WO2019223293A1 - 骨架整合核苷类似物药物的功能性核酸及其衍生物和应用 - Google Patents

骨架整合核苷类似物药物的功能性核酸及其衍生物和应用 Download PDF

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WO2019223293A1
WO2019223293A1 PCT/CN2018/121076 CN2018121076W WO2019223293A1 WO 2019223293 A1 WO2019223293 A1 WO 2019223293A1 CN 2018121076 W CN2018121076 W CN 2018121076W WO 2019223293 A1 WO2019223293 A1 WO 2019223293A1
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nucleic acid
integrated
nucleoside analog
functional nucleic
skeleton
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French (fr)
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张川
马媛
牟全兵
朱新远
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上海交通大学
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    • 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
    • C12N15/1135—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 against oncogenes or tumor suppressor genes
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    • A61K47/69—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
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    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/318—Chemical structure of the backbone where the PO2 is completely replaced, e.g. MMI or formacetal

Definitions

  • the invention belongs to the technical field of biomedicine, and particularly relates to a functional nucleic acid of a skeleton-integrated nucleoside analogue drug and a derivative thereof, and a preparation method and application thereof.
  • tumor cells develop drug resistance, such as enhanced anti-apoptotic capacity and increased drug pumping rate (Nat. Rev. Cancer 2012, 12, 494-501.).
  • tumor cells will overexpress anti-apoptotic proteins in order to avoid the harm of chemotherapy drugs, thereby inhibiting the anti-tumor activity of chemotherapy drugs.
  • tumor cells can also up-regulate the expression of drug transporters.
  • Transporters have the ability to pump chemotherapeutic drugs out of cells. Therefore, up-regulation of transporters can reduce the drug concentration in tumor cells, thereby reducing drug efficacy.
  • nano drug delivery systems for the coordinated delivery of gene drugs and chemotherapy drugs (Nat. Nanotechnol. 2011, 6, 658-667.). This is because the nano delivery system has a long half-life and is stable in vivo. Good sex, fast cell uptake, and small side effects (Nat. Biotechnol. 2016, 34, 414-418.). So far, researchers have developed many kinds of nano delivery vehicles, such as micelles (J.Am.Chem.Soc.2016,138,10834-10837.), Liposomes (Proc.Natl.Acad.Sci USA 2010, 107, 10737-10742.), And nucleic acid assemblies (J. Am. Chem. Soc. 2013, 135, 18644-18650.) And the like.
  • nanocarrier-based gene drug and chemotherapeutic drug delivery systems have been carefully designed, due to the large differences in the physicochemical properties of both gene drugs and chemotherapeutic drugs, these nanocooperative delivery systems still face many challenges, including:
  • the first object of the present invention is to provide a functional nucleic acid of a skeleton-integrated nucleoside analog drug, to realize the combined delivery of gene drugs and chemical drugs, to solve the problems existing in the existing combined delivery technology, including: (1) gene drugs and The hydrophilicity and hydrophobicity of chemotherapeutic drugs are often greatly different, so it is difficult to design an ideal nanocarrier to efficiently load two substances at the same time; (2) the amount and proportion of gene drugs and chemotherapeutic drugs in the carrier are difficult to accurately control; (3) The release time sequence of gene drugs and chemotherapy drugs is difficult to precisely control.
  • a second object of the present invention is to provide a method for preparing a functional nucleic acid of the above-mentioned skeleton-integrated nucleoside analog drug.
  • a third object of the present invention is to provide a functional nucleic acid derivative based on the above-mentioned skeleton-integrated nucleoside analog drug, so as to achieve efficient delivery of the functional nucleic acid of the skeleton-integrated nucleoside analog drug in a cell.
  • a fourth object of the present invention is to provide a method for preparing a functional nucleic acid derivative based on the aforementioned skeleton-integrated nucleoside analog drug, so as to realize efficient delivery of the functional nucleic acid of the skeleton-integrated nucleoside analog drug in a cell.
  • a fifth object of the present invention is to provide a functional nucleic acid of a skeleton-integrated nucleoside analogue drug and a derivative thereof for use in preparing a nucleic acid drug and a chemotherapeutic drug based on gene therapy, chemotherapy combined treatment of diseases.
  • a functional nucleic acid of a skeleton-integrated nucleoside analog drug wherein after the nucleoside analog drug is chemically modified, it is integrated into the backbone of the functional nucleic acid by replacing natural nucleotides in the functional nucleic acid structure.
  • the chemical modification method of the nucleoside analog drug is that the nucleoside analog drug is modified by phosphoramidite or triphosphate.
  • the nucleoside analog drug is chemically modified, it is integrated into the functional nucleic acid backbone through solid-phase synthesis technology or in vitro enzyme transcription technology or PCR amplification technology.
  • the gene-to-drug ratio can be precisely adjusted by adjusting the number of natural nucleotides replaced by the drug, which solves the problem that the loading amount and ratio of the gene drug and the chemotherapeutic drug in the carrier are difficult to accurately control.
  • the functional nucleic acid in the functional nucleic acid of the backbone-integrated nucleoside analog drug is selected from the group consisting of antisense DNA, small interfering RNA (siRNA), messenger RNA (mRNA), One of micro RNA, long non-coding RNA (lncRNA), small hairpin RNA (shRNA), guide RNA for gene editing (sgRNA, etc.), and circular RNA (circRNA), but it is not limited to one These several functional nucleic acids.
  • the backbone integrates a functional nucleic acid of a nucleoside analog drug, wherein the nucleoside analog drug is selected from one of the following drugs:
  • Purine analogs may be mercaptopurine, thioguanine, azathioprine, or 8-Azaguanin;
  • Guanosine analogs which may be Nelarabine or Forodesine;
  • Cytidine analogs can be Cytarabine, Ancitabine, Gemcitabine, Enocitabine, 5-Azacytidine or Deoxyazacytidine (Decitabine);
  • Adenosine analogs can be Fludarabine, Cladribine, Clofarabine or Acadesine;
  • the uridine analog can be Fluorouracil, Carmofur, Tegafur, 5'-Deoxy-5-fluorouridine, Capecitabine or Fluorine Floxuridine.
  • the technical scheme of the present invention is not limited to the aforementioned nucleoside analog drugs.
  • the present invention also provides a method for preparing a functional nucleic acid of a skeleton-integrated nucleoside analog drug, including one of the following methods:
  • a functional nucleic acid of a skeletal-integrated nucleoside analog drug is prepared by an in vitro enzyme transcription method or a PCR amplification technique.
  • the specific preparation process is selected from one of the following:
  • RNA polymerase A mixed solution of RNA polymerase, template DNA, RNA nucleoside triphosphate monomer, triphosphate monomer of nucleoside analog drug, and reaction buffer was reacted at 37 ° C to prepare functional nucleic acid of backbone integrated nucleoside analog drug. ;
  • a mixed solution of DNA polymerase, template DNA, DNA primer, DNA nucleoside triphosphate monomer, nucleoside analog triphosphate monomer, and reaction buffer was reacted at 37 ° C to prepare a skeleton-integrated nucleoside analog drug. Functional nucleic acid.
  • the functional nucleic acid of the above-mentioned skeleton-integrated nucleoside analog drug of the present invention can be made into various derivatives, including but not limited to:
  • the functional nucleic acid of the skeleton-integrated nucleoside analog drug may also be modified with a polymer or a hydrophobic molecule to obtain a derivative and a self-assembled nanostructure of the derivative; or
  • the functional nucleic acid of the skeleton-integrated nucleoside analog drug can also be obtained by self-assembly with a transfection reagent to obtain a composite nanostructure derivative;
  • the functional nucleic acid of the backbone-integrated nucleoside analogue drug is modified by a polymer or a hydrophobic molecule to obtain a functional nucleic acid derivative, and the functional nucleic acid derivative and a transfection reagent are used to obtain a composite nanoparticle through self-assembly. Derivative of structure.
  • Another aspect of the present invention provides a functional nucleic acid derivative of the above-mentioned skeleton-integrated nucleoside analog drug, which is one of the following:
  • a functional derivative formed by combining a functional nucleic acid of a backbone-integrated nucleoside analog drug with a molecular targeting group formed by combining a functional nucleic acid of a backbone-integrated nucleoside analog drug with a molecular targeting group
  • a composite nanostructure obtained by self-assembly of a functional nucleic acid and a transfection reagent of the skeleton-integrated nucleoside analog drug obtained by self-assembly of a functional nucleic acid and a transfection reagent of the skeleton-integrated nucleoside analog drug;
  • the polymer is selected from one or more of polycaprolactone, polyethylene glycol, and polylactic-glycolic acid copolymer. But it is not limited to the above polymers.
  • the hydrophobic molecule is selected from one or more of the following:
  • Phospholipid molecules such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, diphosphatidylglycerol, sphingomyelin, glycosphingolipid, etc .;
  • Alkanes such as pentane, dodecane, octadecane, etc .
  • Sterol molecules such as cholesterol, cortisol, aldosterone, testosterone, estradiol, and vitamin D.
  • it is not limited to the modification of the functional nucleic acid of the backbone-integrated nucleoside analog drug using only the above-mentioned several kinds of hydrophobic molecules.
  • the transfection reagent is selected from polyethyleneimine, polylysine, chitosan, Lipofectamine TM transfection reagent, Lipofectamine TM 2000 transfection reagent, Lipofectamine TM 3000 transfection reagent, Lipofectamine TM RNAiMAX
  • a transfection reagent gold nanoparticles, iron tetroxide nanoparticles, and silicon dioxide nanoparticles. But it is not limited to the above cationic polymers, cationic liposomes, inorganic nanoparticles, and other transfection reagents that can be used for functional nucleic acid loading and transfection.
  • the method for preparing a functional nucleic acid derivative of the above-mentioned skeleton-integrated nucleoside analog drug provided by the present invention mainly includes:
  • nucleic acid aptamers include nucleic acid aptamers, targeting polypeptides, targeted small molecules, and other modified backbone-integrated nucleoside analogue drugs, or
  • the polymer is modified to obtain a functional nucleic acid derivative of a backbone-integrated nucleoside analog drug; wherein the polymer is selected from, for example, one of polycaprolactone, polyethylene glycol, and polylactic acid-glycolic acid copolymer, but
  • the present invention is not limited to using only the aforementioned polymers; or
  • hydrophobic molecule Modification using a hydrophobic molecule to obtain a functional nucleic acid derivative of a backbone-integrated nucleoside analog drug, wherein the hydrophobic molecule is selected from the group consisting of phospholipid molecules, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidyl One of glycerol, phosphatidylinositol, diphosphatidylglycerol, sphingomyelin, and sphingolipid; or an alkane molecule, such as one of pentane, dodecane, octadecane, or the like; or a sterol molecule
  • phospholipid molecules such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidyl One of glycerol, phosphatidylinositol, diphosphatidy
  • the molecularly modified products and transfection reagents generate nanomaterials through a self-assembly process.
  • the transfection reagents include polyethyleneimine, polylysine, chitosan, Lipofectamine TM transfection reagent, Lipofectamine TM 2000 transfection reagent, Lipofectamine One of TM 3000 Transfection Reagent, Lipofectamine TM RNAiMAX Transfection Reagent, Gold Nanoparticles, Ferric Tetraoxide Nanoparticles, Silica Nanoparticles, etc. But it is not limited to the above cationic polymers, cationic liposomes, inorganic nanoparticles, and other transfection reagents that can be used for functional nucleic acid loading and transfection.
  • the method of using a polymer modification includes the following steps:
  • a degradable polymer with an azide group at the end synthesized by ring-opening polymerization and then make the degradable polymer and a diphenylcyclooctyne-modified backbone integrated nucleoside analog drug functional nucleic acid Copper-free catalyzed Click reaction to obtain a functional nucleic acid conjugate that can degrade a polymer-backbone-integrated nucleoside analog drug, which is one of the functional nucleic acid derivatives of the backbone-integrated nucleoside analog drug.
  • the present invention also provides a method for preparing an aqueous solution of a functional nucleic acid derivative of a skeleton-integrated nucleoside analog drug, which includes the following steps:
  • the conjugate of functional nucleic acid that can degrade the polymer-framework integrated nucleoside analog drug is dissolved in dimethyl sulfoxide (DMSO), and the solution is dialyzed in ultrapure water for 24 hours to obtain
  • DMSO dimethyl sulfoxide
  • An aqueous solution of a conjugate that degrades a functional nucleic acid of a polymer-backbone-integrated nucleoside analog drug is an aqueous solution of a derivative of a functional nucleic acid of a backbone-integrated nucleoside analog drug.
  • the invention also discloses the use of the functional nucleic acid of the above-mentioned skeleton-integrated nucleoside analogue drug and its derivative in the preparation of nucleic acid drugs and chemotherapeutic drugs based on gene therapy and chemotherapy combined treatment of diseases.
  • the functional nucleic acid and its derivative of the nucleoside analog drug of the skeleton integration of the present invention can achieve precise adjustment of the ratio of genes to drugs, and specifically can be achieved by adjusting the number of natural nucleotides replaced by drugs;
  • the functional nucleic acid and its derivative of the skeletal-integrated nucleoside analog drug of the present invention can realize the sequential function of genes and drugs, and can maximize the effect of synergistic treatment;
  • the present invention successfully designed a carrier capable of efficiently carrying both a gene drug and a chemotherapeutic agent simultaneously.
  • Figure 1 is the tumor size during the treatment of resistant liver orthotopic transplantation tumors in tumor-bearing nude mice by Bcl-2 antisense nucleotides (F-Bcl-2ASO) with fluorouridine integrated into the skeleton in Example 1 of the present invention The change;
  • Fig. 2 is a tumor-bearing liver after orthotopic transplantation of tumor-bearing nude mice with tumor-bearing orthotopically transplanted tumors of Bcl-2 antisense nucleotides (F-Bcl-2ASO) with integrated fluorouridine in Example 1 of the present invention photo;
  • FIG. 3 is a schematic diagram of a fluorouridine-integrated Bcl-2 antisense nucleotide (F-Bcl-2ASO) in Example 1 of the present invention after treatment of a drug-resistant liver orthotopic transplantation tumor in nude mice bearing tumors, and the resistance is down-regulated.
  • F-Bcl-2ASO fluorouridine-integrated Bcl-2 antisense nucleotide
  • FIG. 4 shows that the Bcl-2 / xL antisense nucleotide (F-Bcl-2 / xL ASO) with fluorouridine integrated into the backbone in Example 2 of the present invention down-regulates the expression of drug resistance-related proteins in drug-resistant cells BEL-7402. Case;
  • FIG. 5 is a synthetic route of a spherical nucleic acid SNA (F-Bcl-2ASO) constructed by a Bcl-2 antisense nucleotide incorporating a fluorouridine backbone in Example 3 of the present invention
  • FIG. 6 is a 20% denatured polyacrylamide of a Bcl-2 antisense nucleotide (F-Bcl-2ASO-DBCO) in which a fluorouridine is integrated into a skeleton of a diphenylcyclooctyne (DBCO) modification in Example 3 of the present invention
  • F-Bcl-2ASO-DBCO Bcl-2 antisense nucleotide
  • DBCO diphenylcyclooctyne
  • Example 7 is a synthetic route of polymer 1 (N 3 -PEG-b-PCL 28 ) in Example 3 of the present invention.
  • Example 8 is a nuclear magnetic resonance proton spectrum of polymer 1 (N 3 -PEG-b-PCL 28 ) in Example 3 of the present invention.
  • FIG. 9 is a Bcl-2 antisense nucleotide (F-Bcl-2ASO-DBCO) integrated with fluorouridine and polymer 1 (N 3 -PEG-b-PCL 28 ) and DBCO modified backbone in Example 3 of the present invention Click on the reaction conjugation synthetic roadmap;
  • FIG. 10 is a 1% non-denaturing agarose gel electrophoresis diagram of a spherical nucleic acid SNA (F-Bcl-2ASO) constructed by Bcl-2 antisense nucleotides with fluorouridine integrated into a backbone in Example 3 of the present invention;
  • FIG. 10 is a 1% non-denaturing agarose gel electrophoresis diagram of a spherical nucleic acid SNA (F-Bcl-2ASO) constructed by Bcl-2 antisense nucleotides with fluorouridine integrated into a backbone in Example 3 of the present invention
  • FIG. 11 is a dynamic light scattering diagram of a spherical nucleic acid SNA (F-Bcl-2ASO) constructed by a Bcl-2 antisense nucleotide incorporating a fluorouridine backbone in Example 3 of the present invention
  • FIG. 12 is a transmission electron microscope image of a spherical nucleic acid SNA (F-Bcl-2ASO) constructed by a Bcl-2 antisense nucleotide incorporating a fluorouridine backbone in Example 3 of the present invention
  • FIG. 13 shows the spherical nucleic acid SNA (F-Bcl-2ASO) constructed by Bcl-2 antisense nucleotides with fluorouridine integrated into the framework in Example 3 of the present invention on orthotopically transplanted tumors of drug-resistant liver in nude mice bearing tumors. Changes in tumor size during treatment;
  • FIG. 14 is a diagram showing the effect of orthotopic transplantation of a spherical nucleic acid SNA (F-Bcl-2ASO) constructed by Bcl-2 antisense nucleotides incorporating fluorouridine into a drug-resistant liver in nude mice in Example 3 of the present invention Photo of tumor-bearing liver after treatment;
  • F-Bcl-2ASO spherical nucleic acid SNA
  • FIG. 15 is a diagram illustrating the effect of orthotopic transplantation of a spherical nucleic acid SNA (F-Bcl-2ASO) constructed by Bcl-2 antisense nucleotides incorporating fluorouridine into a drug-resistant liver in nude mice in Example 3 of the present invention. After treatment, down-regulate the expression of drug resistance-related proteins in drug-resistant tumors;
  • F-Bcl-2ASO spherical nucleic acid SNA
  • FIG. 16 is a 20% denatured polyacrylamide gel electrophoresis of a BCO-2 / xL antisense nucleotide (F-Bcl-2 / xL ASO-DBCO) with a DBCO-modified backbone integrated with fluorouridine in Example 4 of the present invention Figure;
  • FIG. 17 is a 1% non-denaturing agarose gel electrophoresis of a spherical nucleic acid SNA (F-Bcl-2 / xL ASO) constructed with Bcl-2 / xL antisense nucleotides integrated with fluorouridine in Example 4 of the present invention Figure;
  • Example 18 is a spherical nucleic acid SNA (F-Bcl-2 / xL ASO) constructed by Bcl-2 / xL antisense nucleotide integrated with fluorouridine in Example 4 of the present invention down-regulates drug resistance in drug-resistant cells BEL-7402 The expression of related proteins.
  • SNA spherical nucleic acid SNA
  • the input sequence 5'-AAF ACF CCG AAC GFG FCA CGF CCFCCF CAC -3 ' in the solid-phase synthesizer, i.e., the backbone may be synthesized fluorouridine scrambled integrated nucleic acid (F-scramble) As a control .
  • the effect of inhibiting proliferation of drug-resistant tumors in vivo by the Bcl-2 antisense nucleotide (F-Bcl-2ASO) integrated with fluorouridine in the skeleton was evaluated by a model of orthotopic liver transplantation of drug-resistant liver in nude mice bearing tumors.
  • the results are shown in Figure 1.
  • the initial size of the drug-resistant liver transplanted tumors in each treatment group was comparable.
  • the tumor size of the F-Bcl-2ASO treatment group was smaller than that of the other control groups at the seventh and fifteenth days after treatment.
  • Figure 2 is a photo of the tumor-bearing liver taken out of the nude mouse after dissecting the nude mice.
  • the white part of the figure is a drug-resistant liver transplantation tumor. From Figure 2, it can be seen that the tumor size of the F-Bcl-2ASO treatment group is the smallest after the treatment, and the remaining groups The tumor size is only slightly smaller than the PBS control group, which means that F-Bcl-2ASO is the most effective among the above drugs.
  • Bcl-2 antisense nucleotide (F-Bcl-2ASO) integrated with fluorouridine in the skeleton can reverse tumor resistance through gene regulation
  • F-Bcl-2ASO Bcl-2 antisense nucleotide integrated with fluorouridine in the skeleton
  • the measurement results are shown in Fig. 3.
  • the Bcl-2 protein band of the F-Bcl-2ASO treatment group was weaker than that of other groups, which indicates that it can down-regulate the expression of drug-resistant proteins in tumor-bearing nude mice. Therefore, F-Bcl- 2ASO can show the effect of gene therapy in animals and can effectively reverse the drug resistance of drug-resistant tumors.
  • the phosphoramidite monomer and DNA phosphoramidite monomer of the fluorouridine drug are placed in the corresponding position of the DNA solid-phase synthesizer, and the corresponding ordinary microporous glass beads (CPG) are added to the reaction column, and 5 'is input.
  • CPG microporous glass beads
  • Bcl-2 / xL antisense nucleotides (F-Bcl-2 / xL ASO) integrated with fluorouridine in the skeleton were incubated with drug-resistant BEL-7402 cells for 10 hours, and then replaced with normal medium for 48 hours; and F- Scrramble and F were used as a control group, and cells were incubated under the same conditions.
  • the equivalent concentration of F was 10 ⁇ M, and drug-resistant cells without any treatment were used as a negative control group.
  • the protein expression of the target protein Bcl-2 in the cells was determined by Western blot analysis. The results are shown in Figure 4. After incubation of F-Bcl-2 / xL and ASO with drug-resistant cells BEL-7402, the expression of Bcl-2 and Bcl-xL proteins associated with drug resistance can be down-regulated. However, F-scramble and F treatments The expression of Bcl-2 protein in the group was not significantly different from that of the blank control group. Therefore, the Bcl-2 / xL antisense nucleotide (F-Bcl-2 / xL ASO) integrated with fluorouridine in the backbone has a certain degree of resistance to reverse drug-resistant tumors.
  • F-Bcl-2 / xL ASO Bcl-2 / xL antisense nucleotide integrated with fluorouridine in the backbone has a certain degree of resistance to reverse drug-resistant tumors.
  • Bcl-2 antisense nucleotides with fluorouridine integrated into the backbone by solid-phase synthesis method, amino-modified microporous glass beads (NH 2 -CPG) were used to prepare the amino-modified fluorouridine integrated with backbone.
  • Bcl-2 antisense nucleotide F-Bcl-2ASO-NH 2 ).
  • the crude product was purified by multiple extractions with ethyl acetate, ethanol precipitation, and centrifugation.
  • F-Bcl-2ASO-DBCO was reconstituted, and the modification of DNA was characterized by gel electrophoresis, as shown in Fig. 6. As can be seen in the figure, the product showed a single band after electrophoresis. Compared with NH 2 , the product moves slowly in gel electrophoresis, which proves the successful preparation of F-Bcl-2ASO-DBCO.
  • N 3 -PEG-OH molecular weight 2000 azide polyethylene glycol hydroxyl
  • Figure 7 Using stannous octoate as a catalyst and molecular weight 2000 azide polyethylene glycol hydroxyl (N 3 -PEG-OH) as an initiator to initiate the ring-opening polymerization of ⁇ -caprolactone to prepare a block polymer N 3 -PEG -b-PCL ( Figure 7).
  • the specific preparation process is as follows: First, 1.0000 g (0.5 mmol) of N 3 -PEG-OH and 1.7121 g (15 mmol) of anhydrous ⁇ -caprolactone are dissolved in anhydrous toluene.
  • the 1 H NMR spectrum of polymer 1 is shown in FIG. 8.
  • the test solvent is CDCl 3.
  • the assignment of each proton peak is as follows: ⁇ (ppm): 4.22 (t, 2H, -OCH 2 CH 2 OC (O) CH 2 CH 2 CH 2 CH 2 CH 2 O-), 4.05 (t, 56H, -C (O) CH 2 CH 2 CH 2 CH 2 CH 2 O-), 3.64 (s, 174H, -OCH 2 CH 2 O- ), 2.30 (t, 56H, -C (O) CH 2 CH 2 CH 2 CH 2 CH 2 O-), 1.64 (m, 112H, -C (O) CH 2 CH 2 CH 2 CH 2 O- ), 1.37 (m, 56H, -C (O) CH 2 CH 2 CH 2 CH 2 O-).
  • Polymer 1 (N 3 -PEG-b-PCL 28 ) is conjugated with a DBCO-modified backbone to integrate fluorouridine Bcl-2 antisense nucleotides (F-Bcl-2ASO-DBCO) via a classical click reaction, such as Figure 9 shows.
  • N 3 -PEG-b-PCL 28 (200 nmol) is dissolved in 1.2 mL of DMSO solution, and F-Bcl-2ASO-DBCO (400 nmol) is dissolved in 30.0 ⁇ L of water. The reaction was shaken at 58 ° C for 48h.
  • the reaction solution was dialyzed in a dialysis bag with a cut-off molecular weight of 10 kDa to remove DMSO.
  • the N 3 -PEG-b-PCL 28 and F-Bcl-2ASO-DBCO conjugates were gradually assembled to form a spherical shape.
  • Nucleic acid structure SNA F-Bcl-2ASO ( Figure 5).
  • the structure of the purified SNA was characterized by agarose gel electrophoresis.
  • the agarose gel concentration was 1%, and the electrophoretic voltage was 90V.
  • the gel was imaged with a gel imaging system.
  • the characterization results are shown in Fig. 10.
  • the SNA (F-Bcl-2ASO) band is located above the F-Bcl-2ASO-DBCO band of the control group, which proves that the Bcl-2 antisense nucleotide constructed by fluorouridine is integrated into the backbone.
  • Successful preparation of spherical nucleic acid SNA F-Bcl-2ASO).
  • a spherical nucleic acid SNA F-scramble constructed by a scrambled nucleic acid integrated with a fluorouridine backbone can be synthesized as a control.
  • the hydration particle size of the spherical nucleic acid SNA (F-Bcl-2ASO) constructed by Bcl-2 antisense nucleotides integrated with fluorouridine in the backbone was characterized by dynamic light scattering experiments. The test results are shown in Fig. 11, and the SNA (F- Bcl-2ASO) had a hydrated particle size of 17.5 nm. In addition, the transmission electron microscope experimental results shown in FIG. 12 confirmed that the morphology of SNA (F-Bcl-2ASO) was spherical.
  • 3D-OSEM three-dimensional ordered subset maximum expectation method
  • the results are shown in Figure 13.
  • the initial size of the drug-resistant liver transplanted tumors in each treatment group was comparable.
  • the tumor size of the SNA (F-Bcl-2ASO) treatment group was smaller than that of the PBS control group at the seventh and fifteenth days after treatment. There are significant differences.
  • Fig. 14 is a photo of a tumor-bearing liver taken out of a nude mouse after dissecting the nude mice.
  • the white part of the picture is a drug-resistant liver transplantation tumor.
  • Figs. The smallest size, the tumor size in the other groups is only slightly smaller than the PBS control group, which means that SNA (F-Bcl-2ASO) is the most effective of the above drugs.
  • Bcl-2 / xL antisense nucleotides with fluorouridine integrated into the skeleton by solid-phase synthesis method amino-modified microporous glass beads (NH 2 -CPG) were used to prepare the amino-modified fluorouridine integrated with the framework.
  • Bcl-2 / xL antisense nucleotide F-Bcl-2 / xL ASO-NH 2 ).
  • the above-mentioned antisense nucleotide sequence was added to a 30% phosphate buffered DMSO mixed solution, 200 equivalents of DBCO-NHS ester was added, and the reaction was performed at room temperature for 24 hours to obtain a DBCO-modified backbone-integrated fluorouridine Bcl-2 / xL antisense nucleotide (F-Bcl-2 / xL ASO-DBCO).
  • the crude product was purified by multiple extractions with ethyl acetate, ethanol precipitation, and centrifugation.
  • F-Bcl-2 / xL ASO-DBCO was reconstituted, and the modification of DNA was characterized by gel electrophoresis, as shown in FIG. 16. It can be seen in Figure 16 that the product appears as a single band after electrophoresis. Compared with F-Bcl-2 / xL ASO-NH 2 , the product moves slower in gel electrophoresis, which proves that F-Bcl-2 / xL ASO- Successful preparation of DBCO.
  • the polymer 1 (N 3 -PEG-b-PCL 28 ) and the DBCO-modified backbone were used to integrate Bcl-2 / xL antisense nucleotides (F-Bcl-2 / xL ASO- DBCO) is conjugated by a classic click reaction.
  • N 3 -PEG-b-PCL 28 (200 nmol) is dissolved in 1.2 mL of DMSO solution, F-Bcl-2 / xL ASO-DBCO (400 nmol) is dissolved in 30.0 ⁇ L water, and the two solutions are mixed After homogenization, the reaction was shaken at 58 ° C for 48h.
  • reaction solution was dialyzed in a dialysis bag with a cut-off molecular weight of 10 kDa to remove DMSO.
  • the structure of the purified SNA (F-Bcl-2 / xLSO) was characterized by agarose gel electrophoresis.
  • the agarose gel concentration was 1%, and the electrophoretic voltage was 90V.
  • the image was imaged with a gel imaging system.
  • the SNA (F-Bcl-2 / xL ASO) band is located above the F-Bcl-2 / xL ASO-DBCO band in the control group.
  • Successful preparation of spherical nucleic acid SNA (F-Bcl-2 / xL ASO) constructed by sense nucleotides.
  • Spherical nucleic acid SNA (F-Bcl-2 / xLASO) constructed by Bcl-2 / xL antisense nucleotides integrated with fluorouridine in the skeleton reverses tumor resistance through gene regulation
  • Spherical nucleic acid SNA F-Bcl-2 / xLASO constructed with Bcl-2 / xL antisense nucleotides integrated with fluorouridine in the skeleton was incubated with drug-resistant BEL-7402 cells for 10 hours, and then replaced with normal medium for 48 hours SNA (F-scramble) and F were used as a control group, and the cells were incubated under the same conditions.
  • the equivalent concentration of F was 10 ⁇ M, and drug-resistant cells without any treatment were used as a negative control group.
  • the spherical nucleic acid SNA (F-Bcl-2 / xLASO) constructed by Bcl-2 / xL antisense nucleotides integrated with fluorouridine in the backbone can well reverse the drug resistance of drug-resistant tumors.
  • the functional nucleic acid and its derivative of the skeletal integration nucleoside analog drug of the present invention can enter the cell efficiently, and use the functional nucleic acid of the skeletal integration nucleoside analog drug to perform gene regulation function.
  • the functional nucleic acid of the nucleoside analog drug can be degraded by the nuclease and the active ingredient of the nucleoside analog drug can be released by the nuclease, thus playing the role of chemotherapy. Therefore, functional nucleic acids and derivatives of nucleoside analogue drugs integrated with the framework can simply and efficiently realize combined therapy of gene therapy and chemotherapy, and avoid complicated synthetic steps.
  • T7RNA polymerase Y639F was added to a transcription reaction solution for synthetic RNA.
  • the transcription reaction solution contained template DNA (1 ⁇ g), ATP (5 mM), CTP (5 mM), GTP (5 mM), 5-FdUTP (5 mM), and DTT (10 mM). ) And reaction buffer, followed by incubation at 37 ° C for 6h. After the reaction was completed, the product was purified by denaturing a polyacrylamide gel, and the product was recovered, and the target RNA segment was precipitated with ice ethanol at -20 ° C. After centrifuging and reconstituting the RNA, store it in a -80 ° C refrigerator for later use.

Abstract

本发明属于生物医药技术领域，具体公开了一种骨架整合有核苷类似物药物的功能性核酸及其衍生物，以及它们的制备方法，其中，所述衍生物由骨架整合了核苷类似物药物的功能性核酸与聚合物、疏水性分子、转染试剂中的一种缀合或者自组装所得。与现有技术相比，本发明的骨架整合核苷类似物药物的功能性核酸及其衍生物可以高效进入细胞，并利用骨架整合核苷类似物药物的功能性核酸发挥基因调控功能；随后，骨架整合核苷类似物药物的功能性核酸可以被核酸酶降解并释放核苷类似物药物的活性成分，因此起到化学治疗的作用。因此，骨架整合核苷类似物药物的功能性核酸及其衍生物可以简单高效地实现基因治疗、化学治疗的联合治疗，并避免了复杂的合成步骤。

Description

骨架整合核苷类似物药物的功能性核酸及其衍生物和应用 技术领域
本发明属于生物医药技术领域，特别涉及骨架整合核苷类似物药物的功能性核酸及其衍生物、它们的制备方法和应用。
背景技术
化疗过程中，肿瘤细胞产生耐药性是当今癌症治疗领域面临的一个巨大挑战(Nat.Rev.Cancer 2013,13,714-726.)。
导致肿瘤细胞产生耐药性的原因很多，如抗凋亡能力增强、药物泵出速率增加等(Nat.Rev.Cancer 2012,12,494-501.)。例如，化疗时肿瘤细胞为了逃避化疗药物的伤害，会过量表达抗凋亡蛋白，从而抑制化疗药物的抗肿瘤活性。此外，肿瘤细胞也可以上调药物转运蛋白的表达，转运蛋白具有将化疗药物泵出细胞外的能力，因而转运蛋白上调会降低肿瘤细胞内的药物浓度，从而降低药效。
为了解决肿瘤耐药性问题，目前主要采用基因和化疗联合治疗的方法，首先利用基因治疗逆转耐药相关基因的表达，使得肿瘤细胞恢复对化疗药物的敏感性，从而促使化疗药物有效抑制肿瘤增殖(Proc.Natl.Acad.Sci.U.S.A.2010,107,10737-10742.)。
然而，基因药物和化疗药物直接联合使用时存在一定的问题，包括：两者的血液半衰期都较短，基因在体内不稳定且细胞内摄效率低，缺乏肿瘤靶向性，具有潜在的毒副作用等(Nat.Rev.Genet.2014,15,541-555.)。
为了改善上述问题，近年来科研人员尝试将纳米药物输送系统用于基因药物和化疗药物的协同递送(Nat.Nanotechnol.2011,6,658-667.)，这是因为纳米递送系统具有半衰期长、体内稳定性好、细胞摄取速度快而且副作用小等优点(Nat.Biotechnol.2016,34,414-418.)。到目前为止，科研工作者已发展了许多种类的纳米递送载体，如胶束(J.Am.Chem.Soc.2016,138,10834-10837.)、 脂质体(Proc.Natl.Acad.Sci.U.S.A.2010,107,10737-10742.)、以及核酸组装体(J.Am.Chem.Soc.2013,135,18644-18650.)等。
尽管这些基于纳米载体的基因药物和化疗药物协同递送系统都经过精心设计，但由于基因药物和化疗药物两者理化性质差异较大，这些纳米协同递送系统仍然面临诸多挑战，包括：
(1)基因药物和化疗药物的亲疏水性往往差异较大，因此难以设计一个理想的纳米载体同时高效包载两种物质；
(2)基因药物和化疗药物在载体中的包载量和比例难以精准控制；
(3)基因药物和化疗药物的释放时间顺序难以精确调控。
上述困难会限制基因-化疗联合治疗在耐药肿瘤治疗中的应用。
发明内容
本发明的第一目的是提供骨架整合核苷类似物药物的功能性核酸，实现基因药物和化学药物的联合输送，以解决现有联合输送技术中存在的问题，包括：(1)基因药物和化疗药物的亲疏水性往往差异较大，因此难以设计一个理想的纳米载体同时高效包载两种物质；(2)基因药物和化疗药物在载体中的包载量和比例难以精准控制；(3)基因药物和化疗药物的释放时间顺序难以精确调控。
本发明的第二目的是提供上述的骨架整合核苷类似物药物的功能性核酸的制备方法。
本发明的第三目的是提供一种基于上述骨架整合核苷类似物药物的功能性核酸的衍生物，实现骨架整合核苷类似物药物的功能性核酸在细胞内的高效递送。
本发明的第四目的是提供一种基于上述骨架整合核苷类似物药物的功能性核酸衍生物的制备方法，实现骨架整合核苷类似物药物的功能性核酸在细胞内的高效递送。
本发明的第五目的是提供一种骨架整合核苷类似物药物的功能性核酸及 其衍生物在制备基于基因疗法、化学疗法联合治疗疾病的核酸类药物和化疗药物中的用途。
本发明的技术方案如下：
一种骨架整合核苷类似物药物的功能性核酸，其中，所述核苷类似物药物经过化学修饰后，通过替换功能性核酸中的天然核苷酸，被整合进入所述功能性核酸的骨架结构。
优选地，所述的核苷类似物药物化学修饰方法为核苷类似物药物经过亚磷酰胺修饰或者三磷酸修饰。
优选地，所述的核苷类似物药物经过化学修饰后，通过固相合成技术或者体外酶转录技术或者PCR扩增技术整合进入功能性核酸骨架。
优选地，通过调整被药物取代的天然核苷酸的数目实现基因与药物的比例精准可调，解决了基因药物和化疗药物在载体中的包载量和比例难以精准控制的问题。
优选地，所述的骨架整合核苷类似物药物的功能性核酸中的所述功能性核酸选自反义寡核苷酸(antisense DNA)、小干扰RNA(siRNA)、信使RNA(mRNA)、微小RNA(micro RNA)、长链非编码RNA(lncRNA)、小发夹RNA(shRNA)、用于基因编辑向导RNA(sgRNA等)、环状RNA(circRNA)中的一种，但不局限于这几种功能性核酸。
优选地，所述的骨架整合核苷类似物药物的功能性核酸，其中的核苷类似物药物选自下列药物中的一种:
嘌呤类似物，可以是巯嘌呤(Mercaptopurine)、硫鸟嘌呤(Tioguanine)、硫唑嘌呤(Azathioprine)或8-氮杂鸟嘌呤(8-Azaguanin)；
鸟苷类似物，可以是奈拉滨(Nelarabine)或呋咯地辛(Forodesine)；
胞苷类似物，可以是阿糖胞苷(Cytarabine)、环胞苷(Ancitabine)、吉西他滨(Gemcitabine)、依诺他宾(Enocitabine)、氮杂胞苷(5-Azacytidine)或脱氧氮杂胞苷(Decitabine)；
腺苷类似物，可以是氟达拉滨(Fludarabine)、克拉屈滨(Cladribine)、氯法 拉滨(Clofarabine)或阿卡地辛(Acadesine)；
尿苷类似物，可以是氟尿嘧啶(Fluorouracil)、卡莫氟(Carmofur)、替加氟(Tegafur)、脱氧氟尿苷(5'-Deoxy-5-fluorouridine)、卡培他滨(Capecitabine)或氟尿苷(Floxuridine)。
但本发明的技术方案不局限于上述这几种核苷类似物药物。
本发明还提供一种上述的骨架整合核苷类似物药物的功能性核酸的制备方法，包括以下方法中的一种：
利用核苷类似物药物的亚磷酰胺单体，通过固相合成方法制备骨架整合核苷类似物药物的功能性核酸；或
利用核苷类似物药物的三磷酸单体，通过体外酶转录方法或者PCR扩增技术制备骨架整合核苷类似物药物的功能性核酸。在优选实施例中，具体制备过程选自如下的一种：
将要合成的功能性核酸的序列输入固相合成仪中，在普通CPG或者氨基修饰的CPG表面制备骨架整合核苷类似物药物的功能性核酸；或
将RNA聚合酶、模板DNA、RNA核苷三磷酸单体、核苷类似物药物的三磷酸单体、反应缓冲液的混合溶液在37℃反应，制备骨架整合核苷类似物药物的功能性核酸；
将DNA聚合酶、模板DNA、DNA引物、DNA核苷三磷酸单体、核苷类似物药物的三磷酸单体、反应缓冲液的混合溶液在37℃反应，制备骨架整合核苷类似物药物的功能性核酸。
本发明上述的骨架整合核苷类似物药物的功能性核酸，可以制成各种衍生物，包括但不限于：
1.由所述骨架整合核苷类似物药物的功能性核酸与分子靶向基团结合形成的功能化衍生物，所述分子靶向基团例如是核酸适配体、靶向多肽、靶向小分子等；
2、所述骨架整合核苷类似物药物的功能性核酸还可以使用聚合物或疏水性分子修饰，从而得到衍生物及所述衍生物的自组装纳米结构；或者
3、所述骨架整合核苷类似物药物的功能性核酸还可以与转染试剂通过自组装得到复合纳米结构的衍生物；或者
4、使所述骨架整合核苷类似物药物的功能性核酸被聚合物或疏水性分子修饰，得到功能性核酸衍生物，并使该功能性核酸衍生物与转染试剂通过自组装得到复合纳米结构的衍生物。
本发明另一方面提供一种上述的骨架整合核苷类似物药物的功能性核酸的衍生物，为以下的一种：
由所述骨架整合核苷类似物药物的功能性核酸与分子靶向基团结合形成的功能化衍生物；或
所述骨架整合核苷类似物药物的功能性核酸由聚合物或疏水性分子修饰得到的衍生物及其自组装纳米结构；或者
所述骨架整合核苷类似物药物的功能性核酸与转染试剂通过自组装得到的复合纳米结构；或者
所述骨架整合核苷类似物药物的功能性核酸被聚合物或疏水性分子修饰后的产物与转染试剂通过自组装得到的复合纳米结构。
在一些实施方式中，所述聚合物选自聚己内酯、聚乙二醇、聚乳酸-羟基乙酸共聚物中的一种或几种。但不局限于上述几种聚合物。
在一些实施方式中，所述疏水性分子选自如下的一种或几种：
磷脂分子，例如磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸、磷脂酰甘油、磷脂酰肌醇、二磷脂酰甘油、鞘磷脂、鞘糖脂等；
烷烃类分子，例如戊烷、十二烷、十八烷等；
固醇类分子，例如胆固醇、皮质醇、醛固酮、睾丸酮、雌二醇以及维生素D等。但不局限于仅使用上述几种疏水性分子对骨架整合核苷类似物药物的功能性核酸进行修饰。
在一些实施方式中，所述转染试剂选自聚乙烯亚胺、聚赖氨酸、壳聚糖、Lipofectamine TM转染试剂、Lipofectamine TM2000转染试剂、Lipofectamine TM3000转染试剂、Lipofectamine TMRNAiMAX转染试剂、金纳米粒子、四氧化 三铁纳米粒子、二氧化硅纳米粒子中的一种或几种。但不局限于上述几种阳离子聚合物、阳离子脂质体、无机纳米粒子以及其他可用于功能性核酸负载和转染的转染试剂。
本发明提供的上述骨架整合核苷类似物药物的功能性核酸的衍生物的制备方法，主要包括：
使用靶向分子，包括核酸适配体、靶向多肽、靶向小分子等修饰得到的骨架整合核苷类似物药物的功能性核酸的衍生物，或
使用聚合物修饰，得到骨架整合核苷类似物药物的功能性核酸的衍生物；其中聚合物选自例如聚己内酯、聚乙二醇、聚乳酸-羟基乙酸共聚物其的一种，但本发明不局限于仅使用上述几种聚合物；或
使用疏水性分子修饰，得到骨架整合核苷类似物药物的功能性核酸的衍生物，其中所述疏水性分子选自：磷脂分子，例如磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸、磷脂酰甘油、磷脂酰肌醇、二磷脂酰甘油、鞘磷脂、鞘糖脂中的一种；或者烷烃类分子，例如戊烷、十二烷、十八烷等中的一种；或者固醇类分子，例如胆固醇、皮质醇、醛固酮、睾丸酮、雌二醇以及维生素D中的一种，但本发明不局限于仅使用上述几种疏水性分子对所述骨架整合核苷类似物药物的功能性核酸进行修饰；或
使用转染试剂与所述骨架整合核苷类似物药物的功能性核酸自组装生成复合纳米结构的衍生物；或将所述骨架整合核苷类似物药物的功能性核酸或者使用聚合物或疏水性分子修饰后的产物与转染试剂通过自组装过程生成纳米材料，其中转染试剂包括聚乙烯亚胺、聚赖氨酸、壳聚糖、Lipofectamine TM转染试剂、Lipofectamine TM2000转染试剂、Lipofectamine TM3000转染试剂、Lipofectamine TMRNAiMAX转染试剂、金纳米粒子、四氧化三铁纳米粒子、二氧化硅纳米粒子等其中的一种。但不局限于上述几种阳离子聚合物、阳离子脂质体、无机纳米粒子以及其他可用于功能性核酸负载和转染的转染试剂。
在优选实施方式中，上述的骨架整合核苷类似物药物的功能性核酸的衍生物的制备方法中，使用聚合物修饰的方法包括以下步骤：
提供以开环聚合的方式合成的末端带有叠氮基团的可降解高分子，然后 使该可降解高分子与二苯基环辛炔修饰的骨架整合核苷类似物药物的功能性核酸进行无铜催化点击(Click)反应，得到可降解高分子-骨架整合核苷类似物药物的功能性核酸的缀合物，即为骨架整合核苷类似物药物的功能性核酸的衍生物中的一种。
本发明还提供一种上述的骨架整合核苷类似物药物的功能性核酸的衍生物的水溶液的制备方法，包括以下步骤：
将可降解高分子-骨架整合核苷类似物药物的功能性核酸的缀合物溶解在二甲基亚砜(DMSO)中，再将该溶液置于超纯水中透析24小时，制得可降解高分子-骨架整合核苷类似物药物的功能性核酸的缀合物的水溶液，即为骨架整合核苷类似物药物的功能性核酸的衍生物的水溶液。
本发明还公开了上述骨架整合核苷类似物药物的功能性核酸及其衍生物在制备基于基因疗法、化学疗法联合治疗疾病的核酸类药物和化疗药物中的用途。
与现有技术相比，本发明的有益效果如下：
第一、本发明的骨架整合核苷类似物药物的功能性核酸及其衍生物，可以实现基因与药物的比例精准可调，具体可以通过调整被药物取代的天然核苷酸数目实现；
第二、本发明的骨架整合核苷类似物药物的功能性核酸及其衍生物，可以实现基因和药物程序性依次发挥作用，能最大程度增加协同治疗的效果；
第三、本发明成功的设计了一个载体能够同时高效包载基因药物和化疗药物两种物质。
当然，实施本发明的任一产品并不一定需要同时达到以上所述的所有优点。
附图说明
图1为本发明实施例1中骨架整合氟尿苷的Bcl-2反义核苷酸(F-Bcl-2ASO)对荷瘤裸鼠中的耐药肝脏原位移植瘤的治疗过程中肿瘤尺寸的变化；
图2为本发明实施例1中骨架整合氟尿苷的Bcl-2反义核苷酸(F-Bcl-2ASO)对荷瘤裸鼠中的耐药肝脏原位移植瘤的治疗后荷瘤肝脏照片；
图3为本发明实施例1中骨架整合氟尿苷的Bcl-2反义核苷酸(F-Bcl-2ASO)对荷瘤裸鼠中的耐药肝脏原位移植瘤的治疗后，下调耐药肿瘤中耐药相关蛋白表达量的情况；
图4为本发明实施例2中骨架整合氟尿苷的Bcl-2/xL反义核苷酸(F-Bcl-2/xL ASO)下调耐药细胞BEL-7402中耐药相关蛋白的表达量的情况；
图5为本发明实施例3中骨架整合氟尿苷的Bcl-2反义核苷酸构筑的球形核酸SNA(F-Bcl-2ASO)的合成路线；
图6为本发明实施例3中二苯基环辛炔(DBCO)修饰的骨架整合氟尿苷的Bcl-2反义核苷酸(F-Bcl-2ASO-DBCO)的20％变性聚丙烯酰胺凝胶电泳图；
图7为本发明实施例3中聚合物1(N 3-PEG-b-PCL 28)的合成路线；
图8为本发明实施例3中聚合物1(N 3-PEG-b-PCL 28)的核磁共振氢谱图；
图9为本发明实施例3中聚合物1(N 3-PEG-b-PCL 28)与DBCO修饰的骨架整合氟尿苷的Bcl-2反义核苷酸(F-Bcl-2ASO-DBCO)点击反应缀合的合成路线图；
图10为本发明实施例3中骨架整合氟尿苷的Bcl-2反义核苷酸构筑的球形核酸SNA(F-Bcl-2ASO)的1％非变性琼脂糖凝胶电泳图；
图11为本发明实施例3中骨架整合氟尿苷的Bcl-2反义核苷酸构筑的球形核酸SNA(F-Bcl-2ASO)的动态光散射图；
图12为本发明实施例3中骨架整合氟尿苷的Bcl-2反义核苷酸构筑的球形核酸SNA(F-Bcl-2ASO)的透射电镜图；
图13为本发明实施例3中骨架整合氟尿苷的Bcl-2反义核苷酸构筑的球形核酸SNA(F-Bcl-2ASO)对荷瘤裸鼠中的耐药肝脏原位移植瘤的治疗过程中肿瘤尺寸的变化；
图14为本发明实施例3中骨架整合氟尿苷的Bcl-2反义核苷酸构筑的球形核酸SNA(F-Bcl-2ASO)对荷瘤裸鼠中的耐药肝脏原位移植瘤的治疗后荷瘤 肝脏照片；
图15为本发明实施例3中骨架整合氟尿苷的Bcl-2反义核苷酸构筑的球形核酸SNA(F-Bcl-2ASO)对荷瘤裸鼠中的耐药肝脏原位移植瘤的治疗后，下调耐药肿瘤中耐药相关蛋白表达量的情况；
图16为本发明实施例4中DBCO修饰的骨架整合氟尿苷的Bcl-2/xL反义核苷酸(F-Bcl-2/xL ASO-DBCO)的20％变性聚丙烯酰胺凝胶电泳图；
图17为本发明实施例4中骨架整合氟尿苷的Bcl-2/xL反义核苷酸构筑的球形核酸SNA(F-Bcl-2/xL ASO)的1％非变性琼脂糖凝胶电泳图；
图18为本发明实施例4中骨架整合氟尿苷的Bcl-2/xL反义核苷酸构筑的球形核酸SNA(F-Bcl-2/xL ASO)下调耐药细胞BEL-7402中耐药相关蛋白的表达量的情况。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应该理解，这些实施例仅用于说明本发明，而不用于限定本发明的保护范围。在实际应用中本领域技术人员根据本发明做出的改进和调整，仍属于本发明的保护范围。
实施例1骨架整合氟尿苷的Bcl-2反义核苷酸(F-Bcl-2ASO)
1.1 骨架整合氟尿苷的Bcl-2反义核苷酸(F-Bcl-2 ASO)的合成
在DNA固相合成时，本实施例中将反义核苷酸中的核苷酸T全部替换为抗肿瘤药物氟尿苷(F)。具体地，
将氟尿苷药物的亚磷酰胺单体、DNA亚磷酰胺单体置于DNA固相合成仪对应位置，反应柱中加入普通微孔玻璃小球(CPG)，输入5’-CAG CGF GCG CCA FCC FFC CC AFCC FCCFCC-3’序列信息，加入催化、盖帽、氧化以及脱保护试剂，合成含氟尿苷序列后，经氨解、氮吹、制备色谱分离纯化、脱保护、浓缩即可得F-Bcl-2ASO序列。
此外，按照相同的方法，在固相合成仪中输入5’-AAF ACF CCG AAC GFG  FCA CGF CCFCCF CAC-3’的序列，即可以合成出 骨架整合氟尿苷的乱序核酸 (F-scramble)作为对照。
1.2骨架整合氟尿苷的Bcl-2反义核苷酸(F-Bcl-2ASO)对荷瘤裸鼠中的耐药肝脏原位移植瘤的治疗效果
本实施例中通过荷瘤裸鼠的耐药肝脏原位移植瘤模型评估骨架整合氟尿苷的Bcl-2反义核苷酸(F-Bcl-2ASO)的体内抑制耐药肿瘤增殖效果。
具体地，通过尾静脉注射PBS和等当量氟尿苷、F-Bcl-2ASO、F-scramble(其中注射氟尿苷的当量浓度为5mg/kg bw)，用磁共振成像和西门子Inveon Research Workplace软件分析，通过三维有序子集最大期望值法(3D-OSEM)，手动圈出每层肿瘤部位后，重构图像并计算肿瘤尺寸，治疗结束后解剖取出荷瘤肝脏并拍照记录耐药肝脏原位移植瘤直观大小。
结果如图1所示，各治疗组耐药肝脏移植瘤的起始尺寸相当，治疗到第七天和十五天时F-Bcl-2ASO治疗组的肿瘤尺寸小于其余对照组的肿瘤尺寸。
图2为治疗结束后解剖裸鼠取出的荷瘤肝脏照片，图中偏白色部位为耐药肝脏移植瘤，由图2可见治疗结束后F-Bcl-2ASO治疗组的肿瘤尺寸最小，其余组别肿瘤尺寸只是略小于PBS对照组，也就是说F-Bcl-2ASO是上述药物中疗效最好的。
1.3骨架整合氟尿苷的Bcl-2反义核苷酸(F-Bcl-2ASO)通过基因调控逆转肿瘤耐药
为了验证骨架整合氟尿苷的Bcl-2反义核苷酸(F-Bcl-2ASO)能否通过基因调控逆转肿瘤耐药，本实施例中在治疗结束后，将肿瘤组织取出，迅速提取总蛋白，采用蛋白质印迹分析测定皮下耐药肿瘤中耐药蛋白表达量。
测定结果如图3所示，F-Bcl-2ASO治疗组的Bcl-2蛋白条带比其他组别弱，这说明它能下调荷瘤裸鼠肿瘤内耐药蛋白的表达，因此F-Bcl-2ASO能在动物体内表现出基因治疗效果，能有效逆转耐药肿瘤的耐药性。
实施例2骨架整合氟尿苷的Bcl-2/xL反义核苷酸(F-Bcl-2/xL ASO)
2.1 骨架整合氟尿苷的Bcl-2/xL反义核苷酸(F-Bcl-2/xL ASO)的合成
在DNA固相合成时，本实施例中将反义核苷酸中的核苷酸T全部替换为抗肿瘤药物氟尿苷(F)。
具体地，将氟尿苷药物的亚磷酰胺单体、DNA亚磷酰胺单体置于DNA固相合成仪对应位置，反应柱中加入对应的普通微孔玻璃小球(CPG)，输入5’-AAG GCA FCC CAG CCF CCG FF CCFCCFCCF A-3’序列信息，加入催化、盖帽、氧化以及脱保护试剂，合成含氟尿苷序列后，经氨解、氮吹、制备色谱分离纯化、脱保护、浓缩即可得F-Bcl-2/xL ASO序列。
2.2骨架整合氟尿苷的Bcl-2/xL反义核苷酸(F-Bcl-2/xL ASO)通过基因调控逆转肿瘤耐药
骨架整合氟尿苷的Bcl-2/xL反义核苷酸(F-Bcl-2/xL ASO)与耐药BEL-7402细胞共孵育10h，随后更换为正常培养基培养48小时；而F-scramble和F用作对照组，遵循同样的条件与细胞孵育，其中F的当量浓度为10μM，未做任何处理的耐药细胞作为阴性对照组。
在提取总蛋白后，经蛋白质印迹分析测定细胞中目标蛋白Bcl-2的蛋白表达量。结果如图4所示，F-Bcl-2/xL ASO与耐药细胞BEL-7402一同孵育后，可下调耐药相关Bcl-2和Bcl-xL蛋白的表达量，然而F-scramble和F处理组中Bcl-2蛋白表达量与空白对照组比较无明显差异。因此，骨架整合氟尿苷的Bcl-2/xL反义核苷酸(F-Bcl-2/xL ASO)具有一定的逆转耐药肿瘤的耐药性。
实施例3骨架整合氟尿苷的Bcl-2反义核苷酸构筑的球形核酸SNA(F-Bcl-2ASO)
3.1 DBCO修饰的骨架整合氟尿苷的Bcl-2反义核苷酸(F-Bcl-2 ASO-DBCO)的合成
在通过固相合成方法制备骨架整合氟尿苷的Bcl-2反义核苷酸时，使用氨基修饰的微孔玻璃小球(NH 2-CPG)，制备得到氨基修饰的骨架整合氟尿苷的Bcl-2反义核苷酸(F-Bcl-2ASO-NH 2)。
将上述反义核苷酸序列在含30％磷酸盐缓冲液的DMSO混合溶液中，加入200当量的DBCO-NHS ester，室温反应24h即可得到DBCO修饰的骨架整合氟尿苷的Bcl-2反义核苷酸(F-Bcl-2ASO-DBCO)(图5)。
上述粗产物采用乙酸乙酯多次萃取、乙醇沉淀、离心等步骤纯化。
最后，将F-Bcl-2ASO-DBCO复溶，用凝胶电泳表征DNA的修饰情况，如图6所示，图中可见，产物在电泳后呈现为单一条带，与F-Bcl-2ASO-NH 2相比，产物在凝胶电泳中移动速度较慢，证明F-Bcl-2ASO-DBCO的成功制备。
3.2聚合物1的合成
用辛酸亚锡作催化剂、分子量2000的叠氮聚乙二醇羟基(N 3-PEG-OH)作引发剂引发ε-己内酯的开环聚合反应即可制备嵌段聚合物N 3-PEG-b-PCL(图7)。具体制备过程如下所述：首先将N 3-PEG-OH 1.0000g(0.5mmol)和无水ε-己内酯1.7121g(15mmol)溶于无水甲苯中。接着通过注射器加入催化量的辛酸亚锡于120℃氮气氛围中反应24h。结束反应后，旋蒸抽干溶剂，剩余混合物复溶于二氯甲烷中，通过冰乙醚沉淀，过滤和真空干燥即可得到白色粉末状产物聚合物1，通过核磁共振氢谱表征，其分子式为N 3-PEG-b-PCL 28。
聚合物1的 1H NMR谱图如图8所示，测试溶剂为CDCl 3，各质子峰的归属如下：δ(ppm):4.22(t,2H,-OCH 2CH 2OC(O)CH 2CH 2CH 2CH 2CH 2O-),4.05(t,56H,-C(O)CH 2CH 2CH 2CH 2CH 2O-),3.64(s,174H,-OCH 2CH 2O-),2.30(t,56H,-C(O)CH 2CH 2CH 2CH 2CH 2O-),1.64(m,112H,-C(O)CH 2CH 2CH 2CH 2CH 2O-),1.37(m,56H,-C(O)CH 2CH 2CH 2CH 2CH 2O-)。
3.3骨架整合氟尿苷的Bcl-2反义核苷酸构筑的球形核酸SNA(F-Bcl-2ASO)的合成
聚合物1(N 3-PEG-b-PCL 28)与DBCO修饰的骨架整合氟尿苷的Bcl-2反义核苷酸(F-Bcl-2ASO-DBCO)通过经典的点击反应缀合，如图9所示。
合成过程如下：首先，将N 3-PEG-b-PCL 28(200nmol)溶于1.2mL的DMSO溶液中，F-Bcl-2ASO-DBCO(400nmol)溶于30.0μL水中，两溶液混合均匀后 于58℃振荡反应48h。
反应结束后，将反应液置于截留分子量为10kDa的透析袋中透析，除去DMSO，透析过程中N 3-PEG-b-PCL 28与F-Bcl-2ASO-DBCO缀合物会逐步组装形成球形核酸结构SNA(F-Bcl-2ASO)(图5)。
未参与点击反应的过量上述反义核苷酸用截留分子量为100kDa的超滤管超滤去除。
纯化后的SNA(F-Bcl-2ASO)用琼脂糖凝胶电泳表征其结构的形成，琼脂糖凝胶浓度为1％，电泳电压为90V，电泳结束后用凝胶成像系统成像。表征结果如图10所示，SNA(F-Bcl-2ASO)条带位于对照组F-Bcl-2ASO-DBCO条带的上方，证明骨架整合氟尿苷的Bcl-2反义核苷酸构筑的球形核酸SNA(F-Bcl-2ASO)的成功制备。
此外，按照相同的方法，可以合成出骨架整合氟尿苷的乱序核酸构筑的球形核酸SNA(F-scramble)作为对照。
骨架整合氟尿苷的Bcl-2反义核苷酸构筑的球形核酸SNA(F-Bcl-2ASO)的水合粒径通过动态光散射实验表征，测试结果如图11所示，可见SNA(F-Bcl-2ASO)的水合粒径为17.5nm。此外，图12所示的透射电镜实验结果证实，SNA(F-Bcl-2ASO)的形貌为球形。
3.4骨架整合氟尿苷的Bcl-2反义核苷酸构筑的球形核酸SNA(F-Bcl-2ASO)对荷瘤裸鼠中的耐药肝脏原位移植瘤的治疗效果
本实施例中通过荷瘤裸鼠的耐药肝脏原位移植瘤模型评估SNA(F-Bcl-2ASO)的体内抑制耐药肿瘤增殖效果。
在本实施例中通过尾静脉注射PBS和等当量氟尿苷、SNA(F-Bcl-2ASO)、SNA(F-scramble)以及反义核苷酸构筑的球形核酸与氟尿苷的混合物SNA(Bcl-2ASO)/F(其中注射氟尿苷的当量浓度为5mg/kg bw)，用磁共振成像和西门子Inveon Research Workplace软件分析，通过三维有序子集最大期望值法(3D-OSEM)，手动圈出每层肿瘤部位后，重构图像并计算肿瘤尺寸，治疗结束后解剖取出荷瘤肝脏并拍照记录耐药肝脏原位移植瘤直观大小。
结果如图13所示，各治疗组耐药肝脏移植瘤的起始尺寸相当，治疗到第七天和十五天时SNA(F-Bcl-2ASO)治疗组的肿瘤尺寸小于PBS对照组的肿瘤尺寸，存在显著差异。
图14为治疗结束后解剖裸鼠取出的荷瘤肝脏照片，图中偏白色部位为耐药肝脏移植瘤，由图13及图14可见治疗结束后SNA(F-Bcl-2ASO)治疗组的肿瘤尺寸最小，其余组别肿瘤尺寸只是略小于PBS对照组，也就是说SNA(F-Bcl-2ASO)是上述药物中疗效最好的。
3.5骨架整合氟尿苷的Bcl-2反义核苷酸构筑的球形核酸SNA(F-Bcl-2ASO)通过基因调控逆转肿瘤耐药
为了验证骨架整合氟尿苷的Bcl-2反义核苷酸构筑的球形核酸SNA(F-Bcl-2ASO)能否通过基因调控逆转肿瘤耐药，治疗结束后，将肿瘤组织取出，迅速提取总蛋白，采用蛋白质印迹分析测定皮下耐药肿瘤中耐药蛋白表达量。
结果如图15所示，SNA(F-Bcl-2ASO)治疗组的Bcl-2蛋白条带均比其他组别弱，这说明SNA(F-Bcl-2ASO)治疗组能下调荷瘤裸鼠的肿瘤内耐药蛋白的表达，因此SNA(F-Bcl-2ASO)能在动物体内表现出优异的基因治疗效果，能有效逆转耐药肿瘤的耐药性。
实施例4骨架整合氟尿苷的Bcl-2/xL反义核苷酸构筑的球形核酸SNA(F-Bcl-2/xL ASO)
4.1  DBCO修饰的骨架整合氟尿苷的Bcl-2/xL反义核苷酸的合成
在通过固相合成方法制备骨架整合氟尿苷的Bcl-2/xL反义核苷酸时，使用氨基修饰的微孔玻璃小球(NH 2-CPG)，制备得到氨基修饰的骨架整合氟尿苷的Bcl-2/xL反义核苷酸(F-Bcl-2/xL ASO-NH 2)。
将上述反义核苷酸序列在含30％磷酸盐缓冲液的DMSO混合溶液中，加入200当量的DBCO-NHS ester，室温反应24h即可得到DBCO修饰的骨架整合氟尿苷的Bcl-2/xL反义核苷酸(F-Bcl-2/xL ASO-DBCO)。
粗产物采用乙酸乙酯多次萃取、乙醇沉淀、离心等步骤纯化。
最后，将F-Bcl-2/xL ASO-DBCO复溶，用凝胶电泳表征DNA的修饰情况，如图16所示。图16中可见，产物在电泳后呈现为单一条带，与F-Bcl-2/xL ASO-NH 2相比，产物在凝胶电泳中移动速度较慢，证明F-Bcl-2/xL ASO-DBCO的成功制备。
4.2骨架整合氟尿苷的Bcl-2/xL反义核苷酸构筑的球形核酸SNA(F-Bcl-2/xL ASO)的合成
使用实施例3中的聚合物1(N 3-PEG-b-PCL 28)与DBCO修饰的骨架整合氟尿苷的Bcl-2/xL反义核苷酸(F-Bcl-2/xL ASO-DBCO)通过经典的点击反应缀合。
合成过程如下：首先，将N 3-PEG-b-PCL 28(200nmol)溶于1.2mL的DMSO溶液中，F-Bcl-2/xL ASO-DBCO(400nmol)溶于30.0μL水中，两溶液混合均匀后于58℃振荡反应48h。
反应结束后，将反应液置于截留分子量为10kDa的透析袋中透析，除去DMSO，透析过程中N 3-PEG-b-PCL 28与F-Bcl-2/xL ASO-DBCO缀合物会逐步组装形成球形核酸结构SNA(F-Bcl-2/xL ASO)。
未参与点击反应的过量上述反义核苷酸用截留分子量为100kDa的超滤管超滤去除。
纯化后的SNA(F-Bcl-2/xL ASO)用琼脂糖凝胶电泳表征其结构的形成，琼脂糖凝胶浓度为1％，电泳电压为90V，电泳结束后用凝胶成像系统成像。如图17所示，SNA(F-Bcl-2/xL ASO)条带位于对照组F-Bcl-2/xL ASO-DBCO条带的上方，证明骨架整合氟尿苷的Bcl-2/xL反义核苷酸构筑的球形核酸SNA(F-Bcl-2/xL ASO)的成功制备。
4.3骨架整合氟尿苷的Bcl-2/xL反义核苷酸构筑的球形核酸SNA(F-Bcl-2/xL ASO)通过基因调控逆转肿瘤耐药
骨架整合氟尿苷的Bcl-2/xL反义核苷酸构筑的球形核酸SNA(F-Bcl-2/xLASO)与耐药BEL-7402细胞共孵育10h，随后更换为正常培养基培养48小时；而SNA(F-scramble)和F用作对照组，遵循同样的条件与 细胞孵育，其中F的当量浓度为10μM，未做任何处理的耐药细胞作为阴性对照组。
在提取总蛋白后，经蛋白质印迹分析测定细胞中目标蛋白Bcl-2和Bcl-2/xL的蛋白表达量。结果如图18所示，图18中可见，SNA(F-Bcl-2/xL ASO)与耐药细胞BEL-7402一同孵育后，可显著下调耐药相关Bcl-2和Bcl-xL蛋白的表达量，然而SNA(F-scramble)和F处理组中Bcl-2蛋白表达量与空白对照组比较无明显差异。因此，骨架整合氟尿苷的Bcl-2/xL反义核苷酸构筑的球形核酸SNA(F-Bcl-2/xL ASO)能够很好的逆转耐药肿瘤的耐药性。
与现有技术相比，本发明的骨架整合核苷类似物药物的功能性核酸及其衍生物可以高效进入细胞，并利用骨架整合核苷类似物药物的功能性核酸发挥基因调控功能；随后，骨架整合核苷类似物药物的功能性核酸可以被核酸酶降解并释放核苷类似物药物的活性成分，因此起到化学治疗的作用。因此，骨架整合核苷类似物药物的功能性核酸及其衍生物可以简单高效地实现基因治疗、化学治疗的联合治疗，并避免了复杂的合成步骤。
实施例5骨架整合氟尿苷的PLK1小干扰RNA(F-siPLK1)
5.1 骨架整合氟尿苷的PLK1小干扰RNA(F-siPLK1)的合成
在体外酶转录时，本实施例中将siRNA中的核苷酸U全部替换为抗肿瘤药物氟尿苷(F)。具体地，
将T7RNA聚合酶Y639F加入到合成RNA的转录反应液中，转录反应液包含模板DNA(1μg)、ATP(5mM)、CTP(5mM)、GTP(5mM)、5-FdUTP(5mM)、DTT(10mM)以及反应缓冲液，接着在37℃孵育6h。反应结束后，通过变性聚丙烯酰胺凝胶切胶纯化、回收产物，并用冰乙醇在-20℃下沉淀目标RNA链段。经离心、复溶RNA后将其存储在-80℃冰箱中备用。
实施例6骨架整合吉西他滨的Bcl-2反义核苷酸(G e-Bcl-2ASO)
6.1 骨架整合吉西他滨的Bcl-2反义核苷酸(G e-Bcl-2 ASO) 的合成
在DNA固相合成时，本实施例中将反义核苷酸中的核苷酸T全部替换为抗肿瘤药物吉西他滨(G e)。具体地，
将吉西他滨药物的亚磷酰胺单体、DNA亚磷酰胺单体置于DNA固相合成仪对应位置，反应柱中加入普通微孔玻璃小球(CPG)，输入5’-G eAGG eGTGG eGG eG eATG eG eTTG eG eG eATG eG eTG eG eTG eG e-3’序列信息，加入催化、盖帽、氧化以及脱保护试剂，合成含氟尿苷序列后，经氨解、氮吹、制备色谱分离纯化、脱保护、浓缩即可得G e-Bcl-2ASO序列。
以上公开的本发明优选实施例只是用于帮助阐述本发明。优选实施例并没有详尽叙述所有的细节，也不限制该发明仅为所述的具体实施方式。显然，根据本说明书的内容，可作很多的修改和变化。本说明书选取并具体描述这些实施例，是为了更好地解释本发明的原理和实际应用，从而使所属技术领域技术人员能很好地理解和利用本发明。本发明仅受权利要求书及其全部范围和等效物的限制。

Claims (12)

  1. 一种骨架整合核苷类似物药物的功能性核酸，其特征在于，所述核苷类似物药物经过化学修饰后，通过替换功能性核酸中的天然核苷酸被整合进入所述功能性核酸的骨架结构。
  2. 如权利要求1所述的骨架整合核苷类似物药物的功能性核酸，其特征在于，所述核苷类似物药物经过亚磷酰胺修饰或者三磷酸修饰。
  3. 如权利要求1所述的骨架整合核苷类似物药物的功能性核酸，其特征在于，所述核苷类似物药物经过化学修饰后，通过固相合成技术、体外酶转录技术或聚合酶链式反应(PCR)扩增技术整合进入所述功能性核酸的骨架架构。
  4. 如权利要求1所述的骨架整合核苷类似物药物的功能性核酸，其特征在于，通过调整被所述核苷类似物药物取代的天然核苷酸的数目实现基因与药物的比例精准可调。
  5. 如权利要求1所述的骨架整合核苷类似物药物的功能性核酸，其特征在于，所述的骨架整合核苷类似物药物的功能性核酸，其中的所述功能性核酸选自反义寡核苷酸、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。
  6. 如权利要求1所述的骨架整合核苷类似物药物的功能性核酸，其特征在于，所述的骨架整合核苷类似物药物的功能性核酸，其中的所述核苷类似物药物选自嘌呤类似物、鸟苷类似物、胞苷类似物、腺苷类似物或尿苷类似物的一种。
  7. 一种权利要求1-6中任一所述的骨架整合核苷类似物药物的功能性核酸的制备方法，其特征在于，包括以下步骤中的一种：
    利用核苷类似物药物的亚磷酰胺单体，通过固相合成方法制备骨架整合核苷类似物药物的功能性核酸；或
    利用核苷类似物药物的三磷酸单体，通过体外酶转录方法或PCR扩增方法制备骨架整合核苷类似物药物的功能性核酸。
  8. 如权利要求7所述的骨架整合核苷类似物药物的功能性核酸的制备方 法，其特征在于，具体制备过程为如下方法中的一种：
    将要合成的功能性核酸的序列输入固相合成仪中，在普通微孔玻璃小球或者氨基修饰的微孔玻璃小球表面制备骨架整合核苷类似物药物的功能性核酸；或
    将RNA聚合酶、模板DNA、RNA核苷三磷酸单体、核苷类似物药物的三磷酸单体、反应缓冲液的混合溶液在37℃反应，制备骨架整合核苷类似物药物的功能性核酸；
    或将DNA聚合酶、模板DNA、DNA引物、DNA核苷三磷酸单体、核苷类似物药物的三磷酸单体、反应缓冲液的混合溶液在37℃反应，制备骨架整合核苷类似物药物的功能性核酸。
  9. 一种权利要求1-6中任一所述的骨架整合核苷类似物药物的功能性核酸的衍生物，其特征在于，为以下的一种：
    由所述骨架整合核苷类似物药物的功能性核酸与分子靶向基团结合形成的功能化衍生物；或
    所述骨架整合核苷类似物药物的功能性核酸由聚合物或疏水性分子修饰得到的衍生物及其自组装纳米结构；或者
    所述骨架整合核苷类似物药物的功能性核酸与转染试剂通过自组装得到的复合纳米结构；或者
    所述骨架整合核苷类似物药物的功能性核酸被聚合物或疏水性分子修饰后的产物与转染试剂通过自组装得到的复合纳米结构。
  10. 一种权利要求9中所述的骨架整合核苷类似物药物的功能性核酸的衍生物的制备方法，其特征在于，主要包括：
    使用靶向分子，修饰得到骨架整合核苷类似物药物的功能性核酸的衍生物，或
    使用聚合物修饰而得到骨架整合核苷类似物药物的功能性核酸的衍生物；或
    使用疏水性分子修饰而得到骨架整合核苷类似物药物的功能性核酸的衍 生物，或
    使用转染试剂与所述骨架整合核苷类似物药物的功能性核酸自组装生成复合纳米结构的衍生物；或
    将所述骨架整合核苷类似物药物的功能性核酸或者使用聚合物或疏水性分子修饰后的产物与转染试剂通过自组装过程生成纳米材料。
  11. 根据权利要求10所述的骨架整合核苷类似物药物的功能性核酸的衍生物的制备方法，其特征在于，使用聚合物修饰的方法包括以下步骤：
    提供以开环聚合的方式合成的末端带有叠氮基团的可降解高分子，然后使该可降解高分子与二苯基环辛炔修饰的骨架整合核苷类似物药物的功能性核酸进行无铜催化点击反应，得到可降解高分子-骨架整合核苷类似物药物的功能性核酸的缀合物，即为骨架整合核苷类似物药物的功能性核酸的衍生物中的一种。
  12. 一种权利要求1-6中任一所述的骨架整合核苷类似物药物的功能性核酸及权利要求9所述的骨架整合核苷类似物药物的功能性核酸的衍生物在制备基于基因疗法、化学疗法联合治疗疾病的核酸类药物和化疗药物中的用途。
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