CN110559445A - Novel efficient intelligent targeted drug loading system - Google Patents
Novel efficient intelligent targeted drug loading system Download PDFInfo
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- CN110559445A CN110559445A CN201810565680.XA CN201810565680A CN110559445A CN 110559445 A CN110559445 A CN 110559445A CN 201810565680 A CN201810565680 A CN 201810565680A CN 110559445 A CN110559445 A CN 110559445A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/66—Phosphorus compounds
- A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
The invention provides a drug carrier, a drug composition and application. The drug carrier includes: an aptamer sequence; (ii) a variable sequence which can form a G-quadruplex structure; the two ends of the connecting sequence are respectively connected with the aptamer sequence and the variable structure sequence; wherein a portion of the aptamer sequence is complementarily paired with at least a portion of the linker sequence. The drug carrier has the effects of specifically recognizing target cells and accurately releasing drugs, provides a more accurate targeted drug delivery mode for efficient treatment of tumors, and is suitable for large-scale application.
Description
Technical Field
The invention relates to the field of biomedicine. Specifically, the invention relates to a novel efficient intelligent targeted drug loading system. More particularly, the invention relates to pharmaceutical carriers, pharmaceutical compositions and uses.
Background
In the face of the global severe anti-cancer situation, how to reduce the toxic and side effects of the drugs to relieve the pain of patients and prolong the life cycle of the patients while improving the treatment effect of the diseases becomes a significant social problem. At present, although various first-line anticancer drugs have better anticancer effect clinically, the first-line anticancer drugs also have serious toxic and side effects on human bodies.
The anthraquinone antibiotic doxorubicin (Dox) is a broad-spectrum antitumor drug, is mainly used for clinically treating solid tumors such as lung cancer, breast cancer, thyroid cancer and the like, and easily enters tumor cells through a cell membrane, interacts with DNA and is embedded between G-C base sequences of the tumor cells to cause DNA cracking. However, the chemical drugs have low targeting property, cannot accurately identify tumor cells, can damage normal cells while inhibiting cancer cells, and bring serious side effects to human bodies. Meanwhile, multidrug resistance is also one of the major obstacles to the success of chemotherapy, and overexpression of P-glycoprotein (P-gp) in drug-resistant cells induces increased drug efflux and decreased intracellular drug accumulation.
The metal platinum complex cisplatin is a broad-spectrum antitumor drug approved by FDA in 1978, and is clinically used for treating lung cancer, bladder cancer, leukemia and the like. Cisplatin is stable under the condition of high chloride ion concentration, can diffuse through cell membranes after entering a human body, and is embedded into cells between two bases in a DNA single strand to form a closed chelate ring so as to inhibit the replication of DNA. Due to its low selectivity, cisplatin has serious side effects such as nephrotoxicity, ototoxicity and neurotoxicity, while exerting its therapeutic effect. Platinum drugs use organic cation transporter 2(OCT-2) as an absorption mechanism, which then forms adducts with DNA, triggering oxidative stress, DNA damage, and apoptosis. However, such drugs have the same effect in other tissues expressing OCT transporters, e.g., causing nephrotoxicity in the kidney. Meanwhile, the drug uptake of cells is reduced, and the over-expression of the copper-transported P-type adenosine triphosphatase causes drug resistance of tumor cells, so that the treatment effect of the cisplatin is greatly reduced.
Therefore, the development of a drug carrier system which can efficiently target tumor cells and automatically release drugs in the tumor cells is necessary.
Disclosure of Invention
The present invention aims to solve at least to some extent at least one of the technical problems of the prior art.
It should be noted that the present invention has been completed based on the following findings of the inventors:
At present, the drug release mechanism of the targeting drug-carrying system comprises enzyme cleavage, pH concentration difference, chemical bond breakage and the like, and the drug-carrying system for releasing the drug according to the ion concentration difference inside and outside cells is not reported. The oligonucleotide sequence Hu22(5'-AGGGTTAGGGTTAGGGTTAGGG-3') exhibits different configurations in solutions of different ion concentrations: the compound is in an intramolecular antiparallel G-quadruplex structure in a high-sodium solution and can be combined with a medicament; and the compound is in an intramolecular parallel G-quadruplex structure in a high potassium solution, and can release drugs. The human body has a high potassium environment inside the cells and a high sodium environment outside the cells.
In view of the above, the inventors designed a drug carrier based on the specific recognition ability of the aptamer to tumor cells and the characteristic of the conformation transition of Hu 22G-quadruplex in different ion concentration environments inside and outside cells. The drug carrier comprises a variable structure sequence capable of forming a G-quadruplex, an aptamer sequence and a connecting sequence for connecting the variable structure sequence and the aptamer, wherein the variable structure sequence has the effect of intelligently releasing drugs, the aptamer sequence can specifically and targetedly identify tumor cells, the combination of the variable structure sequence and the aptamer sequence can realize targeting and releasing at the same time, the drug carrier is simple and efficient, the damage of anti-cancer drugs to normal cells is avoided, a more accurate targeting drug delivery mode is provided for efficient treatment of tumors, and the drug carrier is suitable for large-scale application.
to this end, in one aspect of the invention, a pharmaceutical carrier is provided. According to an embodiment of the invention, the drug carrier comprises: an aptamer sequence; (ii) a variable sequence which can form a G-quadruplex structure; the two ends of the connecting sequence are respectively connected with the aptamer sequence and the variable structure sequence; wherein a portion of the aptamer sequence is complementarily paired with at least a portion of the linker sequence.
The inventor designs a drug carrier based on the specific recognition capability of the aptamer on tumor cells and the characteristic of the configuration transformation of Hu 22G-quadruplex in environments with different ion concentrations inside and outside cells. The drug carrier comprises a variable structure sequence capable of forming a G-quadruplex, an aptamer sequence and a connecting sequence for connecting the variable structure sequence and the aptamer, wherein the variable structure sequence has the effect of intelligently releasing drugs, the aptamer sequence can specifically and targetedly identify tumor cells, the combination of the variable structure sequence and the aptamer sequence can realize targeting and releasing at the same time, the drug carrier is simple and efficient, the damage of anti-cancer drugs to normal cells is avoided, a more accurate targeting drug delivery mode is provided for efficient treatment of tumors, and the drug carrier is suitable for large-scale application.
According to an embodiment of the present invention, the above drug carrier may further have the following additional technical features:
According to an embodiment of the invention, the allosteric sequence has the amino acid sequence of SEQ ID NO: 1. Therefore, a G-quadruplex structure can be formed, the configuration can be converted inside and outside cells, and the purpose of combining and releasing the drug can be achieved.
according to an embodiment of the invention, the aptamer is selected from the group consisting of non-small cell lung cancer cell aptamer S6, nucleolin aptamer AS1411, mucin 1 aptamer Apt, tyrosine aptamer sgc8, and prostate specific membrane antigen aptamer a 10. Therefore, a better specific target recognition effect is achieved.
According to an embodiment of the invention, the connection sequence comprises: 2-10 bases that are complementarily paired to the aptamer sequence; and at least one of 0 to 20 adenine, cytosine and thymine. Therefore, the variable structure sequence can form a G-quadruplex and change the structure inside and outside the cell, and the effect of accurately releasing the drug is realized; aptamers can also be made to form a loop structure to ensure that the aptamers specifically recognize the target cells.
In yet another aspect of the invention, a pharmaceutical composition is provided. According to an embodiment of the invention, the pharmaceutical composition comprises: a pharmaceutical carrier as described above; and a medicament. The pharmaceutical composition disclosed by the invention can specifically act on target cells, avoids damage to other normal cells, can realize the effect of accurately releasing the drugs, provides a more accurate targeted drug delivery mode for efficient treatment of tumors, and is suitable for large-scale application.
According to an embodiment of the invention, the drug is selected from at least one of the following: drugs having a conjugated plane, preferably drugs having a benzene ring and/or a naphthalene ring; drugs with positive charges or metal ions; a drug having an amino group. Thereby, an effect of precisely releasing the drug is achieved.
According to an embodiment of the invention, the drug is selected from at least one of the following: anthraquinone antibiotics, cyclophosphamide, cisplatin, 5-fluorouracil and paclitaxel. Thereby, an effect of precisely releasing the drug is achieved.
According to an embodiment of the invention, the allosteric sequence is conjugated to the drug. Thereby, the effect of accurately releasing the drug by the variable structure sequence is realized.
According to an embodiment of the invention, the binding is covalent bonding. Thereby, the effect of accurately releasing the drug by the variable structure sequence is realized.
According to an embodiment of the invention, the drug is separated from the drug carrier when the pharmaceutical composition is in an environment with a potassium ion concentration of at least 100mM, and the drug is bound to the drug carrier when the pharmaceutical composition is in an environment with a sodium ion concentration of at least 100 mM.
In a further aspect of the invention, the invention provides the use of a pharmaceutical carrier as hereinbefore described for the preparation of a pharmaceutical composition. The drug carrier has the effects of specifically recognizing target cells and accurately releasing drugs, provides a more accurate targeted drug delivery mode for efficient treatment of tumors, and is suitable for large-scale application.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 shows a schematic structural view of a drug carrier according to an embodiment of the present invention;
FIG. 2 shows a spectrum according to an embodiment of the invention;
FIG. 3 shows a schematic representation of the analysis of cytostatic effect of a drug-loaded system according to one embodiment of the invention;
FIG. 4 shows a CD map of circular and single stranded S6 under physiological buffer conditions, according to one embodiment of the invention;
FIG. 5 is a schematic diagram showing an analysis of the effect of the presence or absence of a linker sequence on the binding of the Hu22 sequence to a drug (Dox), wherein A is a linker-free sequence, according to one embodiment of the present invention; b is a connecting sequence.
Detailed Description
The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.
The invention provides a drug carrier, a drug composition and application of the drug carrier in preparation of the drug composition. Which will be described in detail below, respectively.
Drug carrier
in one aspect of the invention, a drug carrier is provided. According to an embodiment of the present invention, referring to fig. 1, a drug carrier includes: an aptamer sequence 100, a variable construct sequence 200, and a linker sequence 300. The drug carrier has the effects of specifically recognizing target cells and accurately releasing drugs, thereby avoiding damage to other normal cells, providing a more accurate targeted drug delivery mode for efficient treatment of tumors, and being suitable for large-scale application. Which will be described in detail below.
according to an embodiment of the present invention, one end of the aptamer 100 is connected to the reconfigurable sequence 200 through a linker sequence 300. The inventor finds that if the aptamer is directly connected with the variable structure sequence, the respective functions are affected, namely, the effect of the aptamer on specifically recognizing target cells is poor, and the aptamer is easy to act on non-specific cells to cause normal cell damage; leading to poor configuration transformation effect of the variable configuration sequence in different ion concentration environments inside and outside cells, thereby failing to accurately release the drug.
According to an embodiment of the invention, the variable sequence has the sequence of SEQ ID NO: 1. The oligonucleotide sequence Hu22(5'-AGGGTTAGGGTTAGGGTTAGGG-3', namely the nucleotide sequence shown in SEQ ID NO: 1) presents different configurations in solutions with different ion concentrations: in high sodium solution, it has an intramolecular antiparallel G-quadruplex structure to release the drug, and in high potassium solution, it has an intramolecular parallel G-quadruplex structure to bind the drug. The human body has a high potassium environment inside the cells and a high sodium environment outside the cells. Furthermore, the characteristic of the configuration transformation of Hu 22G-quadruplex in environments with different ion concentrations inside and outside cells is utilized to achieve the effects of carrying the drug into the cells and accurately releasing the drug in the cells.
In the present invention, the type of the aptamer is not limited strictly, and an aptamer capable of specifically recognizing a target cell can be selected for different target cell characteristics according to actual conditions. According to a specific embodiment of the present invention, the aptamer 100 is selected from the group consisting of non-small cell lung cancer cell aptamer S6, nucleolin aptamer AS1411, mucin 1(MUC-1) aptamer Apt, tyrosine aptamer (Protein kinase-7) sgc8, Prostate Specific Membrane Antigen (PSMA) aptamer a 10. This enables the target cell to be specifically recognized.
According to an embodiment of the invention, a portion of the aptamer sequence 100 is complementarily paired with at least a portion of the linker sequence 300 such that the aptamer forms a loop structure. The inventors found that, when the aptamer exists in a free-coiled single-chain form, the aptamer is easily bound to proteins, metal ions, and the like in cells, and a G-quadruplex structure is easily formed, which affects the specific recognition of target cells, thereby causing damage to normal cells. Then, the inventors designed a base sequence complementary to the partial sequence of the aptamer at the junction sequence so that the aptamer forms a loop structure, thereby improving its ability to specifically recognize a target cell.
According to an embodiment of the invention, the connection sequence 300 comprises: 2-10 bases that are complementary paired with the aptamer sequence; and at least one of 0 to 20 adenine, cytosine and thymine. Therefore, the aptamer can form a ring structure so as to specifically recognize the target cell, and the characteristics of the variable structure sequence are not influenced, so that the effect of accurately releasing the medicament is realized.
Pharmaceutical composition
In yet another aspect of the invention, a pharmaceutical composition is provided. According to an embodiment of the invention, the pharmaceutical composition comprises: a pharmaceutical carrier as described above; and a medicament. The pharmaceutical composition disclosed by the invention can specifically act on target cells, avoids damage to other normal cells, can realize the effect of accurately releasing the drugs, provides a more accurate targeted drug delivery mode for efficient treatment of tumors, and is suitable for large-scale application.
The type of drug in the present invention is not particularly limited, as long as it can bind to the allosteric sequence of a drug carrier extracellularly and can be separated intracellularly. According to a particular embodiment of the invention, the drug is selected from at least one of the following: drugs having a conjugated plane, preferably drugs having a benzene ring and/or a naphthalene ring; drugs with positive charges or metal ions; a drug having an amino group. According to another embodiment of the invention, the drug is selected from at least one of the following: anthraquinones antibiotics, Cyclophosphamide (CTX), cisplatin (DDP), 5-fluorouracil (5-Fu) and paclitaxel (TAX). Therefore, the compound can be combined with a drug carrier to enter cells, and can realize accurate drug release in the cells. Meanwhile, the specific recognition of the target cells by the aptamer is not influenced.
according to embodiments of the invention, one end of the allosteric sequence is linked to a linker sequence and the other end can bind a drug. Specifically, the binding may be performed by a covalent bond. Therefore, the characteristic that the variable structure sequence is changed in the environments with different ion concentrations inside and outside the cell is utilized to realize the effects of combining the medicine outside the cell and accurately releasing the medicine inside the cell.
According to an embodiment of the invention, the binding is covalent bonding. Thus, covalent linkage is employed to facilitate binding and release of the drug.
According to an embodiment of the present invention, the drug is separated from the drug carrier when the pharmaceutical composition is in an environment having a potassium ion concentration of at least 100mM, and the drug is bound to the drug carrier when the pharmaceutical composition is in an environment having a sodium ion concentration of at least 100 mM. The variable structure sequence on the drug carrier is in an intramolecular antiparallel G-quadruplex structure in a high sodium solution and can be combined with a drug, and is in an intramolecular parallel G-quadruplex structure in a high potassium solution and can release the drug. The human body has a high potassium environment inside the cells and a high sodium environment outside the cells. Therefore, the characteristic that the variable structure sequence of the drug carrier is changed in the environment with different ion concentrations inside and outside the cell is utilized to realize the effects of combining the drug outside the cell and accurately releasing the drug inside the cell.
It will be appreciated by those skilled in the art that the features and advantages previously described for the pharmaceutical carrier are equally applicable to the pharmaceutical composition and will not be described in detail herein.
Use of
in a further aspect of the invention, the invention provides the use of a pharmaceutical carrier as hereinbefore described for the preparation of a pharmaceutical composition. The drug carrier has the effects of specifically recognizing target cells and accurately releasing drugs, provides a more accurate targeted drug delivery mode for efficient treatment of tumors, and is suitable for large-scale application.
It will be appreciated by those skilled in the art that the features and advantages previously described for the pharmaceutical carrier and pharmaceutical composition apply equally to this use and will not be described in any further detail herein.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
EXAMPLE 1 determination of the configuration of the oligonucleotide Hu22 sequence in different buffer systems by circular dichroism spectroscopy
(1) The Hu22 sequence was dissolved separately in two solutions, one solution having the same potassium ion concentration as the cell sap (140mM K)+,10mM Na+) The other solution had the same sodium ion concentration as the extracellular solution (145mM Na)+,5mM K+) And the change of the configuration of Hu22 is verified by circular dichroism spectrum and fluorescence spectrum.
(2) As shown in FIG. 2A, Hu22 in the high potassium solution shows a negative peak at about 240nm, indicating that Hu22 is a mixed G-quadruplex structure with parallel G-quadruplex predominating inside the cell. Whereas Hu22 in the high-sodium solution showed a negative peak at approximately 260nm and positive peaks at 240nm and 290nm (FIG. 2B), indicating that Hu22 exists extracellularly as a mixed G-quadruplex structure dominated by an antiparallel G-quadruplex structure.
Example 2 construction of a Brand-new intelligent targeting non-Small cell Lung cancer drug delivery System
1. The aptamer S6 capable of specifically recognizing the non-small cell lung cancer cell is linked with the Hu 22G-quadruplex capable of generating configuration transformation in different ion concentration environments inside and outside the cell through a nucleic acid sequence 5'-GGCCACTTTTT-3' (SEQ ID NO: 2) to form a novel intelligent targeted drug-loading system.
S6：5'-GTGGCCAGTCACTCAATTGGGTGTAGGGGTGGGGATTGTGGGTTG-3'(SEQ ID NO：3)
Hu22：5'-AGGGTTAGGGTTAGGGTTAGGG-3'(SEQ ID NO：1)
Hu22-(T)5GGCCAC-S6：5'-GTGGCCAGTCACTCAATTGGGTGTAGGGGTGGGGATT GTGGGTTGGGCCACTTTTTAGGGTTAGGGTTAGGGTTAGGG-3' (SEQ ID NO: 4) (Note: the sequence complementary to S6 at the horizontal line)
2. And performing in-vitro cytotoxic activity test on the constructed novel intelligent targeted drug-loaded system on a biological level through an MTT (methyl thiazolyl tetrazolium) experiment. The experimental cell was a 549.
cells were seeded in 96-well cell culture plates at 200. mu.L per well and after 24 hours of culture, different concentrations of test samples (DDP alone, Dox alone, Hu22- (T) alone) were added5GGCCAC-S6、Hu22-(T)5GGCCAC-S6 and DDP mixtures and Hu22- (T)5GGCCAC-S6 and Dox mixture). A solvent control was also set, with 3 parallel wells for each sample concentration. After 72 hours of drug action, 20. mu.L of MTT (5mg/mL) solution was added to each well, the culture was continued for 4 hours, the supernatant was discarded, and DMSO was added to each wellStanding for 15min at 200 μ L, and measuring absorbance at 490nm wavelength with enzyme labeling instrument.
Cell inhibition (%) was 1- (mean OD of administered cells/mean OD of solvent control cells) × 100%
3. as shown in FIG. 3A, when the drug carrier is loaded with Dox, Hu22- (T)5GGCCAC-S6-Dox has stronger inhibition ratio on A549 cells than Dox alone. When the drug carrier is loaded with DDP (FIG. 3B), when the working concentration is more than 4 mu M, Hu22- (T)5IC of GGCCAC-S6-DDP for A549 tumor cells50IC of value 4.9. mu.M, DDP alone50The value was 6.5. mu.M. Thus, Hu22- (T) is shown5GGCCAC-S6-DDP has stronger inhibiting effect on A549 cells than DDP alone.
example 3 construction of a novel Intelligent targeting leukemia drug delivery System
1. PTK-7 nucleic acid aptamer sgc8 capable of specifically recognizing Acute Myelocytic Leukemia (AML) and Hu 22G-quadruplex capable of generating configuration transformation in different ion concentration environments inside and outside cells are connected through a section of nucleic acid sequence to form a novel intelligent targeted drug-loading system.
Sgc8：5'-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA-3'(SEQ ID NO：5)；
Hu22：5'-AGGGTTAGGGTTAGGGTTAGGG-3'；
Hu22-(T)2CGGCGGCGCAGCAGTTAGAT-Sgc8：5'-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGACGGCGGCGCAGCAGTTAGATTTAGGGTTAGGGTTAGGGTTAGGG-3'(SEQ ID NO：6)
2. And performing in-vitro cytotoxic activity test on the constructed novel intelligent targeted drug-loaded system on a biological level through an MTT (methyl thiazolyl tetrazolium) experiment. The experimental cells are acute myelocytic leukemia cell strain CMK and human normal umbilical vein endothelial HUVEC cells. The method comprises the following steps:
Cells were seeded in 96-well cell culture plates at 200. mu.L per well and after 24 hours of culture, test samples (DDP alone, 5-Fu alone, Hu22- (T) alone) were added at different concentrations2CGGCGGCGCAGCAGTTAGAT-Sgc8、Hu22-(T)2CGGCGGCGCAGCAGTTAGAT-Sgc8 and DDP mixtures and Hu22- (T)2CGGC GGCGCAGCAGTTAGAT-Sgc8 and 5-Fu mixtures). At the same time setting solutionControl group, 3 parallel wells per sample concentration. After the drug acts on the cells for 72 hours, 20 mu L of MTT (5mg/mL) solution is added into each hole, the cells are continuously cultured for 4 hours, the supernatant is discarded, 200 mu L of DMSO is added into each hole, the cells are kept stand for 15min, and the absorbance value is measured at the 490nm wavelength of an enzyme-labeling instrument.
The cell inhibition ratio (%) × 1- (mean OD of administered cells/mean OD of solvent control cells) × 100%.
TABLE 1 IC of different drug delivery systems and drugs for leukemia cells CMK and human normal umbilical vein endothelial cells50Value of
The results of the experiment are shown in table 1. As can be seen, the single antitumor drug DDP or 5-Fu has stronger inhibition effect on the proliferation of tumor cells CMK and human normal vein endothelial cells.
After the drug carrier is combined with the two drugs, the drug-carrying system obviously enhances the inhibition effect on the proliferation of tumor cells on one hand, and obviously reduces the toxic and side effects on normal cells on the other hand. The toxic and side effects on normal cells after drug loading are far lower than those of DDP or 5-Fu.
Example 4 construction of novel Intelligent targeting human Breast cancer drug Carrier
1. Nucleolin aptamer AS1411 over-expressed on the surface of a human breast cancer cell and Hu 22G-quadruplex capable of generating configuration transformation in different ion concentration environments inside and outside the cell are linked through a nucleic acid sequence to form a novel intelligent targeted drug-loading system.
AS1411：5'-GGTGGTGGTGGTTGTGGTGGTGGTGG-3'(SEQ ID NO：7)；
Hu22：5'-AGGGTTAGGGTTAGGGTTAGGG-3'；
Hu22-(T)20CC-AS1411：5'-GGTGGTGGTGGTTGTGGTGGTGGTGGCCTTTTTTTT
TTTTTTTTTTTTAGGGTTAGGGTTAGGGTTAGGG-3'(SEQ ID NO：8)
2. And performing in-vitro cytotoxic activity test on the constructed novel intelligent targeted drug-loaded system on a biological level through an MTT (methyl thiazolyl tetrazolium) experiment. The experimental cells are human breast cancer MCF-7 and human normal umbilical vein endothelial HUVEC cells. The method comprises the following steps:
Cells were seeded in 96-well cell culture plates at 200. mu.L per well, and after 24 hours of culture, test samples (CTX alone, TAX alone, Hu22- (T) alone) were added at different concentrations20CC-AS1411、Hu22-(T)20CC-AS1411 and CTX blends and Hu22- (T)20CC-AS1411 and TAX mixture). A solvent control was also set, with 3 parallel wells for each sample concentration. After the drug acts on the cells for 72 hours, 20 mu L of MTT (5mg/mL) solution is added into each hole, the cells are continuously cultured for 4 hours, the supernatant is discarded, 200 mu L of DMSO is added into each hole, the cells are kept stand for 15min, and the absorbance value is measured at the 490nm wavelength of an enzyme-labeling instrument.
the cell inhibition ratio (%) × 1- (mean OD of administered cells/mean OD of solvent control cells) × 100%.
TABLE 2 IC of different drug delivery systems and drugs on human breast cancer MCF-7 and human normal umbilical vein endothelial HUVEC cells50value of
The results of the experiment are shown in table 2. Therefore, the single antitumor drug CTX or TAX has stronger inhibition effect on the proliferation of human breast cancer MCF-7 and human normal vein endothelial cells. After the drug-carrying system is combined with the two drugs, the inhibition effect of the drug-carrying system on the proliferation of tumor cells is obviously enhanced on one hand, and the toxic and side effects of the drug-carrying system on normal cells are obviously reduced on the other hand. The toxic and side effects on normal cells after drug loading are far lower than those of CTX or TAX.
example 5 closed and single-stranded structures of aptamer S6:
Aptamer S6 is a guanine (G) -rich oligonucleotide sequence containing 45 bases (SEQ ID NO: 2). S6 can spontaneously form a G-quadruplex structure (S6G-quadruplex) under the induction of metal ions in a physiological buffer solution. The inventors have found that the formation of a G-quadruplex structure affects the recognition effect on target cells.
When the head end of the S6 sequence is formed into a loop by base complementation, the G-quadruplex structure cannot be formed, and the Single-Stranded structure (S6Single-Stranded) is always maintained (as shown in FIG. 4). Furthermore, the inventors designed a base sequence complementary to the aptamer on the linker sequence, so that the aptamer forms a single-stranded loop structure, thereby achieving the effect of specifically recognizing the target cell.
Example 6 Effect of the ligation sequence
1. Experimental group 1: the aptamer S6 capable of specifically recognizing the non-small cell lung cancer cell is connected with the Hu 22G-quadruplex capable of generating configuration transformation in different ion concentration environments inside and outside the cell through a section of connecting sequence (5'-TTTTTCACCGG-3') to form a novel intelligent targeted drug-loading system.
Experimental group 2: the S6 is directly connected with Hu 22G-quadruplex to construct a drug-carrying system.
2. The constructed drug-loaded system was tested for in vitro cytotoxic activity at the biological level by MTT assay. The experimental cell was a 549.
Cells were seeded in 96-well cell culture plates at 200. mu.L per well and after 24 hours of culture, the drug-loaded systems of experimental groups 1 and 2 were added, respectively. A solvent control was also set, with 3 parallel wells for each sample concentration. After the drug acts on the cells for 72 hours, 20 mu L of MTT (5mg/mL) solution is added into each hole, the cells are continuously cultured for 4 hours, the supernatant is discarded, 200 mu L of DMSO is added into each hole, the cells are kept stand for 15min, and the absorbance value is measured at the 490nm wavelength of an enzyme-labeling instrument.
Cell inhibition (%) was 1- (mean OD of administered cells/mean OD of solvent control cells) × 100%
The results are shown in FIG. 5. As can be seen in FIG. 2, the fluorescence quenching of Dox bound to Hu22 with the linker sequence was greater than that of Dox bound to Hu22 without the linker sequence. The binding constant of Dox and Hu22 sequence without the connecting sequence is calculated to be 4.31 multiplied by 10 by a binding constant formula4M-1And the binding constant with Hu22 with the connecting sequence is 7.82 multiplied by 104M-1Indicating that the presence of the linker sequence may directly affect the binding of the Hu22 sequence to the drug.
example 7 construction of a novel Intelligent targeting leukemia drug delivery System
1. The Hu22- (T) obtained in step 1 of example 3 was constructed by linking a PTK-7 nucleic acid aptamer sgc8 capable of specifically recognizing Acute Myeloid Leukemia (AML) with a Hu 22G-quadruplex capable of undergoing configuration transition in environments of different ion concentrations inside and outside cells via a nucleic acid sequence2CGGCGGCGCAGCAGTTAGAT-Sgc8 novel intelligent targeting drug-loaded system.
2. And performing in-vitro cytotoxic activity test on the constructed novel intelligent targeted drug-loaded system on a biological level through an MTT (methyl thiazolyl tetrazolium) experiment. The experimental cells are acute myelocytic leukemia cell strain CMK and human normal umbilical vein endothelial HUVEC cells.
Cells were seeded in 96-well cell culture plates at 200. mu.L per well and after 24 hours of culture, different concentrations of test samples (carmustine alone (CA), mechlorethamine alone (MH), Hu22- (T) alone) were added2CGGCGGCGCAGCAGTTAGAT-Sgc8、Hu22-(T)2CGGCGGCGCAGCAGTTAGAT-Sgc8 and CA mixture and Hu22- (T)2CGGCGGCGCAGCAGTTAGAT-Sgc8 and MH mixture). A solvent control was also set, with 3 parallel wells for each sample concentration. After the drug acts on the cells for 72 hours, 20 mu L of MTT (5mg/mL) solution is added into each hole, the cells are continuously cultured for 4 hours, the supernatant is discarded, 200 mu L of DMSO is added into each hole, the cells are kept stand for 15min, and the absorbance value is measured at the 490nm wavelength of an enzyme-labeling instrument.
Cell inhibition (%) was 1- (mean OD of administered cells/mean OD of solvent control cells) × 100%
TABLE 3 IC of different drug delivery systems and drugs for leukemia cells CMK and human normal umbilical vein endothelial cells50Value of
The results of the experiment are shown in table 3. It can be seen that the single antitumor drug CA or MH has strong inhibition effect on the proliferation of tumor cell CMK and human normal vein endothelial cells.
The drug carrier and the two drugs are combined to form a drug-carrying system, and the inhibition rate of the drug-carrying system on the tumor cells is not improved but is reduced. The conjecture is that the binding capacity between CA or MH and Hu22 is too strong, so that after a medicine carrying system enters tumor cells, the medicine can not be effectively released from a medicine carrier, and the curative effect of the medicine is influenced.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
SEQUENCE LISTING
<110> chemical research institute of Chinese academy of sciences
<120> a novel efficient intelligent targeted drug-loading system
<130> PIDC3182224
<160> 8
<170> PatentIn version 3.5
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Claims (10)
1. A drug carrier, comprising:
An aptamer sequence;
(ii) a variable sequence which can form a G-quadruplex structure; and
A connecting sequence, wherein two ends of the connecting sequence are respectively connected with the aptamer sequence and the variable structure sequence;
Wherein a portion of the aptamer sequence is complementarily paired with at least a portion of the linker sequence.
2. The nucleic acid of claim 1, wherein the allosteric sequence has the amino acid sequence of SEQ ID NO: 1.
3. The nucleic acid of claim 1, wherein the aptamer is selected from the group consisting of non-small cell lung cancer cell aptamer S6, nucleolin aptamer AS1411, mucin 1 aptamer Apt, tyrosine aptamer sgc8, and prostate specific membrane antigen aptamer a 10.
4. The nucleic acid of claim 1, wherein the linker sequence comprises:
2-10 bases that are complementarily paired to the aptamer sequence; and
0 to 20 of at least one of adenine, cytosine and thymine.
5. A pharmaceutical composition, comprising:
a pharmaceutical carrier according to any one of claims 1 to 4; and
A medicine is provided.
6. The pharmaceutical composition of claim 5, wherein the drug is selected from at least one of the following:
Drugs having a conjugated plane, preferably drugs having a benzene ring and/or a naphthalene ring;
Drugs with positive charges or metal ions;
A drug having an amino group.
7. the pharmaceutical composition of claim 5, wherein the drug is selected from at least one of the following: anthraquinone antibiotics, cyclophosphamide, cisplatin, 5-fluorouracil and paclitaxel.
8. The pharmaceutical composition of claim 5, wherein the allosteric sequence binds to the drug;
Optionally, the binding is covalent bonding.
9. The pharmaceutical composition of claim 5, wherein the drug is separated from the drug carrier when the pharmaceutical composition is in an environment having a potassium ion concentration of at least 100mM,
The drug is associated with the drug carrier when the pharmaceutical composition is in an environment having a sodium ion concentration of at least 100 mM.
10. Use of a pharmaceutical carrier according to any one of claims 1 to 4 in the preparation of a pharmaceutical composition according to any one of claims 5 to 9.
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| CN113491773A (en) * | 2020-04-03 | 2021-10-12 | 湖南大学 | Artemisinin derivative aptamer drug conjugate and preparation method and application thereof |
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| CN114934047A (en) * | 2022-06-24 | 2022-08-23 | 中国科学院化学研究所 | High-drug-loading-rate targeted drug loading system and construction method and application thereof |
| CN115990264A (en) * | 2022-12-21 | 2023-04-21 | 中国科学院基础医学与肿瘤研究所(筹) | PTK7 targeted aptamer coupled drug |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113491773A (en) * | 2020-04-03 | 2021-10-12 | 湖南大学 | Artemisinin derivative aptamer drug conjugate and preparation method and application thereof |
| CN113491773B (en) * | 2020-04-03 | 2022-09-30 | 湖南大学 | Artemisinin derivative aptamer drug conjugate and preparation method and application thereof |
| CN114574496A (en) * | 2020-12-01 | 2022-06-03 | 上海交通大学医学院附属仁济医院 | Aptamer sgc8 modified by nucleoside derivative |
| CN114574496B (en) * | 2020-12-01 | 2024-04-09 | 上海交通大学医学院附属仁济医院 | Nucleoside derivative modified aptamer sgc8 |
| CN114934047A (en) * | 2022-06-24 | 2022-08-23 | 中国科学院化学研究所 | High-drug-loading-rate targeted drug loading system and construction method and application thereof |
| CN114934047B (en) * | 2022-06-24 | 2023-11-03 | 中国科学院化学研究所 | High drug-loading targeted drug-loading system and construction method and application thereof |
| CN115990264A (en) * | 2022-12-21 | 2023-04-21 | 中国科学院基础医学与肿瘤研究所(筹) | PTK7 targeted aptamer coupled drug |
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