WO2020150915A1 - 核酸-药物结合物、药物递送系统及其制备方法和应用 - Google Patents
核酸-药物结合物、药物递送系统及其制备方法和应用 Download PDFInfo
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- A61K47/6927—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
- A61K47/6929—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
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Definitions
- the invention belongs to the field of biomedicine, and particularly relates to a nucleic acid-drug conjugate based on nucleic acid phosphorothioate modification, a drug delivery system, and a preparation method and application thereof.
- Chemotherapy is one of the important methods of tumor treatment.
- most chemotherapeutic drugs have defects such as poor water solubility, non-targeting, high blood clearance and even serious toxic side effects, resulting in low bioavailability (Nat. Rev. Cancer) 2006,6,789.), and long-term use will produce drug resistance, which brings certain limitations to their clinical application.
- nano-drug delivery systems based on polymers or inorganic nanoparticles to improve the properties of chemotherapeutic drugs and promote their therapeutic effects, such as micelles, vesicles, and lipids. Plastids, albumin nanoparticles, microbubbles, etc. (Science 2004, 303, 1818.).
- nano-carrier encapsulated chemotherapy drugs mainly include physical embedding methods and chemical binding methods.
- the drug conjugate strategy has become another hot spot in drug delivery. .
- materials used for drug conjugates including polymers (J.
- nucleic acid can meet the three conditions of biocompatibility, biodegradability and low immunogenicity at the same time, and has other specific molecular characteristics such as targeting and specific molecules.
- the recognition function and the precise controllability of nanostructures formed by self-assembly have attracted more and more attention in the field of biomedicine.
- nucleic acid nanostructures J.Polym.Sci.2017,35,1.
- polyhedrons and origami structures for drug delivery is also endless, such as the use of doxorubicin (DOX) to insert DNA double helix structure.
- DOX doxorubicin
- the delivery of DOX has achieved good anti-tumor effects both in vivo and in vitro.
- this nucleic acid insertion method to physically encapsulate drugs not only limits the drugs, but also their in vivo stability remains to be studied.
- nucleic acids and chemotherapeutics into DNA nanostructures through functional groups modified at the ends of nucleic acid sequences or through polymers linked to DNA to achieve drug delivery.
- the former is limited by the number of functional groups and the drug loading is relatively low, while the latter may cause certain biocompatibility problems due to the polymer chain segment, and these two methods often require more complicated synthetic processes.
- the first objective of the present invention is to provide a nucleic acid-drug conjugate based on nucleic acid phosphorothioate modification to achieve accurate and controllable grafting and efficient delivery of chemotherapeutics, and to solve the following shortcomings of the current drug conjugate delivery system : (1) The three major requirements of carrier material biocompatibility, in vivo degradability and low immunogenicity can not be met at the same time; (2) It is difficult to precisely control the drug grafting site and drug loading; (3) It can be grafted There are some limitations on the number of drugs that can not achieve the universality of nucleic acid as a carrier material, and it is difficult to reduce the cost of nucleic acid drug delivery; (4) a more complicated synthesis process is required; (5) the reasonable and simple construction of a multifunctional drug delivery system cannot be achieved.
- the second objective of the present invention is to provide a drug delivery system, which is a drug-loaded nano-system formed by self-assembly of the aforementioned nucleic acid-drug conjugate.
- the third object of the present invention is to provide a method for preparing the above-mentioned nucleic acid phosphorothioate modified nucleic acid-drug conjugate.
- the fourth object of the present invention is to provide a method for preparing the above-mentioned drug delivery system.
- the fifth object of the present invention is to provide a use of the nucleic acid-drug conjugate and drug delivery system based on the above in the preparation of tumor therapeutic drugs based on nucleic acid as a carrier.
- the sixth object of the present invention is to provide a drug, which includes the drug delivery system formed by the aforementioned nucleic acid-drug conjugate.
- a nucleic acid-drug conjugate based on phosphorothioate modified nucleic acid comprising a phosphorothioate modified nucleic acid backbone and a drug molecule grafted on the nucleic acid backbone, and the grafting is through sulfur on the nucleic acid backbone.
- the phosphorothioate group reacts with the modified group on the drug molecule that can react electrophilically with the phosphorothioate.
- the nucleic acid backbone and the drug molecule form a functional nucleic acid-drug conjugate (ie, a nucleic acid-drug conjugate), and the nucleic acid-drug conjugate can be self-assembled to obtain a drug-loaded nanosystem.
- the site and number of the phosphorothioate modification can be adjusted and controlled as required, and the phosphorothioate is continuously modified at a certain end of the nucleic acid sequence, and/or in the nucleic acid sequence Selective modification of the intermediate base sequence, the modification mode is multiple modification or single modification.
- the phosphorothioate modified nucleic acid is prepared by a solid-phase synthesis method, so that the position and quantity of the thiomodification can be adjusted.
- sequence and segment types of the oligonucleotides of the phosphorothioate modified nucleic acid backbone can be independently designed, and can be further assembled through molecular recognition to obtain a controllable DNA nanostructure, the nucleic acid-drug combination
- Both the substance and its assembly structure can be used as a new type of drug delivery system to prepare a controllable drug delivery system.
- the nucleic acid backbone can be designed with different positions and numbers of thiomodifications according to different requirements, for example, nucleic acids used for the assembly of gels and tetrahedral structures, due to the consideration of steric hindrance after modification and their base
- the effect of pairing is to set a thiomodification site every 2 to 3 bases on the nucleic acid backbone; the nucleic acid used for micellar assembly considers the mechanism of micellar assembly and tends to carry out continuous thiomodification at one end of the nucleic acid sequence , And further prepare a block-type nucleic acid containing a phosphodiester bond and a phosphorothioate bond.
- the drug molecule introduces a group capable of electrophilic reaction with phosphorothioate through a simple esterification or acylation reaction.
- the drug molecule also introduces a cleavable responsive chemical bond
- the cleavable responsive chemical bond may be a disulfide bond, an acylhydrazone bond, an ester bond, etc., but is not limited to the responsive chemical bond listed above.
- the group that is modified on the drug molecule and can react electrophilically with phosphorothioate is selected from one or more of the following: 1) Bromine-containing or iodine-containing functional groups, such as iodine Substituted or brominated acetyl compound, ⁇ -bromo- ⁇ , ⁇ -unsaturated carbonyl, benzyl bromide or bromomaleimide; 2) maleimide group; 3) aziridinyl sulfonamide group , But not limited to the above.
- the drug molecule is selected from anticancer drugs such as paclitaxel, camptothecin, cisplatin, docetaxel, chlorambucil, methotrexate, doxorubicin, cisplatin prodrugs, etc., but not Limited to the above, cancer-targeted drug molecules such as erlotinib, imatinib, gefitinib, sorafenib, etc., but not limited to the above listed ones.
- anticancer drugs such as paclitaxel, camptothecin, cisplatin, docetaxel, chlorambucil, methotrexate, doxorubicin, cisplatin prodrugs, etc.
- cancer-targeted drug molecules such as erlotinib, imatinib, gefitinib, sorafenib, etc., but not limited to the above listed ones.
- the drug molecule is a bromo-modified drug molecule containing a disulfide bond.
- carbonylethyl bromide and benzyl bromide structures can be introduced through a simple chemical reaction to achieve integration of the nucleic acid backbone modified with the phosphorothioate backbone. Further reaction, and the introduction of disulfide bonds in the process can also achieve redox release of the drug.
- this process is not limited to carbonyl ethyl bromide and benzyl bromide structures.
- the drug molecules grafted onto the nucleic acid skeleton are functional drug molecules, fluorescent probe molecules, or cell targeting molecules.
- the drug molecule is an anti-tumor drug.
- the drug molecules of the present invention are not limited to anti-tumor drugs. Drugs for the treatment of other diseases or drug molecules for imaging can also be modified by this method; in some specific embodiments, the anti-tumor drugs are Tonine or paclitaxel.
- the anti-tumor drugs of the present invention are not limited to camptothecin and paclitaxel. Other modifiable drugs are, for example, cisplatin, docetaxel, chlorambucil, methotrexate, doxorubicin and the like.
- the type and sequence of the nucleic acid backbone are not limited.
- both deoxyribonucleic acid sequences and ribonucleic acid sequences can be selected; in terms of sequence requirements, non-functional common base sequences can be selected, including simple nucleic acid sequences composed of one base and those that can be used in nucleic acids Complicated nucleic acid sequence assembled with precise structure; functional nucleic acid sequence can also be selected, the functional nucleic acid sequence is selected from antisense nucleic acid sequence, aptamer sequence, nuclease sequence, small interfering RNA, messenger RNA, microRNA, long One of stranded non-coding RNA, small hairpin RNA, guide RNA for gene editing, and circular RNA.
- the nucleic acid molecule grafted with the drug maintains its base complementary pairing properties, and by matching other functional nucleic acid sequences with this property, it gives the nucleic acid-drug conjugate drug delivery system targeting and imaging functions to prepare multifunctional nucleic acids- A drug conjugate drug delivery system, wherein the functional nucleic acid for pairing is selected from nucleic acid aptamers, antisense nucleic acid sequences, fluorescent molecule modified nucleic acid sequences, functional polypeptide modified nucleic acid sequences, and targeted galactose modified nucleic acids A kind of sequence.
- the present invention also provides a drug delivery system, which is a drug-loaded nano-system formed by self-assembly of the aforementioned nucleic acid-drug conjugate.
- the form of the assembly prepared by using the aforementioned nucleic acid-drug conjugate is not limited, for example:
- Simple nucleic acid-drug macromolecular prodrugs can be prepared by design; the design method can be: reduce the number of phosphorothioates or select small molecules with strong hydrophilicity;
- DNA nanostructures can be designed and prepared, such as drug-loaded nucleic acid polyhedron structures, and are not limited to DNA tetrahedrons, and DNA origami structures of different sizes can be constructed; not limited to DNA, RNA nanostructures can also be selected.
- the design method can be: select a DNA or RNA sequence of a specific sequence, perform thiomodification at a specific position in the nucleic acid backbone, and assemble through base complementary pairing after binding to the drug;
- DNA nanogels can be prepared by design; the design method can be: by selecting a DNA or RNA sequence with a specific sequence, selecting a DNA or RNA sequence with a specific sequence, and preparing Y by thiomodification at a specific position in the nucleic acid backbone Type or i-type assembled bodies, and then further assemble to prepare drug-loaded nanogels;
- the drug-loaded micellar spherical nucleic acid can be prepared by design; the method of design can be: select ordinary or functional nucleic acid sequence, continuously thiomodify at one end, prepare one end of phosphodiester bond structure and one end of phosphorothioate structure Block-type nucleic acids are modified with drugs at the thio-modification site; because the hydrophilic shell of micellar spherical nucleic acids maintains the nature of complementary base pairing, this property can give micellar spherical nucleic acids targeting and imaging Features;
- the above-mentioned drug molecules used for the preparation of precisely assembled DNA nanostructures can choose molecules with weak hydrophobicity and small molecular weight as much as possible, and can reduce the number of thiomodifications of the nucleic acid backbone; and the drug molecules used for micelle assembly A highly hydrophobic molecule, and can increase the number of thiomodifications of the nucleic acid backbone.
- the present invention provides a method for preparing the aforementioned nucleic acid phosphorothioate modified nucleic acid-drug conjugate, which mainly includes the following steps:
- the first step is to prepare a thiomodified nucleic acid molecule; preferably, the thiomodified nucleic acid molecule is prepared by a solid-phase synthesis method, so that the position and quantity of the thiomodification can be adjusted;
- a drug molecule containing a group that can react electrophilically with phosphorothioate is prepared by a chemical reaction method; in a preferred embodiment, the drug molecule further contains a cleavable responsive chemical bond, and more preferably, the drug molecule The broken responsive chemical bond is a disulfide bond, and the electrophilic reaction group is a bromo-modified group.
- a bromo-modified drug molecule containing a disulfide bond by a chemical reaction method, one of the following methods can be used Species: Under the protection of argon, the disulfide bond is connected to the camptothecin drug molecule through triphosgene, and then through the reaction with bromoacetyl bromide, bromocamptothecin drug molecule is obtained; or, through 4-bromomethylbenzene
- the esterification reaction of methanol and dithiodipropionic acid obtains a disulfide bond-containing carboxylic acid structure, and then the esterification reaction of the carboxylic acid and the paclitaxel hydroxyl group obtains the benzyl bromide structure-modified paclitaxel drug molecule;
- the second step is to prepare the nucleic acid-drug conjugate.
- the modified drug molecule is dissolved in an organic solvent, and then an appropriate amount of nucleic acid molecule is added for the reaction.
- the drug molecule in this reaction is greatly excessive relative to the phosphorothioate.
- the reaction is complete After removing the excess small molecules, the nucleic acid-drug conjugate can be obtained after drying.
- the treatment process after the reaction can be: adding a certain volume of aqueous solution (the volume of the aqueous solution can be determined by limited experiments, not detailed here), and then removing excess small molecules by ethyl acetate extraction or ethanol precipitation.
- the nucleic acid-drug conjugate can be obtained after the water phase is dried.
- the grafting efficiency of drug molecules can be controlled by controlling the concentration of nucleic acid molecules, the ratio of drug molecules and phosphorothioate, and whether the reaction solution contains a salt solution.
- the organic solvent is selected from one of the following: the reaction system used when grafting camptothecin is a mixed system of dimethyl sulfoxide and phosphate buffer (volume ratio 4:1); The reaction system used for paclitaxel is dimethyl sulfoxide.
- the reaction temperature and time are: react at 50°C-58°C overnight or longer, but the reaction temperature and time can be changed and are not limited thereto.
- the present invention provides a method for preparing the aforementioned nucleic acid-drug conjugate drug delivery system, which is selected from one of the following methods:
- Preparation of drug-loaded nucleic acid polyhedron structure or nucleic acid gel select nucleic acid sequences that can be complementary paired, modify the nucleic acid-drug conjugate obtained by the above method after phosphorothioate modification at the characteristic site, and place the complementary paired nucleic acid sequence in TAE/Mg 2+ solution was mixed in proportion and prepared by annealing method; taking camptothecin as an example: Four nucleic acid-camptothecin prodrugs with different sequences were mixed in 1 ⁇ TAE/Mg 2+ solution buffer Mix in equimolar amounts, place it at 90°C for 5 minutes and then quickly cool to 4°C to obtain camptothecin-loaded DNA nanotetrahedrons; or
- Preparation of drug-loaded micellar spherical nucleic acid select a segment of continuous phosphodiester bond and a segment of continuous phosphorothioate bond modified block-type nucleic acid, according to the above method to obtain the nucleic acid-drug conjugate, use the dialysis method to prepare drug-loaded Micelles, in which the nucleic acid block that has not been modified by phosphorothioate is used as the hydrophilic shell of the micelle, and the hydrophobic drug is used as the core; specifically, taking paclitaxel as an example: the resulting block structure of nucleic acid-paclitaxel is combined The substance is dissolved in dimethyl sulfoxide, added with an equal volume of water and dialyzed in water overnight to obtain a micellar spherical nucleic acid containing paclitaxel;
- Preparation of drug-loaded multifunctional spherical nucleic acid select the nucleic acid sequence containing the complementary pairing part of the continuous phosphodiester bond block, this nucleic acid sequence has fluorescent molecule modification, targeting aptamer modification, targeting polypeptide modification, and targeting small molecules
- the modification function is to prepare a multifunctional drug-loaded spherical nucleic acid that integrates targeting, imaging, gene therapy and chemotherapy by annealing the micelles prepared in (3) above.
- a functional nucleic acid sequence (targeting or fluorescent modification) containing a complementary pairing portion of the phosphodiester bond block in the spherical nucleic acid is selected, and prepared by an annealing method to integrate targeting, imaging, gene therapy and chemotherapy.
- a multifunctional drug-loaded spherical nucleic acid is selected, and prepared by an annealing method to integrate targeting, imaging, gene therapy and chemotherapy.
- the present invention provides a use of the nucleic acid-drug conjugate and drug delivery system based on the above in preparing nucleic acid nanomedicine and chemotherapeutic drugs based on gene therapy and chemotherapy combined treatment of diseases.
- the present invention provides a drug, which includes the above-mentioned nucleic acid-drug conjugate or a drug delivery system formed thereof.
- the drug is a tumor treatment drug.
- nucleic acid-drug conjugates are obtained, and the drug delivery system is obtained through self-assembly of nucleic acid-drug conjugates, which realizes the purpose of using nucleic acid to deliver drugs.
- thio-modified oligonucleotides are obtained by solid-phase synthesis, without tedious chemical synthesis, and the site and quantity of thio-modification can be adjusted and controlled according to needs, for the later drug loading and drug loading sites Achieved the purpose of precise design;
- the required DNA nanostructure can be obtained by independently designing the sequence and segment types of oligonucleotides, and then a controllable drug delivery system can be prepared;
- the drug-carrying system uses biocompatible nucleic acid as the material, which has lower immunogenicity to the organism, lower metabolic burden, and no toxic side effects;
- This drug-carrying system can further introduce targeting groups and functional nucleic acid sequences to prepare a multifunctional nucleic acid drug-carrying system to achieve the purpose of combining gene therapy and chemotherapy; it can also realize a DNA nanostructure integrating tumor imaging ;
- Figure 1 shows the synthetic route of the bromocamptothecin prodrug and bromopaclitaxel molecules in Example 1 and Example 2;
- Figure 2 is a 1 H NMR spectrum of the prodrug compound 1 in Example 1;
- Figure 3 is an LC-MS spectrum of the prodrug compound 1 in Example 1;
- Figure 4 is a 1 H NMR spectrum of the prodrug compound 2 in Example 1;
- Figure 5 is an LC-MS spectrum of the prodrug compound 2 in Example 1;
- Figure 6 is a UV-Vis spectrophotometric diagram of the camptothecin-modified oligonucleotide prodrug in Example 1;
- Figure 7 is a denaturing gel electrophoresis diagram of the camptothecin-modified oligonucleotide prodrug in Example 1;
- Example 8 is a matrix-assisted laser desorption ionization time-of-flight mass spectrum of the camptothecin-modified oligonucleotide prodrug in Example 1;
- Figure 9 is a 1 H NMR spectrum of DTDP-Bz-Br in Example 2.
- Figure 10 is a 1 H NMR spectrum of PTX-Bz-Br in Example 2.
- Figure 11 is a mass spectrum of PTX-Bz-Br in Example 2.
- Figure 12 is a 31 P NMR spectrum chart of DNA-b-PTX-g-DNA in Example 2;
- Example 15 is a data diagram of hydrodynamic diameter of the DNA tetrahedral origami structure modified by camptothecin in Example 3;
- 16 is an atomic force microscope photograph of the DNA tetrahedral origami structure modified by camptothecin in Example 3;
- 17 is a schematic diagram of the toxicity evaluation of the DNA tetrahedral origami structure modified by camptothecin on cancer cells in Example 3;
- Fig. 18 is a schematic diagram of the cancer cell apoptosis achieved by the camptothecin-modified DNA tetrahedral origami structure in Example 3;
- Figure 19 is a 20% deformed gel electrophoresis diagram of PTX-SNA in Example 4.
- Fig. 21 is a transmission electron microscope image of PTX-SNA in Example 4.
- Figure 22 is a 1% agarose gel electrophoresis diagram of FAM/AS1411/PTX-SNA of the multifunctional spherical nucleic acid in Example 4;
- Figure 23 is an agarose gel electrophoresis diagram of the spherical nucleic acid AS1411/Bcl-2-PTX-SNA where the targeted gene and chemotherapy coexist in Example 4;
- Fig. 24 is an in vitro anti-tumor and reversal of tumor multidrug resistance of various micellar spherical nucleic acids carrying paclitaxel in Example 4.
- the invention provides a nucleic acid-drug conjugate based on nucleic acid phosphorothioate backbone modification, a drug delivery system, and their preparation method and application.
- the invention belongs to the field of biomedicine, and specifically discloses a nucleic acid-drug conjugate based on phosphorothioate modified nucleic acid, a drug delivery system, and a preparation method thereof.
- the nucleic acid-drug conjugate is formed by grafting the phosphorothioate in the nucleic acid phosphorothioate backbone with a drug molecule modified with an electrophilic reactive group that can react with it, wherein different targets can be used to The nucleic acid sequence including the functional nucleic acid is selected.
- the nucleic acid-drug conjugate can be self-assembled into a drug-containing nanocarrier for drug delivery.
- the phosphorothioate nucleic acid backbone used in the present invention can be achieved by simple solid-phase synthesis technology.
- the drug grafting through the phosphorothioate group can precisely control the drug molecules on the nucleic acid backbone.
- the grafting site and its assembly behavior, this method has universal applicability to chemotherapy drugs.
- the present invention uses biocompatible and biodegradable water-soluble nucleic acid macromolecules as a carrier, which can significantly improve the physical and chemical properties and in vivo distribution properties of chemotherapeutic drugs and promote their therapeutic effects. It can also achieve combined therapy of gene therapy and chemotherapy and avoid Complex synthesis and modification steps.
- oligonucleotide sequences with complementary bases are selected, and the thio modification site is in the middle of the backbone, and the number of phosphorothioate groups is TET-A, TET-C: 7; TET-B, TET-D: 8 pcs.
- the sequences of the four oligonucleotides are as follows:
- TET-A 5’-ACATTC*CTAAG*TCTGAAACATTAC*AGCT*TGCT*ACACGAGAAGAGC*CGCC*ATAGTA-3’;
- TET-B 5’-TATCA*CCAG*GCAG*TTGACAGTGTAGC*AAGC*TGTA ATAGATGCG*AGGG*TCCA*ATAC-3’;
- TET-C 5’-TCA ACTG*CCTG*GTGATA AAACGACAC*TACG*ACTA*TGGC*GGCT*CTTC-3’;
- TET-D 5’-TTCAG*ACTT*AGGA*ATGTGCTTCCC*ACGT*AGTG*TCGTTTGTA TTGG*ACCC*TCGCAT-3’;
- camptothecin bromide prodrug compound 2 Take camptothecin bromide prodrug compound 2 and dissolve it in 80 ⁇ L dimethyl sulfoxide solution, add 20 ⁇ L phosphorothioate-modified oligonucleotide phosphate buffer, the oligonucleotide concentration is 350 ⁇ M (phosphorothioate group and The ratio of compound 2 is 1:50), placed at 55° C., shaking for 20 hours. After the reaction, the excess compound 2 in the reaction was extracted with ethyl acetate multiple times, evaporated to dryness, and re-dissolved in ultrapure water to obtain the camptothecin-modified oligonucleotide prodrug.
- the obtained 4 kinds of base complementary camptothecin modified oligonucleotide prodrug solutions were detected by UV spectrophotometer.
- the characteristic absorption peak of camptothecin molecule appeared at 365nm, as shown in the figure 6 shown.
- the DNA sequence used in this example is a block-type DNA nucleic acid sequence, near the 5'end is a phosphodiester bond modified DNA (26 bases), and near the 3'end is a continuous phosphorothioate bond modified DNA (19 Bases, 18 phosphorothioate bonds).
- the nucleic acid sequence is as follows:
- the synthesis method is: dissolving PTX-Bz-Br in DMSO, adding DNA, and shaking the reaction overnight at 55°C. Then after adding water, extracting with ethyl acetate to remove the excess PTX-Bz-Br in the reaction, concentrating and evaporating to dryness, the block nucleic acid-paclitaxel molecule is obtained. The successful grafting was verified by NMR phosphorus spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, as shown in Figures 12 and 13.
- FIG. 14 Compared with the unmodified DNA polyhedron, the size has increased, as shown in Figure 14. Shown. Dynamic light scattering detection shows that the hydrated particle size of the drug-modified DNA polyhedron is about 24 nm, which is about 10 nm higher than that of the unmodified DNA polyhedron, as shown in Figure 15.
- Figure 16 is a drug-modified DNA polyhedron photographed by an atomic force microscope with a particle size of about 18 nm.
- the prepared DNA polyhedron modified by thio-oligonucleotides can be effectively taken up by tumor cells to produce a cancer cell killing effect similar to or even better than the original drug.
- the DNA polyhedron modified by thio-oligonucleotide drugs of the present invention can kill tumor cells by inducing apoptosis of cancer cells.
- the Annexin V-FITC/PI method was used to detect tumor cell apoptosis.
- the results are shown in Figure 18.
- the cells co-incubated with the drug-modified polyhedron can achieve It has a good effect of inducing apoptosis of cancer cells and causes a similar rate of apoptosis to the original drug. It is proved that the way of drug delivery through sulfur-modified oligonucleotides can achieve rapid drug release, induce tumor cell apoptosis, and ultimately achieve the purpose of treating cancer.
- the nucleic acid sequence is as follows:
- PolyA 20 -FAM 5'-AAAAAAAAAAAAAAAAAA-FAM-3';
- PolyA 20- AS1411 5'-AAAAAAAAAAAAAAAAAAGGTGGTGGTGGTTGTGGTGGTGGTGG-3'.
- the Bcl-2-b-( PS DNA-g-PTX) and PO T 26 -b-( PS DNA-g-PTX) were dialyzed together in proportion to the obtained SNA, and mixed with polyA 20- AS1411 in proportion to 1 ⁇ TAE /Mg 2+ , and then annealed from 65°C to room temperature to get AS1411/Bcl-2-PTX-SNA with targeted genes and chemotherapy.
- the assembly was characterized by 0.5% agarose gel, as shown in Figure 23.
- micellar spherical nucleic acid conjugated with paclitaxel In vitro antitumor and reversal of tumor multidrug resistance of micellar spherical nucleic acid conjugated with paclitaxel
- PTX-SNAs prepared in Examples 4.1-4.3 were incubated with tumor cells for 72 hours, the cell survival rate was detected by MTT, and the results are shown in Figure 24.
- MCF-7 and HeLa cells are sensitive tumor cells, and L929 cells are normal cells used to characterize the anti-tumor effect of targeting AS1411/PTX-SNA; HeLa/PTX are paclitaxel-resistant cells used to characterize AS1411/Bcl- 2-PTX-SNA reverses the effect of tumor resistance.
- Experimental results show that AS1411/PTX-SNA modified by targeting molecules has a better targeting function on tumor cells, and the tumor cell killing effect is better than that of non-targeted PTX-SNA.
- the above-mentioned embodiment of the present invention firstly obtains the DNA tetrahedral origami structure of the drug-carrying system, and the hydrated nanometer particle size is 20 nanometers.
- the extremely hydrophobic camptothecin drug molecule is accurately and accurately grafted into the oligonucleotide chain segment, which improves the solubility of the camptothecin drug and forms a water-soluble nano-prodrug, which is realized through a pre-designed DNA sequence
- the assembly of the polyhedral structure achieves the structural controllability of the drug-carrying system.
- the above-mentioned embodiment of the present invention also obtained drug-loaded micelle type spherical nucleic acid with a hydrated particle size of 68 nanometers; the paclitaxel drug was accurately grafted onto the phosphorothioate group of the nucleic acid, using phosphodiester bonds and sulfur
- the phosphodiester bond block type DNA structure and the hydrophobic nature of paclitaxel are used to prepare the micelle structure with the drug as the core and the phosphodiester bond nucleic acid block as the outer shell, and the prepared micelle has a drug loading as high as 53%.
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Abstract
Description
Claims (24)
- 一种基于硫代磷酸酯修饰核酸的核酸-药物结合物,其特征在于,包括硫代磷酸酯修饰的核酸骨架与接枝在所述核酸骨架上的药物分子,所述接枝通过所述核酸骨架上的硫代磷酸酯基团与所述药物分子上修饰的可与硫代磷酸酯发生亲电反应的基团进行反应实现。
- 如权利要求1所述的核酸-药物结合物,其特征在于,所述硫代磷酸酯修饰的核酸骨架上,硫代修饰的位点和数量能够根据需要调整和控制,硫代磷酸酯在核酸序列的某一端进行连续修饰,和/或在核酸序列的中间碱基序列选择性修饰,修饰方式为多修饰或单修饰。
- 如权利要求1或2所述的核酸-药物结合物,其特征在于,通过固相合成方法来制备硫代磷酸酯修饰的核酸骨架。
- 如权利要求1或2所述的核酸-药物结合物,其特征在于,所述硫代磷酸酯修饰的核酸骨架的寡核苷酸的序列和链段种类能够自主设计,并能够进一步通过分子识别组装得到可控的DNA纳米结构,所述的核酸-药物结合物及其组装结构均能够用作新型药物递送系统。
- 如权利要求2所述的核酸-药物结合物,其特征在于,用于凝胶和四面体结构组装的核酸,在核酸骨架上每隔2到3个碱基设置一个硫代修饰位点;而用于胶束组装的核酸,在核酸序列的一端进行连续硫代修饰,进而制备含有磷酸二酯键和硫代磷酸酯键的嵌段型核酸。
- 如权利要求1所述的核酸-药物结合物,其特征在于,所述药物分子通过简单的酯化或者酰化反应引入可与硫代磷酸酯发生亲电反应的基团。
- 如权利要求1或6所述的核酸-药物结合物,其特征在于,所述药物分子还引入了可断裂的响应型化学键。
- 如权利要求1所述的核酸-药物结合物,其特征在于,所述的修饰在药物分子上并可与硫代磷酸酯发生亲电反应的基团选自以下的一种或几种:1)含溴或含碘的功能性基团;2)马来酰亚胺基团;3)吖丙啶基磺胺基团。
- 如权利要求1所述的核酸-药物结合物,其特征在于,所述药物分子选自抗癌药物或癌症靶向药物分子的一种或几种。
- 如权利要求1所述的核酸-药物结合物,其特征在于,所述核酸骨架上接枝的药物分子为功能性药物分子或荧光探针分子、细胞靶向分子。
- 如权利要求1所述的核酸-药物结合物,其特征在于,所述核酸骨架的种类选自脱氧核糖核酸序列或核糖核酸序列;所述核酸骨架的序列,选自如下的一种或几种:非功能性的普通碱基序列,包括由一种碱基构成的单纯核酸序列和可用于核酸精确结构组装的复杂核酸序列;功能性核酸序列,所述功能性核酸序列选自反义核酸序列、核酸适配体序列、核酸酶序列、小干扰RNA、信使RNA、微小RNA、长链非编码RNA、小发夹RNA、用于基因编辑向导RNA、环状RNA中的一种。
- 如权利要求1所述的核酸-药物结合物,其特征在于,接枝药物后的核酸分子保持其碱基互补配对的性质,通过此性质配对其他功能性核酸序列赋予核酸-药物结合物药物输送体系靶向和成像功能,制备多功能性的核酸-药物结合物药物输送体系,其中,用于配对的功能性核酸选自核酸适配体、反义核酸序列、荧光分子修饰的核酸序列、功能性多肽修饰的核酸序列、靶向半乳糖修饰的核酸序列等的一种。
- 一种药物递送系统,其特征在于,为由权利要求1-12中任一所述的核酸-药物结合物自组装形成的载药纳米体系。
- 如权利要求13所述的药物递送系统,其特征在于,根据所选药物分子和核酸序列的不同,通过不同方法能够制备不同的药物递送体系,所述的药物递送体系选自简单的链状核酸-药物大分子药物、精确组装的DNA纳米结构、DNA纳米凝胶、载药胶束型球形核酸纳米结构、载药核酸多面体结构或载药核酸水凝胶。
- 如权利要求14所述的药物递送系统,其特征在于,上述用于制备精确组装的DNA纳米结构的药物分子选择疏水性弱、分子量小的分子,和/或减少核酸骨架硫代修饰的数量;而用于胶束组装的药物分子选择疏水性强的分子,和/或可增加核酸骨架硫代修饰的数量。
- 一种权利要求1-12中任一所述的核酸-药物结合物的制备方法,其特征在于,包括:第一步、制备硫代修饰的核酸分子;通过化学反应方法制备含有可与硫代磷酸酯发生亲电反应的基团的药物分子;以上制备核酸分子和制备药物分子的步骤没有先后顺序;第二步、制备核酸-药物结合物,将修饰的药物分子溶于有机溶剂中,然后加入适量的核酸分子进行反应,此反应中的药物分子相对于硫代磷酸酯基团是大大过量的,反应完之后除去多余的小分子,干燥后即可得到核酸-药物结合物。
- 如权利要求16所述的核酸-药物结合物的制备方法,其特征在于,通过固相合成方法制备硫代修饰的核酸分子。
- 如权利要求16所述的核酸-药物结合物的制备方法,其特征在于,所述药物分子的修饰采用如下方法中的一种:(1)通过两步酯化反应制备含有二硫键的羰乙基溴的药物分子;或(2)通过两步酯化反应制备含有二硫键的苄溴修饰的药物分子。
- 如权利要求16所述的核酸-药物结合物的制备方法,其特征在于,所述药物分子还引入了可断裂的响应型化学键,所述可断裂的响应型化学键为二硫键,所述与硫代磷酸酯发生亲电反应的基团为溴代修饰基团;通过化学反应方法制备含有二硫键的溴代修饰的药物分子,使用以下方法中的一种:在氩气保护下,通过三光气将二硫键接入喜树碱药物分子,再通过与溴乙酰溴的反应,得到溴代喜树碱药物分子;或,通过4-溴甲基苯甲醇与二硫代二丙酸的酯化反应得到含有二硫键的羧酸结构,再通过羧酸和紫杉醇2’位羟基的酯化反应得到苄溴结构修饰的紫杉醇药物分子。
- 如权利要求16所述的核酸-药物结合物的制备方法,其特征在于,通过控制核酸分子浓度、药物分子与硫代磷酸酯基团的比例以及反应溶液中是否含有盐溶液来控制药物分子的接枝效率。
- 如权利要求16所述的核酸-药物结合物的制备方法,其特征在于,所述有机溶剂选自如下的一种:接枝喜树碱时所用的反应体系是二甲基亚砜与磷酸缓冲液的混合体系中,体积比4:1;接枝紫杉醇所用的反应体系是二甲基亚砜;所述反应的温度和时间是:在50℃-55℃反应,过夜或更长。
- 一种权利要求13-15中任一所述的核酸-药物结合物药物递送系统的制备方法,其特征在于,选自以下方法中的一种:(1)载药核酸大分子药物分子制备:采用直接溶解法制备,将核酸-药物结合物直接溶于水溶液或盐溶液,制备载药核酸大分子前药;(2)载药核酸多面体结构或核酸凝胶的制备:选择可互补配对的核酸序列,在特点位点进行硫代磷酸酯修饰后按照权利要求16-21中任一所述的方法得到核酸-药物结合物,将互补配对的核酸序列在TAE/Mg 2+溶液中按比例混合,通过退火的方法制备;(3)载药胶束型球形核酸的制备:选择一段连续磷酸二酯键和一段连续硫代磷酸酯键修饰的嵌段型核酸,按照权利要求16-21中任一所述的方法得到核酸-药物结合物,利用透析法制备载药胶束,其中,未经过硫代磷酸酯修饰的核酸嵌段作为胶束的亲水外壳,疏水性药物作为内核;(4)载药多功能球形核酸的制备:选择含有与连续磷酸二酯键嵌段互补配对部分的核酸序列,此核酸序列具有荧光分子修饰、靶向适配体修饰、靶向多肽修饰、靶向小分子修饰功能,通过将上述(3)中所制备的胶束,通过退火的方法制备集靶向、成像、基因治疗和化疗于一身的多功能载药球形核酸。
- 一种权利要求1-12中任一所述的核酸-药物结合物及权利要求13-16中任一所述的药物递送系统在制备基于基因疗法、化学疗法联合治疗疾病的核酸纳米药物和化疗药物中的用途。
- 一种药物,其特征在于,包括权利要求1-12中任一所述的核酸-药物结合物或权利要求13-16中任一所述的药物递送系统。
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