μL。从而，每只小鼠接种1×107个U118MG胶质瘤细胞。
用PBS将上述制备的缀合物AP2配制为1.94mg/mL的溶液。用PBS将缀合物20分别配制为0.625mg/mL、1.25mg/mL以及2.06mg/mL的溶液(均以适配体的量计算)；用10％DMSO+90％PBS(体积比)的混合溶液将MMAE(购自上海麦克林公司，批号C12886583)溶解为0.038mg/mL的溶液。
将细胞接种当天记为D1，分别于D8、D12、D16和D20各给药一次。
将36只小鼠随机分为以下6组，每组6只小鼠：
对于空白对照组1，采用尾静脉注射方式给予PBS，单次给药容积为10μL/g；
对于对照组2，采用尾静脉注射方式给予上述缀合物AP2溶液，单次给药容积为10μL/g，单次给药剂量为15.5mg/kg；
对于对照组3，采用尾静脉注射方式给予上述MMAE溶液，单次给药容积为10μL/g，单次给药剂量为0.3mg/kg；
对于测试组4，采用尾静脉注射方式给予上述浓度为0.625mg/mL的缀合物20溶液，单次给药容积为10μL/g，单次给药剂量为5mg/kg(以适配体质量计)，其中包含MMAE的剂量相当于0.3mg/kg；
对于测试组5，采用尾静脉注射方式给予上述浓度为2.06mg/mL的 缀合物20溶液，单次给药容积为10μL/g，单次给药剂量为16.5mg/kg(以适配体质量计)，其中包含MMAE的剂量相当于1mg/kg；
对于测试组6，采用皮下注射方式给予上述浓度为1.25mg/mL的缀合物20溶液，单次给药容积为5μL/g，单次给药剂量为5mg/kg(以适配体质量计)，其中包含MMAE的剂量相当于0.3mg/kg。
[3]检测
通过体外测量的方式测定测量肿瘤的长径和短径。肿瘤体积根据公式1/2(长径×短径2)进行计算。在D8首次给药前，测定各组肿瘤体积并记录平均肿瘤体积，D16开始分别测量并记录各组肿瘤体积，每周测量两次。
图10是示出了各组小鼠中肿瘤体积随时间变化的折线图。由图10结果可知，在仅给予PBS与AP2的空白对照组1和对照组2中，肿瘤体积迅速增加；仅给予MMAE的对照组3中肿瘤体积增加速度有所降低，表明MMAE本身对肿瘤增殖显示出抑制效果。进一步地，MMAE含量与对照组3相当的测试组4和6在测试期间肿瘤体积均显著小于对照组3，显示出比单独给予MMAE的对照组3更加优异的抗肿瘤活性。这表明，本公开提供的缀合物能够有效地将MMAE递送至肿瘤组织，在显示出肿瘤靶向能力的同时，还降低了MMAE分子在其它组织分布带来的毒性风险，且各种给药方式均能够有效抑制肿瘤增殖。此外，进一步提高给药剂量的测试组5的肿瘤体积在测试期间几乎不增加，显示出更加优异的抗肿瘤效果，表明本公开提供的包含小分子药物基团的缀合物能够迅速、稳定地对肿瘤产生抑制作用。
上述结果表明，本公开提供的缀合物能够有效地将对肿瘤具有抑制作用的小分子药物基团靶向递送至肿瘤组织，本公开提供的包含小分子药物基团的缀合物显示出良好的抗肿瘤活性和剂量依赖效应。
实验例12本公开提供的缀合物对正常细胞及肿瘤细胞活力的影响
本实验例考察了制备得到的缀合物19对正常细胞及肿瘤细胞活力的影响。
本实验的U118-MG神经胶质瘤细胞购自吉尼欧；SVGp12人星形胶质细胞购自中科院上海细胞库；CCK8试剂盒购自同仁化学研究所。
分别选取对数生长的U118-MG细胞和SVGp12细胞，每孔5000个种于CCK8试剂盒的96孔板中，37下5％CO2培养24h，镜下观察细胞融合度80％时，得到待检测U118-MG细胞和待检测SVGp12细胞。
用添加有10％的胎牛血清(FBS，GIBCO公司)的DMEM完全培养基(MACGENE公司，货号CM15019)梯度分别稀释AP1以及缀合物19和酵母tRNA，分别获得以下缀合物溶液：AP1溶液1(浓度为0nM)；AP1溶液2(浓度为50nM)；AP1溶液3(浓度为200nM)；AP1溶液4(浓度为800nM)；以及以下缀合物溶液：缀合物19溶液1(浓度为0nM)；缀合物19溶液2(浓度为25nM)；缀合物19溶液2(浓度为25nM)；缀合物19溶液3(浓度为50nM)；缀合物19溶液4(浓度为100nM)；缀合物19溶液5(浓度为200nM)；缀合物19溶液6(浓度为400nM)；缀合物19溶液7(浓度为800nM)，并且，以上每一缀合物溶液中分别还包含终浓度为100μg/ml的酵母tRNA。
将AP1溶液1、AP1溶液2、AP1溶液3和AP1溶液4分别加入待检测U118-MG细胞中，37避光孵育72h。每组实验重复5次，依次分别记为空白对照组12A1、测试组12A2、12A3和12A4。
将AP1溶液1、AP1溶液2、AP1溶液3和AP1溶液4分别加入待检测SVGp12细胞中，37避光孵育72h。每组实验重复5次，依次分别记为空白对照组12B1、测试组12B2、12B3和12B4。
将缀合物19溶液1、缀合物19溶液2、缀合物19溶液3、缀合物19溶液4、缀合物19溶液5、缀合物19溶液6以及缀合物19溶液7分别加入待检测U118-MG细胞中，37避光孵育72h。每组实验重复5次，依次分别记为空白对照组12C1和测试组12C2-12C7。
将缀合物19溶液1、缀合物19溶液2、缀合物19溶液3、缀合物19溶液4、缀合物19溶液5、缀合物19溶液6以及缀合物19溶液7分别加入待检测SVGp12细胞中，37避光孵育72h。每组实验重复5次，依次分别记为空白对照组12D1和测试组12D2-12D7。
使用CC8试剂盒，按照说明书的方法在紫外吸收光谱中，检测波长450nm处检测上述各测试组和对照组的紫外吸光度，对各组重复实验取平 均值。细胞活力按照以下公式计算：
细胞活力％＝(测试组OD450-空白组OD450)/(对照组OD450-空白
组OD450)×100％
其中，测试组OD450是指450nm处各测试组的吸光度值，对照组OD450是各测试组所对应的空白对照组在450nm处的吸光度值，空白组OA450是紫外检测时所使用的空白比色池在450nm处的吸光度值。结果示于图11A和11B中。
图11A示出了不同浓度的缀合物AP1对U118-MG神经胶质瘤细胞和SVGp12人星形胶质细胞的细胞活力的影响；图11B示出了不同浓度的缀合物19对U118-MG神经胶质瘤细胞和SVGp12人星形胶质细胞的细胞活力的影响。
由图11A结果可知，仅包含诊断剂基团CY5的缀合物AP1在不同浓度下对正常细胞以及肿瘤细胞的细胞活力均无影响。由图11B结果可知，包含siRNA的缀合物19能够显著降低肿瘤细胞的细胞活力，且抑制效力随着使用剂量的增加而提高。同时，对于正常细胞而言，细胞活力仅略有减小，仍基本维持稳定，表明本公开提供的适配体在缀合siRNA后，不仅能够高效靶向肿瘤细胞并抑制目标mRNA含量，还能不显著影响正常细胞中的相应mRNA水平，具有优异的靶向递送效率和高的安全性。总而言之，上述结果表明，本公开提供的包含功能性寡核苷酸的缀合物能够有效地将对肿瘤具有抑制作用的功能性寡核苷酸如siRNA靶向递送至肿瘤组织，显示出良好的抗肿瘤活性和剂量依赖效应。
实验例13本公开提供的缀合物在小鼠体内的靶向性
本实验例考察了制备得到的缀合物15、缀合物16、缀合物17以及缀合物18在小鼠体内的靶向性。
缀合物15-18包含相同的适配体序列。对于缀合物15，该正义链序列中包含siRNA的正义链，该siRNA正义链5′末端连接有荧光基团Cy5，且3′末端经作为连接基团的GAU三核苷酸亚基连接至本公开提供的适配体。
对于缀合物16，该正义链序列中包含siRNA的正义链，该siRNA正 义链5′末端经作为连接基团的GAU三核苷酸亚基连接至本公开提供的适配体，该适配体的5′末端连接有荧光基团Cy5。
缀合物17是包含双链siRNA和本公开提供的适配体的缀合物，其中适配体经四个依次连接的亚丙基磷酸酯基作为连接基团连接至siRNA正义链的5′末端，并且在适配体的5′末端连接有Cy5荧光基团。
缀合物18与缀合物17结构相似，其中适配体结构与缀合物AP2中的适配体略有不同，参见表1B以及制备例18。
按照实验例4的方法培养U118MG细胞并皮下接种至小鼠。
用PBS将上述制备的缀合物15、缀合物16、缀合物17以及缀合物18分别配制为0.3mg/mL的溶液。
U118MG细胞接种后30天开始给药，给药当天记为D1。实验采用尾静脉注射给药方式，每天给药一次，共三次给药。
将12只小鼠(体重均约为25g)随机分为4组，每组3只小鼠：
对于测试组1，采用缀合物15，单次给药容积为250μL，单次给药剂量为3mg/kg；
对于测试组2，采用缀合物16，单次给药容积为250μL，单次给药剂量为3mg/kg；
对于测试组3，采用缀合物17，单次给药容积为250μL，单次给药剂量为3mg/kg；
对于测试组4，采用缀合物18，单次给药容积为250μL，单次给药剂量为3mg/kg。
[3]检测
于首次给药后24天，使用小动物活体光学成像系统IVIS Lumina Series III对各小鼠进行活体成像。
图12是示出了给予不同缀合物后，小鼠体内的荧光成像结果的图。由图12可知，采用不同连接基团、不同连接方式，以及不同适配体的各个缀合物都富集于肿瘤细胞处。上述结果表明，尽管所包含的适配体通 过不同方式连接至siRNA或适配体的序列发生改变，然而本公开提供的各种缀合物均能够有效地靶向至肿瘤组织。进一步地，与其它缀合物相比，缀合物18的荧光信号更为强烈并且在体内呈现弥散性分布，表明缀合物18在循环系统中持续时间更长而不被快速代谢排出体外，因此可能具有更为持久的靶向作用。
实验例14缀合物在小鼠体内的靶向性
本实验例考察了制备得到的缀合物AP2、AP21-26以及对比AP27-对比AP30在小鼠体内的靶向性。
按照实验例4的方法培养U118MG细胞并皮下接种至24只小鼠(均为雌性)，获得接种U118MG皮下瘤的小鼠。
用DMEM培养基将上述制备的缀合物AP2、AP21-26以及对比AP27-对比AP30分别配制为0.3mg/mL的溶液。
U118MG细胞接种后14天开始给药，给药当天记为D1。实验采用尾静脉注射给药方式，每天给药一次，共三次给药。
将接种U118MG皮下瘤的24只小鼠随机分为12组，每组2只小鼠：
对于7组小鼠，分别向每组中的每只小鼠给予AP2、AP21、AP22、AP23、AP24、AP25或AP26，单次给药容积为10μL/g小鼠体重，计算可知单次给药剂量为3mg/kg，依次分别记为测试组14A-14G；
对于另外4组小鼠，分别向每组中的每只小鼠给予对比AP27、对比AP28、对比AP29或对比AP30，单次给药容积为10μL/g小鼠体重，计算可知单次给药剂量为3mg/kg，依次分别记为对照组14H-14K；
对于另外1组中的2只小鼠，向每只小鼠给予DMEM培养基，给药容积为10μL/g小鼠体重，记为空白对照组15Y。
于首次给药后1h、24h和48h时，使用小动物活体光学成像系统IVIS Lumina Series III对各小鼠进行活体成像。在D5时，处死各组小鼠并取肿瘤组织和肾脏进行荧光成像。
图13A-13C依次分别是示出了给予不同缀合物后1h、24h和48h时，小鼠体内的荧光成像结果的图，其中每一小图内3只小鼠中最左侧的1只 为空白对照组15Y的小鼠。由图13A可知，空白对照组不显示任何荧光信号；与其不同，在给药后1h时，各测试组和对照组的小鼠在皮下肿瘤处均显示出荧光信号；由图13B和13C可知，在给药后24h和48h时，仅测试组14A-14G的小鼠在皮下肿瘤处显示出较强的荧光信号，而对照组14H-14K的小鼠几乎不显示荧光信号或仅显示很弱的荧光信号。进一步地，图13D是示出了D5时处死小鼠后，各组小鼠肿瘤组织和肾脏的荧光信号成像的图，其中，Blank表示空白对照组14Y。由图13D可知，空白对照组14Y和对照组14H-14K的小鼠的肿瘤组织几乎不显示荧光信号或仅显示很弱的荧光信号；与此相对，给予本公开提供的缀合物的测试组14A-14G的小鼠的肿瘤组织均显示出强烈的荧光信号，同时在代谢器官肾脏处仅显示微弱的荧光信号，表明与对照缀合物相比，本公开提供的各种适配体均能够稳定、高效地将荧光基团靶向递送至肿瘤组织；本公开提供的包含诊断剂基团的各种缀合物均能够稳定高效地靶向肿瘤组织，从而有助于对肿瘤的存在进行成功诊断和监控。
实验例15缀合物在小鼠体内的靶向性
本实验例考察了制备得到的缀合物AP2、AP12以及对比AP31-对比AP35在小鼠体内的靶向性。
按照实验例4的方法培养U118MG细胞并皮下接种至16只小鼠(均为雄性)，获得接种U118MG皮下瘤的小鼠。
用DMEM培养基将上述制备的缀合物AP2、AP12以及对比AP31-对比AP35分别配制为0.3mg/mL的溶液。
U118MG细胞接种后21天开始给药，给药当天记为D1。实验采用尾静脉注射给药方式，每天给药一次，共三次给药。
将接种U118MG皮下瘤的16只小鼠随机分为8组，每组2只小鼠：
对于2组小鼠，分别向每组中的每只小鼠给予AP2或AP12，单次给药容积为10μL/g小鼠体重，计算可知单次给药剂量为3mg/kg，依次分别记为测试组15A-15B；
对于另外5组小鼠，分别向每组中的每只小鼠给予对比AP31、对比AP32、对比AP33、对比AP34或对比AP35，单次给药容积为10μL/g小 鼠体重，计算可知单次给药剂量为3mg/kg，依次分别记为对照组15C-15G；
对于另外1组中的2只小鼠，向每只小鼠给予DMEM培养基，给药容积为10μL/g小鼠体重，记为空白对照组15Y。
于首次给药后1h、24h和48h时，使用小动物活体光学成像系统IVIS Lumina Series III对各小鼠进行活体成像。在D6时，处死各组小鼠并取肿瘤组织和肾脏进行荧光成像。
图14A-14C依次分别是示出了给予不同缀合物后1h、24h和48h时，小鼠体内的荧光成像结果的图，其中每一小图内3只小鼠中最左侧的1只为空白对照组15Y的小鼠。由图14A可知，空白对照组不显示任何荧光信号；与其不同，在给药后1h时，各测试组和对照组的小鼠在皮下肿瘤处均显示出荧光信号；由图14B和14C可知，在给药后24h和48h时，仅测试组15A和15B的小鼠在皮下肿瘤处显示出较强的荧光信号，而空白对照组15Y及对照组15C-15G的小鼠完全不显示荧光信号。进一步地，图14D是示出了D6时处死小鼠后，各组小鼠肿瘤组织和肾脏的荧光信号成像的图，其中，Blank表示空白对照组15Y。由图14D可知，空白对照组15Y和对照组15C-15G的小鼠的肿瘤组织完全不显示荧光信号；与此相对，给予本公开提供的缀合物的测试组15A或15B的小鼠的肿瘤组织均显示出强烈的荧光信号，同时在代谢器官肾脏处仅显示微弱的荧光信号，表明与对照缀合物相比，本公开提供的缀合物能够稳定、高效地靶向肿瘤组织，并且不具有式(1)所示序列的各种缀合物均未显示出对肿瘤组织的靶向效果。实验例16缀合物在U118MG人胶质瘤细胞内的分布
按照实验例4的方法培养U118MG人胶质瘤细胞(购自广州吉妮欧生物科技有限公司)。取对数期生长的U118MG人胶质瘤细胞，以(0.25％的胰酶)消化，收集细胞，离心去上清，将细胞重悬于无血清DMEM培养基中制成浓度为1×108cells/mL的细胞培养液。
实验动物NOD-SCID小鼠8只(购自斯贝福(北京)生物技术有限公司)，雄性，12周龄。将上述细胞培养液接种于NOD-SCID小鼠右侧背部皮下位置，接种体积为每只100μL，即每只小鼠接种1×107个细胞。注射 后继续饲养小鼠21天。
用无血清DMEM培养基将缀合物AP2、AP4和对比缀合物AP13溶解成0.3mg/mL浓度(以适配体计)的缀合物溶液。
U118MG细胞接种后21天时，将8只小鼠随机分为4组，每组2只。向各组小鼠给药，给药当天记为D1。实验采用尾静脉注射给药方式，单次给药。
对于3组小鼠，分别向不同组中的每只小鼠给予缀合物AP2、AP4或对比缀合物AP13，单次给药容积为10μL/g小鼠体重，计算可知单次给药剂量为3mg/kg，依次分别记为测试组16a、16b和16c。
对于另外的1组小鼠，分别向每组小鼠给予DMEM，单次给药容积为10μL/g小鼠体重，记为空白对照组16Y。
于给药后24h，安乐死小鼠，收集肿瘤组织固定于4％多聚甲醛溶液中，将肿瘤组织置于15％蔗糖脱水24h，再置于30％蔗糖脱水24h，OCT包埋剂浸润组织后，液氮速冻，使用冷冻切片机(型号POLAR-D-JC，购自樱花精机株式会社)将速冻的组织切片，厚度为10μm，获得肿瘤组织切片。
将肿瘤组织切片在室温下静置5min，使用4％多聚甲醛固定组织15min，以PBS对肿瘤组织切片洗涤3次，每次5mL。使用DAPI避光染色3min，再重复上述洗涤3次的步骤，封片，得到DAPI染色切片。
使用激光共聚焦成像仪(型号LSM 900 Basic Operation，购自卡尔蔡司(上海)管理有限公司)对上述得到的DAPI染色切片进行成像分析，参数选择为Cy5激光强度20％(em 650nm，ex 670nm)，DAPI激光强度1.5％(em 360nm，ex 460nm)，在Best signal模式下拍摄。结果参见图15。
图15是示出了向皮下接种U118MG胶质瘤的小鼠给予不同缀合物后24h时，U118MG胶质瘤细胞的激光共聚焦成像结果的图。实验结果表明与空白对照组DMEM和对比缀合物AP13相比，包含本公开的递送基团的缀合物均能够将诊断剂基团高效、特异性地递送进入U118MG胶质瘤内部，显示出优异的靶向效果和潜在的诊断能力。
实验例17缀合物15和缀合物36对U118MG皮下瘤递送效果
按照实验例4的方法培养U118MG人胶质瘤细胞(购自广州吉妮欧生物科技有限公司)。取对数期生长的U118MG人胶质瘤细胞，以(0.25％的胰酶)消化，收集细胞，离心去上清，将细胞重悬于无血清DMEM培养基中制成浓度为1×108cells/mL的细胞培养液。
实验动物NOD-SCID小鼠9只(购自斯贝福(北京)生物技术有限公司)，雄性，12周龄。将上述细胞培养液接种于NOD-SCID小鼠右侧背部皮下位置，接种体积为每只100μL，即每只小鼠接种1×107个细胞。注射后继续饲养小鼠21天。
用无血清DMEM培养基将缀合物AP2、缀合物15和缀合物36溶解成0.5mg/mL浓度(以适配体计)的缀合物溶液。U118MG细胞接种后21天开始给药，给药当天记为D1。实验采用尾静脉注射给药方式，单次给药。
对于对照组17a，分别向每只小鼠给予缀合物AP2，单次给药容积为10μL/g小鼠体重，计算可知单次给药剂量为5mg/kg。
对于对照组17b，分别向每只小鼠给予缀合物15，单次给药容积为10μL/g小鼠体重，计算可知单次给药剂量为5mg/kg。
对于对照组17c，分别向每只小鼠给予缀合物36，单次给药容积为10μL/g小鼠体重，计算可知单次给药剂量为5mg/kg。
分别于给药后24h(D2)和48h(D3)，使用异氟烷(4L/min新鲜空气流中含有0.41ml/min异氟烷)麻醉小鼠，使用小动物活体光学成像系统IVIS Lumina Series III对各小鼠进行活体成像。
图16A和图16B是示出了给予不同缀合物后，小鼠体内的荧光成像结果。结果表明，在给药后的不同时间时，本公开的各种包含siRNA基团和由本公开的适配体形成的递送基团的缀合物均能够有效地靶向并富集至肿瘤组织，具有优异的成药能力。
实验例18缀合物37和缀合物38进入PANC-1(人胰腺癌细胞)的活性评估
在含10％FBS(Gibco，货号10099-141)的DMEM完全培养基(MACGENE公司，货号CM15019)中，于37在含5％CO2/95％空气的培养箱中培养PANC-1人胰腺癌细胞(购自国家实验细胞资源共享平台)。
取对数期生长的PANC-1人胰腺癌细胞，以(0.25％的胰酶)消化，收集细胞，离心去上清，将细胞重悬于含10％FBS的DMEM培养基中制成浓度为1×105cells/mL的细胞培养液，充分混匀后，24孔板铺板，1mL/well，即细胞1×105cells/well。
用DMEM将缀合物37、缀合物38和缀合物AP2分别溶解成浓度20μM的缀合物溶液。
分别在24孔板的不同培养孔中培养24h后，吸尽上清液，每孔加入900μL含10％FBS的DMEM培养基。分别添加100μL空白对照组DMEM(以下简称为对照组)、缀合物37、缀合物38或缀合物AP2到相应的培养基中，以2μM浓度自由摄取，7天后提取RNA，分别记为对照组以及缀合物9组、缀合物10组和缀合物AP2组(后3组统称为测试组)。
随后，使用用磁珠法组织RNA提取试剂盒(购自武汉纳磁生物科技有限公司，货号NMR0211-20)，按照说明书操作方法提取各孔细胞总RNA，分别得到含总RNA的溶液。
对于每孔细胞，分别取含1μg总RNA的溶液，使用反转录试剂盒(购自Promega Corporation，货号A3500)，以Oligo(dT)18作为引物作为引物，按试剂盒说明书中反转录操作步骤对各孔细胞的总RNA进行反转录。反应结束后，向反转录反应体系中加入DEPC水80μl，得到含cDNA的溶液100μL。
对于每一反转录反应体系，分别取上述含cDNA的溶液5μl做模板，使用：使用SYBR Select Master Mix试剂盒(购自Thermo Fisher Scientific，货号4472908)，按照说明书操作方法在SteponePlusTM实时荧光定量PCR仪(购自Thermo Fisher Scientific)扩增目标基因hSTAT3和内参基因hGAPDH。扩增程序为预变性95-10min，变性95-30s，退火60-30s，延伸72-30s，变性、退火、延伸重复40个循环。熔解程序为仪器默认程序。。
表5：引物信息
对于上述各测试组和对照组，分别进行2次上述定量PCR检测。
对于上述各测试组和对照组，分别进行2次上述定量PCR检测。
采用相对定量2-ΔΔCt法对荧光定量数据进行分析。计算方法如下：
ΔCt(测试组)＝Ct(测试组目标基因)-Ct(测试组内参基因)
ΔCt(对照组)＝Ct(对照组目标基因)-Ct(对照组内参基因)
ΔΔCt(测试组)＝ΔCt(测试组)-ΔCt(对照组平均)
ΔΔCt(对照组)＝ΔCt(对照组)-ΔCt(对照组平均)
抑制率(％)＝(对照组2-ΔΔCt均值-测试组2-ΔΔCt)/对照组2-ΔΔCt均
值×100
实验结果使用Graph Prism软件以Mean±SD值作柱状图直观表示，结果参见图17，其中blank表示对照组。
图17是分别示出了本公开的缀合物37、缀合物38和缀合物AP2在PANC-1人胰腺癌细胞中hSTAT3 mRNA的抑制率的柱状图。根据图17的结果可知，给予缀合物37后，PANC1人胰腺癌细胞中hSTAT3 mRNA的抑制率为33％；给予缀合物38后，PANC1人胰腺癌细胞中hSTAT3 mRNA的抑制率达到44％。上述结果表明，具有不同连接方式，不同连接基团和不同修饰的核苷酸序列的本公开的缀合物均能够经自由摄取进入肿瘤细胞，并且显示出抑制靶mRNA的效果。
实验例19缀合物15-19在U118MG细胞内化评估
按照实验例4的方法培养U118MG人胶质瘤细胞(购自广州吉妮欧 生物科技有限公司)。U118MG细胞以(0.25％的胰酶)消化，以2×105cells/孔培养于12孔板中，5％CO2，37℃细胞培养箱中继续培养24h。
用无血清DMEM培养基将缀合物15、缀合物16、缀合物17、缀合物18和缀合物19溶解成200μM浓度。将10μL化合物加入至990μL细胞培养孔中，即化合物终浓度为2μM。37℃共孵育2h，吸弃上清，PBS洗3次，加入100μL 4％多聚甲醛液固定10min，PBS洗3次，加入100μL DAPI染色液，染色10min后PBS洗3次，激光共聚焦成像。
激光共聚焦成像：参数选择为Cy5激光强度2％(em 650nm，ex 670nm)，DAPI激光强度1.5％(em 360nm，ex 460nm)。Best signal模式拍摄，结果参见图18。
图18是示出了本公开的缀合物在U118MG胶质瘤内的激光共聚焦成像结果的图。
图18的结果显示，采用不同连接方式，不同连接基团和不同修饰的核苷酸序列的本公开的缀合物均能够自由摄取进入肿瘤细胞，并且具有2′-O-十六烷基修饰的缀合物18具有更好的进入肿瘤细胞的能力。
实验例20缀合物15和缀合物18在U118MG细胞内化评估
本实验例对缀合物15、缀合物18和缀合物AP2在体外自由摄取进入U118-MG胶质瘤细胞的能力进行了评价和比较。取对数期生长的如实验例4中培养的U118MG人胶质瘤细胞，以0.25wt％胰酶消化并收集细胞，离心去上清，将细胞重悬于添加10％FBS的DMEM培养基中制成浓度为1×105cells/mL的细胞培养液，充分混匀后，24孔板铺板，1000μL/well，即细胞1×105cells/well。
用DMEM将缀合物15、缀合物18和缀合物AP2分别溶解成浓度40μM的缀合物溶液，记为测试组20a、测试组20b和对照组20c。
培养24h后吸尽培养基，每孔加入900μL无血清的DMEM。加100μL空白对照组DMEM，、缀合物15、缀合物18和缀合物AP2到相应的培养基中，以4μM浓度自由摄取，48h后测试细胞荧光值。
对上述测试组20a、测试组20b和对照组20c，分别取100μL DMEM培养基，在高内涵成像系统中成像。经空白对照组DMEM的平均荧光强 度归一化，即测试组20a、测试组20b和对照组20c荧光强度减去空白对照组的平均荧光强度后进行成像。结果示于图19中。
图19是示出了本公开的缀合物在U118MG胶质瘤内荧光强度结果的柱状图，其中DMEM表示空白对照组。图6的结果表明，本公开提供的缀合物能够有效地将siRNA基团递送进入肿瘤细胞内，从而有利于在肿瘤细胞中高效产生RNAi作用。
实验例21缀合物对U118MG原位瘤模型小鼠体内活性
按照实验例4的方法培养表达Luciferase(Photinus pyralis)报告基因的U118MG人胶质瘤细胞，以下简称U118MG-luc人胶质瘤细胞(购自南京科佰生物科技有限公司)。取对数期生长的U118MG-luc人胶质瘤细胞，以0.25wt％胰酶消化并收集细胞，离心后吸去上清液并将细胞重悬于无血清DMEM培养基中制成细胞密度为4×107cells/mL的细胞培养液。
实验动物Balb/C-nude裸鼠24只(购自斯贝福(北京)生物技术有限公司)，雄性，12周龄。将上述细胞培养液接种于Balb/C-nude裸鼠纹状体中，采用小鼠纹状体注射方式，将细胞培养液注射于小鼠右侧纹状体，位置为AP(anteroposterior前后/正位)：1mm，ML(medial lateral内侧)：1.5mm，DV(dorsal ventral背腹侧)：3.5mm，注射体积10μL，即，每只小鼠接种4×105个细胞。原位瘤接种后继续饲养小鼠14天。
用1×DMEM(购自中科迈晨(北京)科技有限公司，批号为K1902200)培养基将缀合物20溶解成1mg/mL浓度(以适配体计)的缀合物溶液。缀合物39和对比缀合物43分别溶解成0.8mg/mL浓度(以适配体计)的缀合物溶液。
接种原位瘤后第15天，使用小动物活体光学成像系统IVIS Lumina Series III对各小鼠进行活体成像，根据脑部荧光强度分组，每组6只小鼠，给药当天记为D1(即，实验第1天，以下D4、D8等相应表示实验第4天、第8天，以此类推)。
活体成像方法：每只小鼠腹腔注射10μL/g体重浓度15mg/mL D-荧光素钾盐工作液(购自翌圣生物科技(上海)股份有限公司)，注射后10min活体成像(Lumina III小动物活体成像系统)。成像后圈选小鼠脑部荧 光区域(ROI)，软件测定荧光强度(Radiance)。在此条件下，U118MG-luc人胶质瘤细胞中表达的Luciferase(Photinus pyralis)报告基因能够产生荧光响应，因此荧光强度能够反映胶质瘤细胞的增殖数量。荧光强度越高，表明胶质瘤细胞的数量越大。
实验通过皮下注射给药方式，每组小鼠分别于D1、D4、D8和D12给药。于给药前称重，按重量给药。
对于测试组21a，分别向每只小鼠给予缀合物20，单次给药容积为5μL/g小鼠体重，计算可知单次给药剂量为5mg/kg，其中包含MMAE的剂量相当于0.3mg/kg。
对于测试组21b，分别向每只小鼠给予缀合物39，单次给药容积为5μL/g小鼠体重，计算可知单次给药剂量为4mg/kg，其中包含MMAE的剂量相当于0.3mg/kg。
对于对照组21c，分别向每只小鼠给予对比缀合物43，单次给药容积为5μL/g小鼠体重，计算可知单次给药剂量为4mg/kg，其中包含MMAE的剂量相当于0.3mg/kg。
对于空白对照组，分别向每只小鼠给予DMEM培养基，单次给药容积为5μL/g小鼠体重。
按照上述方法，分别于D1、D22、D31和D39对每组小鼠进行活体成像分析，测定荧光强度。结果参见图20所示。
图20是示出了给予本公开提供的缀合物或对照化合物后，U118MG原位瘤模型小鼠肿瘤荧光强度随时间变化的折线图。
从图20的结果可以看出，与给药后D1相比较，随着观察时间的延长空白对照组和对照组肿瘤荧光强度(Radiance)明显升高，表明U118MG人胶质瘤细胞数量明显增加；与此不同，给予本公开提供的缀合物的测试组21a和21b肿瘤荧光强度显著降低，降低幅度最高可达1个数量级，与对照组相比可达2个数量级以上，表明U118MG人胶质瘤细胞数量明显减少，与实验开始时相比可能降至开始时的1/10、甚至可能减少至对照组的1％以下。可见，即使是仅通过皮下给药，包含肿瘤治疗剂基团和由本公开提供的靶向适配体形成的递送基团的缀合物也能够有效穿透血脑屏障并高 效靶向进入脑胶质瘤，并具有良好的抑制肿瘤生长效果，显示出良好的治疗依从性和高效抑制肿瘤的高成药能力。
实验例22缀合物在小鼠体内对U118MG皮下瘤的抑制活性
按照实验例4的方法培养U118MG人胶质瘤细胞(购自广州吉妮欧生物科技有限公司)。取对数期生长的U118MG人胶质瘤细胞，以(0.25％的胰酶)消化，收集细胞，离心去上清，将细胞重悬于无血清DMEM培养基中制成浓度为1×108cells/mL的细胞培养液。
实验动物NOD-SCID小鼠36只(购自斯贝福(北京)生物技术有限公司)，雄性，12周龄。将上述细胞培养液接种于NOD-SCID小鼠右侧背部皮下位置，接种体积为每只100μL，即，每只小鼠接种1×107个细胞。注射后继续饲养小鼠7天。
用无血清DMEM培养基将缀合物20溶解成1mg/mL浓度(以适配体计)的缀合物溶液。缀合物39、缀合物40和缀合物41分别溶解成0.8mg/mL浓度(以适配体计)的缀合物溶液。用10％DMSO+90％无血清DMEM培养基(体积比)将MMAE溶解成0.06mg/mL浓度的溶液。
U118MG细胞接种后7天开始给药，给药当天记为D8。实验采用腹部皮下给药方式，分别于D8、D12、D16和D20各给药一次，共4次给药。
对于空白对照组22a，分别向每组小鼠给予DMEM，单次给药容积为5μL/g小鼠体重；
对于对照组22b，分别向每组小鼠给予MMAE，单次给药容积为5μL/g小鼠体重，计算可知单次给药剂量为0.3mg/kg；
对于测试组22c，分别向每只小鼠给予缀合物20，单次给药容积为5μL/g小鼠体重，计算可知单次给药剂量为5mg/kg，其中包含MMAE的剂量相当于0.3mg/kg；
对于3组小鼠，分别向每组小鼠给予缀合物39、缀合物40或者缀合物41，单次给药容积均为5μL/g小鼠体重，计算可知单次给药剂量为 4mg/kg，其中包含MMAE的剂量相当于0.3mg/kg；依次分别记为测试组22d、22e和22f；
于D16、D20、D25、D29、D33、D36、D41、D48、D60测量肿瘤体积。通过体外测量的方式测定测量肿瘤的长径和短径。肿瘤体积根据公式1/2(长径×短径2)进行计算。实验结束D60取肿瘤组织，称重。图21显示给予不同缀合物后，不同天数时小鼠肿瘤体积随时间变化的折线图的折线图。由图21可知，与对照组22b或者空白对照组相比，给予本公开缀合物的测试组22c-22f的小鼠的肿瘤体积和肿瘤重量均明显降低。上述结果表明包含由这些适配体形成的递送基团的缀合物能够有效地到达肿瘤组织，显示出良好的抗肿瘤活性。
实验例23缀合物长间隔给药对U118MG皮下瘤模型小鼠体内活性
按照实验例4的方法获得接种U118MG皮下瘤的小鼠42只，注射后继续饲养小鼠。
用10％DMSO+90％无血清DMEM培养基(体积比)将MMAE溶解成0.03mg/mL和浓度0.01mg/mL的溶液；将缀合物39溶解成0.5mg/mL浓度和0.165mg/mL浓度(以适配体计)的溶液；对比缀合物44溶解成0.5mg/mL浓度和0.165mg/mL浓度(以适配体计)的溶液。
在接种U118MG细胞后7天，将全部小鼠随机分为7组，每组6只，并向各组小鼠给药，给药当天记为D8。实验采用腹部皮下给药方式，分别于D8、D11、D15、D29、D32和D36各给药一次，共6次给药。于给药前称重，按重量计算给药容积。
对于空白对照组，分别向每只小鼠给予DMEM培养基，单次给药容积为10μL/g小鼠体重。
对于测试组23a，分别向每只小鼠给予浓度0.01mg/mL的MMAE，单次给药容积为10μL/g小鼠体重，计算可知单次给药剂量为0.1mg/kg。
对于测试组23b，分别向每只小鼠给予浓度0.165mg/mL的缀合物39，单次给药容积为10μL/g小鼠体重，计算可知单次给药剂量为1.65mg/kg(对应MMAE剂量为0.1mg/kg)。
对于对照组23c，分别向每只小鼠给予浓度0.165mg/mL的对比缀合 物44，单次给药容积为10μL/g小鼠体重，计算可知单次给药剂量为1.65mg/kg(对应MMAE剂量为0.1mg/kg)。
对于测试组23d，分别向每只小鼠给予浓度0.03mg/mL的MMAE，单次给药容积为10μL/g小鼠体重，计算可知单次给药剂量为0.3mg/kg。
对于测试组23e，分别向每只小鼠给予浓度0.5mg/mL的缀合物39，单次给药容积为10μL/g小鼠体重，计算可知单次给药剂量为5mg/kg(对应MMAE剂量为0.3mg/kg)。
对于对照组23f，分别向每只小鼠给予浓度0.5mg/mL的对比缀合物44，单次给药容积为10μL/g小鼠体重，计算可知单次给药剂量为5mg/kg(对应MMAE剂量为0.3mg/kg)。
于D1、D9、D16、D19、D21、D24、D26、D29、D32、D36、D39、D43、D47、D53、D57、D60、D64、D67、D71、D74、D78、D81、D84、D88、D92、D95和D99测量肿瘤体积，其中，空白对照组在D53时、测试组7a、7b和对照组7c在D60时测量后中止实验。
通过体外测量的方式测定测量肿瘤的长径和短径。肿瘤体积根据公式1/2(长径×短径2)进行计算。实验中止后，取肿瘤组织，称重并计算平均值。结果参见图22所示。
图22是示出了给予不同浓度本公开提供的缀合物或对照化合物后，U118MG皮下瘤模型小鼠肿瘤体积随时间变化的折线图。
由图22结果可知，在空白对照组中，肿瘤体积迅速增加；仅给予MMAE的测试组肿瘤体积增加速度有所降低，表明MMAE本身对肿瘤增殖显示出抑制效果。
进一步地，测试组23b在测试期间肿瘤体积均显著小于与MMAE含量相当的测试组23a和对照组23c；测试组23e在测试期间肿瘤体积均显著小于与MMAE含量相当的测试组23d和对照组23f，显示出比单独给予MMAE测试组23a和23d，以及给予对比缀合物44均更为优异的抗肿瘤活性。实验中止后，给予本公开的缀合物的小鼠中的肿瘤重量也显著低于MMAE组和对照组。上述结果表明，本公开提供的缀合物能够有效地将MMAE递送至肿瘤组织，在显示出肿瘤靶向能力的同时，还降低了 MMAE分子在其它组织分布带来的毒性风险，显示出剂量相关性和优异的抗肿瘤效果。
上述结果表明，本公开提供的缀合物能够有效地将对肿瘤具有抑制作用的小分子药物基团靶向递送至肿瘤组织，显示出良好的抗肿瘤活性和剂量依赖效应。
实验例24缀合物对U118MG皮下瘤模型小鼠体内活性
按照实验例4的方法获得接种U118MG皮下瘤的小鼠42只，注射后继续饲养小鼠。
用10％DMSO+90％无血清DMEM培养基(体积比)将MMAE溶解成0.02mg/mL浓度的溶液；将缀合物20和对比缀合物44溶解成0.33mg/mL浓度(以适配体计)的溶液；将缀合物39和缀合物41溶解成0.26mg/mL浓度(以适配体计)的溶液；将缀合物42溶解成0.23mg/mL浓度(以适配体计)的溶液。
U118MG细胞接种后7天，对上述接种的小鼠进行分组，每组6只，向各组小鼠给药，给药当天记为D8。于给药前称重，按小鼠平均体重每只20g计算给药容积。
实验采用腹部皮下给药方式，分别于D8、D12、D15和D19各给药一次，共4次给药。
对于空白对照组，分别向每只小鼠给予DMEM培养基，单次给药容积为100μL/g。
对于测试组24a，分别向每只小鼠给予MMAE，单次给药容积为100μL，计算可知单次给药剂量为0.1mg/kg。
对于测试组24b，分别向每只小鼠给予缀合物20，单次给药容积为100μL，计算可知单次给药剂量为1.65mg/kg。
对于对照组24c，分别向每只小鼠给予对比缀合物44，单次给药容积为100μL，计算可知单次给药剂量为1.65mg/kg。
对于测试组24d，分别向每只小鼠给予缀合物39，单次给药容积为100μL，计算可知单次给药剂量为1.32mg/kg。
对于测试组24e，分别向每只小鼠给予缀合物41，单次给药容积为100μL，计算可知单次给药剂量为1.32mg/kg。
对于测试组24f，分别向每只小鼠给予缀合物42，单次给药容积为100μL，计算可知单次给药剂量为1.17mg/kg。
上述各测试组和对照组24a-24f中，单次给药剂量对应的MMAE剂量均为0.1mg/kg。
于D1、D9、D16、D19、D22、D26、D30、D36、D40、D43、D47、D50、D54、D57、D61、D64、D68和D71测量肿瘤体积。其中，空白对照组在D54时，8a组(仅给予MMAE)在D64时测量后中止实验。
通过体外测量的方式测定测量肿瘤的长径和短径。肿瘤体积根据公式1/2(长径×短径2)进行计算。中止实验后对各组取肿瘤组织，称重并计算平均值。结果参见图23所示。
图23示出了各组小鼠中肿瘤体积随时间变化的折线图和D72肿瘤重量。由图23结果可知，空白对照组的肿瘤体积迅速增加，其它各组的肿瘤体积增加速度均有降低；而与仅给予MMAE的测试组24a和对照组24c相比，给予本公开的缀合物的各测试组在相当于MMAE单次给药剂量为0.1mg/kg的情况下，肿瘤体积增加速度均显著进一步更为降低。此外，缀合物20、缀合物39、缀合物41和缀合物42在实验终点D72时，较测试组24a肿瘤重量至少减少了58％，显示出更加优异的抗肿瘤效果。
实验例25缀合物对A549皮下瘤模型小鼠体内活性
A549人肺腺癌细胞(购自广州吉尼欧生物有限公司)培养条件为含10％FBS(Gibco，货号10099-141)的DMEM完全培养基(MACGENE公司，货号CM15019)，于37，5％CO2/95％空气的培养箱中培养。以0.25wt％胰酶消化并收集细胞，吸去上清并将细胞重悬于无血清DMEM培养基中制成细胞密度为1×108cells/mL的细胞培养液。
按照实验例4的方法获得接种A549皮下瘤的小鼠30只，注射后继续饲养小鼠。
用无血清DMEM培养基将缀合物20和缀合物39溶解成1mg/mL浓 度(以适配体计)的缀合物溶液。对比缀合物43溶解成0.8mg/mL浓度(以适配体计)的缀合物溶液。10％DMSO+90％无血清DMEM培养基(体积比)MMAE溶解成0.06mg/mL浓度的溶液。
A549人肺癌细胞接种后7天，对全部小鼠进行分组，每组6只，向每只小鼠给药，给药当天记为D8。于给药前称重，按小鼠体重计算给药容积。
对于各组小鼠于D8、D12、D15和D19各给药一次，共给药4次。
对于空白对照组25a，分别向每组小鼠给予DMEM，单次给药容积为5μL/g小鼠体重；
对于对照组25b，分别向每组小鼠给予MMAE，单次给药容积为5μL/g小鼠体重，计算可知单次给药剂量为0.3mg/kg；
对于测试组25c，分别向每只小鼠给予缀合物20，单次给药容积为5μL/g小鼠体重，计算可知单次给药剂量为5mg/kg，其中包含MMAE的剂量相当于0.3mg/kg；
对于测试组25d，分别向每只小鼠给予缀合物39，单次给药容积为5μL/g小鼠体重，计算可知单次给药剂量为5mg/kg，其中包含MMAE的剂量相当于0.3mg/kg；
对于测试组25e，分别向每只小鼠给予对比缀合物43，单次给药容积为5μL/g小鼠体重，计算可知单次给药剂量为5mg/kg，其中包含MMAE的剂量相当于0.3mg/kg；
对于各组小鼠于D46、D50和D54各给药一次，单次给药容积为10μL/g小鼠体重，计算可知单次给药剂量包含MMAE的剂量相当于0.6mg/kg。
于D1、D9、D16、D19、D22、D26、D30、D36、D40、D43、D47、D50、D54和D57测量肿瘤体积，空白对照组于D50测量后中止实验。
通过体外测量的方式测定测量肿瘤的长径和短径。肿瘤体积根据公式1/2(长径×短径2)进行计算。结果参见图24所示。
图24是示出了给予不同浓度本公开提供的缀合物或对照化合物后， A549皮下瘤模型小鼠肿瘤体积随时间变化的折线图。从图24的结果可以看出，空白对照组小鼠肿瘤体积迅速增加，其余各组肿瘤体积增加速度均有所降低；在各个时间段，给予缀合物20和缀合物39的小鼠的肿瘤体积均小于对照组25b和25e。上述结果表明，包含由本公开的适配体形成的递送基团的缀合物能够有效地靶向到达肿瘤组织，并显示出良好的抗肿瘤活性。
以上详细描述了本公开的一些实施方式，但是，本公开并不限于上述实施方式中的具体细节，在本公开的技术构思范围内，可以对本公开的技术方案进行多种简单变型，这些简单变型均属于本公开的保护范围。
另外需要说明的是，在上述一些实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本公开对各种可能的组合方式不再另行说明。
此外，本公开的各种不同的实施方式之间也可以进行任意组合，只要其不违背本公开的思想，其同样应当视为本公开所公开的内容。

Claims (53)

  1. 一种适配体，包含一段连续的核苷酸序列，连接相邻的两个核苷酸的基团独立地为磷酸酯基或者具有修饰基团的磷酸酯基，每个核苷酸选自修饰或未修饰A、U、C或G中的一种，所述连续的核苷酸序列具有式(1)所示的序列：
    5′-T1-S1-Na-S2-Nb-S3-Nc-S4-T2-3′    式(1)
    其中，T1是由1-3个核苷酸组成的基序，T2是由0-15个核苷酸组成的基序，并且T2中不包含与T1完全反向互补的基序；
    S1和S4各自是由3-7个核苷酸组成的基序，S1与S4长度相同并且完全反向互补；
    Na和Nc各自是由1-4个核苷酸组成的基序，Na中的每个核苷酸与Nc中的每个核苷酸均不互补，并且Na和Nc中U的总个数占Na和Nc中全部核苷酸总个数的50％以上；
    S2和S3各自是由1-4个核苷酸组成的基序，S2与S3长度相同并且完全反向互补；
    Nb是由3-6个核苷酸组成的基序，并且Nb两端的核苷酸之间不形成AU或GC互补。
  2. 如权利要求1所述的适配体，其中，所述连续的核苷酸序列的长度为18-50个核苷酸，或者20-40个核苷酸，或者21-36个核苷酸，或者24-32个核苷酸。
  3. 如权利要求1或2所述的适配体，其中，T1由2个核苷酸组成；或者，T1由2个核苷酸组成并且含有至少一个C；或者，按照5′-3′方向，T1为CU、UC或AC。
  4. 如权利要求1或2所述的适配体，其中，T2由0-10个核苷酸组成；或者，按照5′-3′方向，T2由U起始的1-9个核苷酸组成。
  5. 如权利要求1所述的适配体，其中，S1和S4各自由3-5个核苷酸 组成且长度相同；或者，S1和S4形成的反向互补中，GC互补占全部互补数量的至少40％；或者，按照5′-3′方向，S1为GCU且S4为AGC，或S1为GAGU且S4为GCUC，或S1为GGAGU且S4为GCUCU，或S1为UAUGG且S4为CCAUG。
  6. 如权利要求1所述的适配体，其中，所述Na和Nc中核苷酸的数量之和为2-4的整数；或者，所述Na和Nc中核苷酸的数量之和为3或4，并且所述Na和Nc中U的数量之和为2或3；或者，按照5′-3′方向，Na或Nc独立地为U、UU、UC或CU。
  7. 如权利要求1所述的适配体，其中，S2和S3各自由2-3个核苷酸组成且长度相同；或者，S2和S3形成的反向互补中至少包含一个GC互补；或者，按照5′-3′方向，S2为CA且S3为UG，或S2为AC且S3为GU，或S2为GCC且S3为GGU。
  8. 如权利要求1所述的适配体，其中，Nb由4个或5个核苷酸组成；或者，按照5′-3′方向，Nb为GACG、GACGU、GACCG、UACU、GUUG或GAUCU。
  9. 如权利要求1所述的适配体，其中，所述连续的核苷酸序列具有SEQ ID NO：1、SEQ ID NO：2或SEQ ID NO：3所示的序列。
  10. 如权利要求1所述的适配体，所述连续的核苷酸序列具有SEQ ID NO：4所示的核苷酸序列：
    5′-N6GGAGUUCAN1N2N3N4UGN5GCUCN7-3′(SEQ ID NO：4)，
    其中，N1、N2、N3各自独立地为A、U、C和G中的一种；N4为U、C或G或者由U、C或G中的两个组成的基序；N5为U、CU或UU；N6为CU、UC或AC；N7为U、UU或UUN8，N8为由1-15个核苷酸组成的基序。
  11. 如权利要求10所述的适配体，其中，N1、N2、N3和N4组成的基 序N1N2N3N4为GACG、GACGU、GACCG、UACU、GUUG或GAUCU中的一种。
  12. 如权利要求10或11所述的适配体，其中，所述连续的核苷酸序列具有SEQ ID NO：5-11中任意一项所示的核苷酸序列。
  13. 如权利要求10所述的适配体，其中，N8是由1-8个核苷酸组成的基序；或者，按照5′-3′方向，N8的核苷酸序列为CCGAUCUC；或者，所述连续的核苷酸序列具有SEQ ID NO：12-14中的一种所示的序列。
  14. 如权利要求1所述的适配体，其中，所述连续的核苷酸序列中的每个胞嘧啶核苷酸为氟代修饰的胞嘧啶核苷酸，和/或所述连续的核苷酸序列中的每个尿嘧啶核苷酸为氟代修饰的尿嘧啶核苷酸；或者，所述连续的核苷酸序列中的每个核苷酸均为2′-甲氧基修饰的核苷酸；或者，所述连续的核苷酸序列中Nb和S3基序中的一个或多个尿嘧啶核苷酸具有修饰的碱基。
  15. 如权利要求14所述的适配体，其中，所述连续的核苷酸序列具有SEQ ID NO：15-33中的一种所示的核苷酸序列。
  16. 如权利要求1所述的适配体，所述适配体中的至少一个连接相邻的两个核苷酸的基团为硫代磷酸酯基，或者每个连接相邻的两个核苷酸的基团均为硫代磷酸酯基。
  17. 如权利要求16所述的适配体，其中，所述连续的核苷酸序列具有SEQ ID NO：34-39中的一种所示的核苷酸序列。
  18. 一种缀合物，包含一个或多个递送基团和一个或多个功能性基团；所述递送基团由如权利要求1-17中任意一项所述的适配体去除一个或多个氢原子或一个或多个官能团形成；每个所述递送基团独立地与所述功能性基团经共价键连接，或通过连接基团连接，每个所述功能性基团独 立地为诊断剂基团、对肿瘤具有治疗作用的小分子治疗剂基团、对肿瘤具有治疗作用的功能性寡核苷酸基团和递送助剂基团中的一种。
  19. 如权利要求18所述的缀合物，具有如式(101)所示的结构：
    其中，每个RAP基团独立地为具有如式(102)所示的结构的基团：
    式中，每个AP基团相同或不同，独立地表示一个所述递送基团；Rj、每个Rk或每个Ri相同或不同，分别独立地表示共价键或者连接基团，且Ri和Rk二者不同时为共价键；每个n1各自独立地表示0-4的整数；
    每个A0基团相同或不同，独立地表示一个所述功能性基团；m0为1-6的整数；n0为1-6的整数，表示基团共价连接的位点。
  20. 如权利要求19所述的缀合物，其中，m0为1-4的整数，和/或n0为1-3的整数，和/或每个n1独立地为0-1的整数；
    或者，m0为1，和/或n0为1，和/或至少一个或者每个n1为0。
  21. 如权利要求19或20所述的缀合物，其中，每个所述Rk或每个所述Ri独立地为共价键或者长度为1-70个碳原子的直链亚烷基，或者，所述直链亚烷基中的一个或多个碳原子被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、OP(O)2、OP(O)(S)、C5-C8亚糖苷基、C2-C10亚烯基、C2-C10亚炔基、C6-C10亚芳基、C3-C18亚杂环基和C5-C10亚杂芳基；并且其中，所述直链亚烷基可具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C10烷基、C6-C10芳基、C5-C10杂芳基、C1-C10卤代烷基、-OC1-C10烷基、-OC1-C10烷基苯基、-C1-C10烷基-OH、-OC1-C10卤代烷基、-SC1-C10烷基、-SC1-C10烷基苯基、-C1-C10烷基-SH、-SC1-C10卤代烷基、卤素取代基、-OH、 -SH、-NH2、-C1-C10烷基-NH2、-N(C1-C10烷基)(C1-C10烷基)、-NH(C1-C10烷基)、-N(C1-C10烷基)(C1-C10烷基苯基)、-NH(C1-C10烷基苯基)、氰基、硝基、-CO2H、-C(O)O(C1-C10烷基)、-CON(C1-C10烷基)(C1-C10烷基)、-CONH(C1-C10烷基)、-CONH2，-NHC(O)(C1-C10烷基)、-NHC(O)(苯基)、-N(C1-C10烷基)C(O)(C1-C10烷基)、-N(C1-C10烷基)C(O)(苯基)、-C(O)C1-C10烷基、-C(O)C1-C10烷基苯基、-C(O)C1-C10卤代烷基、-OC(O)C1-C10烷基、-SO2(C1-C10烷基)、-SO2(苯基)、-SO2(C1-C10卤代烷基)、-SO2NH2、-SO2NH(C1-C10烷基)、-SO2NH(苯基)、-NHSO2(C1-C10烷基)、-NHSO2(苯基)和-NHSO2(C1-C10卤代烷基)。
  22. 如权利要求21所述的缀合物，其中，每个n1均为0，每个Rj独立地为共价键，或者为以下连接基团的一种或多种的连接组合：C1-C20亚烷基、磷酸酯键、硫代磷酸酯键、酰胺键、酯键、醚键、硫醚键、二硫键、1，2，3-三唑亚基、聚乙二醇亚基、吡咯烷亚基、2-氧代吡咯烷亚基、亚苯基、亚环己基、2-丁二酰亚胺亚基、2-硫代丁二酰亚胺亚基、氨基酸亚基、核苷酸亚基。
  23. 如权利要求22所述的缀合物，其中，每个Ri独立地为共价键、二硫键、亚丙基磷酸酯基、2-硫代丁二酰亚胺亚基、氨基酸亚基、或者GAU三核苷酸亚基中的一种或2种的连接组合。
  24. 如权利要求19-23中任意一项所述的缀合物，其中，Rj为共价键，m0为1。
  25. 如权利要求19-23中任意一项所述的缀合物，其中，Rj为连接基团，所述连接基团Rj包含主链部分、侧链部分和缀合连接部，所述主链部分分别与所述缀合连接部和所述侧链部分连接，每个所述侧链部分分别与所述主链部分和所述RAP基团连接，每个所述缀合连接部分别与所述主链部分和所述功能性基团A0连接，其中，
    所述主链部分为长度为1-70个碳原子的直链亚烷基，或者，所述直链亚烷基中的一个或多个碳原子被选自于以下基团所组成的组中的一个 或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、OP(O)2、C5-C8亚糖苷基、C2-C10亚烯基、C2-C10亚炔基、C6-C10亚芳基、C3-C18亚杂环基和C5-C10亚杂芳基；并且其中，所述直链亚烷基可具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C10烷基、C6-C10芳基、C5-C10杂芳基、C1-C10卤代烷基、-OC1-C10烷基、-OC1-C10烷基苯基、-C1-C10烷基-OH、-OC1-C10卤代烷基、-SC1-C10烷基、-SC1-C10烷基苯基、-C1-C10烷基-SH、-SC1-C10卤代烷基、卤素取代基、-OH、-SH、-NH2、-C1-C10烷基-NH2、-N(C1-C10烷基)(C1-C10烷基)、-NH(C1-C10烷基)、-N(C1-C10烷基)(C1-C10烷基苯基)、-NH(C1-C10烷基苯基)、氰基、硝基、-CO2H、-C(O)O(C1-C10烷基)、-CON(C1-C10烷基)(C1-C10烷基)、-CONH(C1-C10烷基)、-CONH2，-NHC(O)(C1-C10烷基)、-NHC(O)(苯基)、-N(C1-C10烷基)C(O)(C1-C10烷基)、-N(C1-C10烷基)C(O)(苯基)、-C(O)C1-C10烷基、-C(O)C1-C10烷基苯基、-C(O)C1-C10卤代烷基、-OC(O)C1-C10烷基、-SO2(C1-C10烷基)、-SO2(苯基)、-SO2(C1-C10卤代烷基)、-SO2NH2、-SO2NH(C1-C10烷基)、-SO2NH(苯基)、-NHSO2(C1-C10烷基)、-NHSO2(苯基)和-NHSO2(C1-C10卤代烷基)；
    每个所述侧链部分独立地是共价键，或者是长度为1-70个碳原子的直链亚烷基，或者，所述直链亚烷基中的一个或多个碳原子被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、OP(O)2、C5-C8亚糖苷基、C2-C10亚烯基、C2-C10亚炔基、C6-C10亚芳基、C3-C18亚杂环基和C5-C10亚杂芳基；并且，所述直链亚烷基可具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C10烷基、C6-C10芳基、C5-C10杂芳基、C1-C10卤代烷基、-OC1-C10烷基、-OC1-C10烷基苯基、-C1-C10烷基-OH、-OC1-C10卤代烷基、-SC1-C10烷基、-SC1-C10烷基苯基、-C1-C10烷基-SH、-SC1-C10卤代烷基、卤素取代基、-OH、-SH、-NH2、-C1-C10烷基-NH2、-N(C1-C10烷基)(C1-C10烷基)、-NH(C1-C10烷基)、-N(C1-C10烷基)(C1-C10烷基苯基)、-NH(C1-C10烷基苯基)、氰基、硝基、-CO2H、-C(O)O(C1-C10烷基)、-CON(C1-C10烷基)(C1-C10烷基)、-CONH(C1-C10烷基)、-CONH2，-NHC(O)(C1-C10烷基)、-NHC(O)(苯基)、-N(C1-C10烷基)C(O)(C1-C10烷基)、-N(C1-C10烷基)C(O)(苯基)、-C(O)C1-C10烷基、-C(O)C1-C10烷基苯基、-C(O)C1-C10 卤代烷基、-OC(O)C1-C10烷基、-SO2(C1-C10烷基)、-SO2(苯基)、-SO2(C1-C10卤代烷基)、-SO2NH2、-SO2NH(C1-C10烷基)、-SO2NH(苯基)、-NHSO2(C1-C10烷基)、-NHSO2(苯基)和-NHSO2(C1-C10卤代烷基)；
    每个所述缀合连接部独立地为共价键或者以下连接结构的一种或多种的连接组合：C1-C10直链亚烷基、磷酸酯键、硫代磷酸酯键、酰胺键、酯键、醚键、二硫键、1，2，3-三唑亚基、聚乙二醇亚基、吡咯烷亚基、2-氧代吡咯烷亚基、亚苯基、亚环己基、2-丁二酰亚胺亚基、2-硫代丁二酰亚胺亚基、氨基酸亚基、核苷酸亚基。
  26. 如权利要求25所述的缀合物，其中，所述连接基团Rj中的每个所述缀合连接部分别与所述主链部分和一个所述功能性基团A0连接；所述侧链部分为n0个，每个侧链部分分别与所述主链部分和一个所述RAP基团连接。
  27. 如权利要求25或26所述的缀合物，其中，全部所述侧链部分连接至所述主链部分中的同一原子；或者，每个所述侧链部分连接至所述主链部分中的不同原子。
  28. 如权利要求27所述的缀合物，其中，m0为1，所述连接基团Rj包含如式(301)所示的结构：
    其中，k为1-3的整数；LC为所述主链部分，LA为所述侧链部分，LB为所述缀合连接部，表示基团共价连接的位点；
    所述主链部分LC为共价键或2-4价、直链或支链的C1-C25饱和烃基，或者，所述饱和烃基中的一个或多个碳原子被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、OP(O)2、C5-C8亚糖苷基、C2-C5亚烯基、C2-C5亚炔基、C6-C10亚芳基、C3-C8亚杂环基和C5-C10亚杂芳基；其中，所述饱和烃基可具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C5烷基、C6-C10芳基、C5-C10杂 芳基、-O-C1-C5烷基、-OC1-C5烷基苯基、-C1-C5烷基-OH、-SC1-C5烷基、硝基、-C(O)O(C1-C5烷基)、-CON(C1-C5烷基)(C1-C5烷基)、-CONH(C1-C5烷基)、-CONH2，-NHC(O)(C1-C5烷基)、-NHC(O)(苯基)、-N(C1-C5烷基)C(O)(C1-C5烷基)、-N(C1-C5烷基)C(O)(苯基)、-C(O)C1-C5烷基、-C(O)C1-C5烷基苯基、-OC(O)C1-C5烷基、-SO2(C1-C5烷基)、-SO2(苯基)、-SO2NH2、-SO2NH(C1-C5烷基)、-SO2NH(苯基)、-NHSO2(C1-C5烷基)和-NHSO2(苯基)；
    每个所述侧链部分独立地是共价键，或者是长度为1-70个碳原子的直链亚烷基，或者，所述直链亚烷基中的一个或多个碳原子被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、OP(O)2、C5-C8亚糖苷基、C2-C10亚烯基、C2-C10亚炔基、C6-C10亚芳基、C3-C18亚杂环基和C5-C10亚杂芳基；并且，所述直链亚烷基可具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C10烷基、C6-C10芳基、C5-C10杂芳基、C1-C10卤代烷基、-OC1-C10烷基、-OC1-C10烷基苯基、-C1-C10烷基-OH、-OC1-C10卤代烷基、-SC1-C10烷基、-SC1-C10烷基苯基、-C1-C10烷基-SH、-SC1-C10卤代烷基、卤素取代基、-OH、-SH、-NH2、-C1-C10烷基-NH2、-N(C1-C10烷基)(C1-C10烷基)、-NH(C1-C10烷基)、-N(C1-C10烷基)(C1-C10烷基苯基)、-NH(C1-C10烷基苯基)、氰基、硝基、-CO2H、-C(O)O(C1-C10烷基)、-CON(C1-C10烷基)(C1-C10烷基)、-CONH(C1-C10烷基)、-CONH2，-NHC(O)(C1-C10烷基)、-NHC(O)(苯基)、-N(C1-C10烷基)C(O)(C1-C10烷基)、-N(C1-C10烷基)C(O)(苯基)、-C(O)C1-C10烷基、-C(O)C1-C10烷基苯基、-C(O)C1-C10卤代烷基、-OC(O)C1-C10烷基、-SO2(C1-C10烷基)、-SO2(苯基)、-SO2(C1-C10卤代烷基)、-SO2NH2、-SO2NH(C1-C10烷基)、-SO2NH(苯基)、-NHSO2(C1-C10烷基)、-NHSO2(苯基)和-NHSO2(C1-C10卤代烷基)；
    每个所述缀合连接部独立地为共价键或者以下连接结构的一种或多种的连接组合：C1-C10直链亚烷基、磷酸酯键、硫代磷酸酯键、酰胺键、酯键、醚键、二硫键、1，2，3-三唑亚基、聚乙二醇亚基、吡咯烷亚基、2-氧代吡咯烷亚基、亚苯基、亚环己基、2-丁二酰亚胺亚基、2-硫代丁二酰亚胺亚基、氨基酸亚基、核苷酸亚基。
  29. 如权利要求28所述的缀合物，其中，所述缀合物具有如式(305)所示的结构：
  30. 如权利要求28所述的缀合物，其中，所述连接基团Rj具有式(306)所示的结构：
    其中，n306为0-3的整数，每个p306独立地为1-6的整数，表示基团共价连接的位点；由*标出的氧原子与所述RAP基团形成磷酸酯键、醚键或酯键连接；由#标出的氧原子中的至少一个与所述功能性基团A0形成醚键、酯键或磷酸酯键而连接，其余由#标出的氧原子与氢原子连接形成羟基，或者与C1-C3烷基连接形成C1-C3烷氧基。
  31. 如权利要求30所述的缀合物，其中，所述缀合物具有如式(307a)、(307b)或(307c)所示的结构：
  32. 如权利要求28所述的缀合物，其中，所述缀合物具有式(308)所示的结构：
    其中，
    n308为选自1-10的整数；
    每个m308独立地为选自2-10的整数；
    每个R308独立地为H、C1-C10烷基、C1-C10卤代烷基或C1-C10烷氧基；
    每个R3独立地为所述功能性基团A0，或者为所述RAP基团，并且至少一个R3为所述功能性基团A0，且至少一个R3为所述RAP基团；或者，一个R3为所述功能性基团A0，其余R3为所述RAP基团；
    连接至所述功能性基团A0的每个L1表示所述缀合连接部，并且连接至所述RAP的每个L1表示所述侧链部分。
  33. 如权利要求32所述的缀合物，其中，每个L1独立地选自于由基团L4-L23及其任意连接组合所组成的组。
  34. 如权利要求33所述的缀合物，其中，每个L1独立地选自于由基团L4-L9、L13、L14、L18中至少2个的连接组合所组成的组；或者，每个L1独立地为基团L4、L5、L7、L9、L13、L14、L18中至少2个的连接组合。
  35. 如权利要求32-34中任意一项所述的缀合物，其中，每个L1的长度独立地为3-25个原子；或者，每个L1的长度独立地为4-15个原子。
  36. 如权利要求32-35中任意一项所述的缀合物，其中，n308为2-6的整数，2-4个R3为所述RAP基团，其余的R3为所述功能性基团。
  37. 如权利要求32-36中任意一项所述的缀合物，其中，每个m308各自独立地为2-5的整数，和/或每个m308均相等。
  38. 如权利要求32-37中任意一项所述的缀合物，其中，n308为选自2-4的整数；每个m308独立地为选自2-4的整数；每个R308均为H。
  39. 如权利要求38所述的缀合物，其中，一个R3为所述功能性基团A0，其余R3为所述RAP基团。
  40. 如权利要求38或39所述的缀合物，其中，每个L1上同时含有与含氮骨架上的N原子连接的连接位点和与所述功能性基团A0或所述RAP基团连接的连接位点，所述与含氮骨架上的N原子连接的位点与该N原子形成酰胺键；或者，一个或多个L1选自B5、B6、B5′或B6′：

    其中，表示基团共价连接的位点，q2为1-10的整数；或者，q2为1-5的整数。
  41. 如权利要求32-40中任一项所述的缀合物，具有式(403)、(404)、(405)、(406)、(407)、(408)、(409)、(410)、(411)、(412)、(413)、(414)、(415)、(416)、(417)、(418)、(419)、(420)、(421)、(422)、(423)、(424)、(425)、(426)或(427)所示的结构：








  42. 如权利要求19-23中任意一项所述的缀合物，其中，Rj包含核苷酸序列I和核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II各自包含5-25个修饰或未修饰的核苷酸，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补，所述递送基团连接至所述核苷酸序列I，所述功能性基团连接至所述核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II在受试者体内不引发免疫反应或毒性反应。
  43. 如权利要求42所述的缀合物，其中，所述递送基团的3′末端经 磷酸酯键连接至所述核苷酸序列I的5′末端核苷酸的核糖5′位，所述功能性基团连接至所述核苷酸序列II的5′末端核苷酸的核糖5′位；或者，所述功能性基团包含一段核苷酸序列，所述核苷酸序列的3′末端经磷酸酯键连接至所述核苷酸序列II的5′末端核苷酸的核糖5′位。
  44. 如权利要求43所述的缀合物，其中，所述核苷酸序列I和所述核苷酸序列II实质上反向互补或者完全反向互补；或者，所述核苷酸序列I和所述核苷酸序列II的长度相等并且均为10-20个修饰或未修饰的核苷酸；或者，所述核苷酸序列I和所述核苷酸序列II均由17个核苷酸组成并且完全反向互补；或者，所述核苷酸序列I和所述核苷酸序列II分别具有SEQ ID NO：40和SEQ ID NO：41所示的序列：
    5′-GUACAUUCUAGAUAGCC-3′(SEQ ID NO：40)
    5′-GGCUAUCUAGAAUGUAC-3′(SEQ ID NO：41)，
    或者，所述核苷酸序列I和所述核苷酸序列II分别具有SEQ ID NO：42和SEQ ID NO：43所示的序列：
    5′-GmUfAmCfAmUfUfCfUfAmGmAmUfAmGmCfCf-3′
    (SEQ ID NO：42)
    5’-GmGmCfUfAmUfCfUfAmGmAmAmUfGmUfAmCf-3’
    (SEQ ID NO：43)。
  45. 如权利要求19-44中任意一项所述的缀合物，其中，Rj为可裂解的。
  46. 如权利要求18-45中任意一项所述的缀合物，其中，至少一个或全部所述功能性基团为诊断剂基团，每个所述诊断剂基团独立地选自造影剂基团或荧光成像基团。
  47. 如权利要求18-45中任意一项所述的缀合物，其中，至少一个或全部所述功能性基团为对肿瘤具有治疗作用的小分子治疗剂基团，每个所述小分子治疗剂基团独立地选自细胞毒素基团、抗生素基团、血管生成抑制剂或抗体药物基团。
  48. 如权利要求18-45中任意一项所述的缀合物，其中，至少一个或全部所述功能性基团为功能性寡核苷酸基团，所述功能性寡核苷酸基团能够调节肿瘤细胞中癌症相关基因的表达水平。
  49. 如权利要求18-48中任意一项所述的缀合物，其中，至少一个所述功能性基团为递送助剂基团，所述递送助剂基团选自于C10-C30烃基、胆固醇基、磷脂基团中的一种或多种。
  50. 一种药物组合物，包含权利要求18-49中任意一项所述的缀合物以及药学上可接受的载体。
  51. 权利要求1-17中任意一项所述的适配体和/或权利要求18-49中任意一项所述的缀合物和/或权利要求50所述的药物组合物在制备用于诊断和/或治疗肿瘤及肿瘤相关疾病或症状的药物中的应用。
  52. 一种肿瘤及肿瘤相关疾病或症状的诊断和/或治疗的方法，包括向有需要的受试者给予有效量的权利要求18-49中任意一项所述的缀合物和/或权利要求50所述的药物组合物。
  53. 一种试剂盒，包括权利要求18-49中任意一项所述的缀合物和/或权利要求50所述的药物组合物。
PCT/CN2023/088466 2022-04-14 2023-04-14 适配体、缀合物与组合物及制备方法和用途 WO2023198201A1 (zh)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102533770A (zh) * 2012-03-16 2012-07-04 广州市锐博生物科技有限公司 一种核酸分子及其应用
WO2017035340A1 (en) * 2015-08-25 2017-03-02 Alnylam Pharmaceuticals, Inc. Methods and compositions for treating a proprotein convertase subtilisin kexin (pcsk9) gene-associated disorder
CN108289906A (zh) * 2015-11-19 2018-07-17 公立大学法人名古屋市立大学 抗肿瘤性药物递送制剂
CN110945132A (zh) * 2017-12-01 2020-03-31 苏州瑞博生物技术有限公司 一种核酸、含有该核酸的组合物与缀合物及制备方法和用途
CN110997919A (zh) * 2017-12-01 2020-04-10 苏州瑞博生物技术有限公司 双链寡核苷酸、含双链寡核苷酸的组合物与缀合物及制备方法和用途
WO2021204216A1 (zh) * 2020-04-08 2021-10-14 北京瑞博开拓医药科技有限公司 核酸、含有该核酸的药物组合物与siRNA缀合物及制备方法和用途

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102533770A (zh) * 2012-03-16 2012-07-04 广州市锐博生物科技有限公司 一种核酸分子及其应用
WO2017035340A1 (en) * 2015-08-25 2017-03-02 Alnylam Pharmaceuticals, Inc. Methods and compositions for treating a proprotein convertase subtilisin kexin (pcsk9) gene-associated disorder
CN108289906A (zh) * 2015-11-19 2018-07-17 公立大学法人名古屋市立大学 抗肿瘤性药物递送制剂
CN110945132A (zh) * 2017-12-01 2020-03-31 苏州瑞博生物技术有限公司 一种核酸、含有该核酸的组合物与缀合物及制备方法和用途
CN110997919A (zh) * 2017-12-01 2020-04-10 苏州瑞博生物技术有限公司 双链寡核苷酸、含双链寡核苷酸的组合物与缀合物及制备方法和用途
WO2021204216A1 (zh) * 2020-04-08 2021-10-14 北京瑞博开拓医药科技有限公司 核酸、含有该核酸的药物组合物与siRNA缀合物及制备方法和用途

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