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 PDF

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WO2020150915A1
WO2020150915A1 PCT/CN2019/072790 CN2019072790W WO2020150915A1 WO 2020150915 A1 WO2020150915 A1 WO 2020150915A1 CN 2019072790 W CN2019072790 W CN 2019072790W WO 2020150915 A1 WO2020150915 A1 WO 2020150915A1
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nucleic acid
drug
modified
phosphorothioate
molecule
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French (fr)
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张川
张娇
郭园园
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上海交通大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • 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
    • 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
    • A61K47/56—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • 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
    • A61K47/69—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
    • A61K47/6921—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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives

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

一种基于硫代磷酸酯修饰核酸的核酸-药物结合物、药物输送体系及它们的制备方法。所述的核酸-药物结合物是由硫代磷酸酯修饰核酸中的硫代磷酸酯基团与修饰在药物分子上、可与硫代磷酸酯基团发生亲电反应的基团反应结合而成，并且，通过选择包括功能性核酸在内的不同核酸序列，所述的核酸-药物结合物可自组装为各种形式的含药纳米载体进行药物递送。与现有技术相比，以上核酸-药物结合物通过简单的固相合成技术即可达到，并可精确控制药物分子在核酸骨架上的接枝位点及其组装形式，并且此方法对化疗药物具有普适性；可显著改善化疗药物的理化性质和体内分布性质并促进其治疗作用，还可实现基因治疗、化疗的联合治疗。

Description

核酸-药物结合物、药物递送系统及其制备方法和应用 技术领域
本发明属于生物医药领域，特别涉及一种基于核酸硫代磷酸酯修饰的核酸-药物结合物、药物递送系统及其制备方法和应用。
背景技术
化疗是肿瘤治疗的重要手段之一，然而由于大部分化疗药物存在水溶性差、非靶向性、血液清除率高甚至严重的毒副作用等缺陷，导致其生物利用度较低(Nat.Rev.Cancer 2006,6,789.)，并且长期使用会产生耐药性，给它们的临床应用带来一定的局限性。
为了攻克这一难题，在过去几十年里研究者们设计了一系列基于聚合物或无机纳米粒子的纳米药物递送系统来改善化疗药物性质并促进其治疗效果，如胶束、囊泡、脂质体、白蛋白纳米粒、微泡等(Science 2004,303,1818.)。目前，纳米载体包载化疗药物主要包括物理包埋方式和化学结合方式两种，但是由于前者载药量较低且药物容易泄露造成毒副作用，因此，药物结合物策略成为药物输送的另一热点。用于药物结合物的材料有很多，包括聚合物(J.Controlled Release 2016,222,116.)、多肽(Adv.Drug Delivery Rev.2017,110-111,112.)、抗体(Trends Biotechnol.2017,35,466.)等。然而，用于制备药物结合物的材料往往不能同时满足生物相容性、生物可降解性和低免疫原性三个特点，因此限制了其临床转化应用。此外，目前所报道的一些药物结合物，都存在以下问题：药物接枝较少、药物接枝位点和接枝数量无法控制、可接枝的药物分子受限制等。
近年来，由于核酸作为一种天然的生物高分子能够同时满足生物相容性、生物可降解性和低免疫原性三个条件，并具有其他一些特定的分子特征如靶向性、特定的分子识别功能和自组装形成的纳米结构的精确可控性，使其在生物医药领域得到了越来越广泛的关注。
目前，利用核酸纳米结构(J.Polym.Sci.2017,35,1.)如多面体和折纸结构进行药物输送的研究也层出不穷，比如利用阿霉素(DOX)可以插入DNA双螺旋结构的特点进行DOX的输送，在体内外都取得了良好的抗肿瘤效果。但是利用这种核酸插入手段进行物理包载药物不仅对药物有所限制，且其体内稳定性还有待深究。
此外，也有一些研究者将核酸和化疗药物通过核酸序列末端修饰的官能团或者通过与DNA连接的聚合物等接入到DNA纳米结构中，来实现药物输送。然而前者由于受到官能团数量限制载药量较低，而后者由于含有聚合物链段因而可能带来一定的生物相容性问题，并且这两种方式往往需要较为复杂的合成过程。
因而，亟需建立一种对化疗药物普遍适用的、载药量和载药位点精确可控的新型的生物相容的核酸纳米载药体系。
发明内容
本发明的第一目的是提供一种基于核酸硫代磷酸酯修饰的核酸-药物结合物，以实现化疗药物准确可控的接枝和高效递送，解决目前药物结合物输送体系所存在的以下缺点：(1)不能同时满足载体材料的生物相容、体内可降解和低免疫原性的三大要求；(2)难以精确控制药物接枝位点和载药量；(3)对可接枝的药物有所限制，无法实现核酸作为载体材料的普适性，难以降低核酸载药的成本；(4)需要较为复杂的合成过程；(5)无法实现多功能载药体系的合理简单构建。
本发明的第二目的是提供一种药物递送系统，所述药物递送系统为载药纳米体系，由上述的核酸-药物结合物自组装形成。
本发明的第三目的是提供上述的基于核酸硫代磷酸酯修饰的核酸-药物结合物的制备方法。
本发明的第四目的是提供上述的药物递送系统的制备方法。
本发明的第五目的是提供一种基于以上的核酸-药物结合物及药物递送系 统在制备基于核酸作为载体的肿瘤治疗药物中的用途。
本发明的第六目的是提供一种药物，其包括上述的核酸-药物结合物形成的药物递送系统。
本发明的技术方案如下：
一种基于硫代磷酸酯修饰核酸的核酸-药物结合物，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。所述核酸骨架和所述药物分子形成功能性核酸-药物缀合物(即核酸-药物结合物)，所述核酸-药物结合物能够进行自组装得到载药纳米体系。
优选地，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。
优选地，通过固相合成方法来制备硫代磷酸酯修饰的核酸，就能够实现硫代修饰的位点和数量可调。
优选地，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，进而制备可控的药物递送系统。
优选地，所述核酸骨架可根据不同的需求进行不同位点和数量的硫代修饰设计，例如，用于凝胶和四面体结构组装的核酸，因考虑修饰后位阻效应及其对碱基配对的影响，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考虑胶束组装机理，倾向于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。
优选地，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。
优选地，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 响应型化学键可为二硫键、酰腙键、酯键等，但不限于上述所列响应型化学键。
优选地，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，如碘代或溴代的乙酰化合物、γ-溴-α,β-不饱和羰基、苄溴或溴代马来酰亚胺；2)马来酰亚胺基团；3)吖丙啶基磺胺基团，但不限于以上几种。
优选地，所述药物分子选自抗癌药物如紫杉醇、喜树碱、顺铂、多西紫杉醇、苯丁酸氮芥、甲氨喋呤、多柔比星、顺铂前药等，但不局限于上述几种，癌症靶向药物分子如厄洛替尼、伊马替尼、吉非替尼、索拉菲尼等，但不局限于上述所列几种。
优选地，所述药物分子为含有二硫键的溴代修饰的药物分子，例如可以通过简单的化学反应引入羰乙基溴、苄溴结构，以实现与硫代磷酸酯骨架修饰的核酸骨架的进一步反应，并且在此过程中引入二硫键还可以实现药物的氧化还原释放。但此过程中不仅限于羰乙基溴和苄溴结构，其他含有可与硫代磷酸酯发生亲电反应的化合物均可引入；并且此结构也不仅限于引入二硫键，其他方式的可断裂的键也可用来替代二硫键，如酯酶可断裂的酯键、光照可断裂或酸性可断裂的化学键等。
优选地，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。
优选地，所述药物分子为抗肿瘤药物。但，本发明的药物分子不局限于抗肿瘤药物，治疗其他疾病的药物或者用于成像的药物分子也可用这种方法实现核酸修饰；在一些具体的实施例中，所述抗肿瘤药物为喜树碱或紫杉醇。但本发明所述的抗肿瘤药物也不局限于喜树碱和紫杉醇。其他可修饰的药物例如是顺铂、多西紫杉醇、苯丁酸氮芥、甲氨喋呤、多柔比星等。
在本发明中，核酸骨架的种类和序列不进行限制。在种类上既可选择脱氧核糖核酸序列，亦可选择核糖核酸序列；在序列要求上，即可选择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；也可选择功能性核酸序列，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。
优选地，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。
本发明还提供一种药物递送系统，其为由上述的核酸-药物结合物自组装形成的载药纳米体系。
在本发明中，利用上述的核酸-药物结合物所制备的组装体的形式不受限，例如：
可通过设计制备简单的核酸-药物大分子前药；设计的方法可以是：减少硫代磷酸酯的个数或选择亲水性强的小分子；
可通过设计制备精确组装的DNA纳米结构，如载药核酸多面体结构，并且不局限于DNA四面体，还可以构建不同结构大小的DNA折纸结构；也不局限于DNA，RNA纳米结构也可以选择。设计的方法可以是：选择特定序列的DNA或RNA序列，通过在核酸主链中特定位置进行硫代修饰，和药物结合后通过碱基互补配对的方式进行组装；
可通过设计制备DNA纳米凝胶等；设计的方法可以是：通过选择特殊序列的DNA或RNA序列，选择特定序列的DNA或RNA序列，通过在核酸主链中特定位置进行硫代修饰，制备Y型或i型组装小体，再进一步组装制备载药的纳米凝胶；
可通过设计制备载药的胶束型球形核酸；设计的方法可以是：选择普通或者功能性核酸序列，在一端连续进行硫代修饰，制备一端磷酸二酯键结构和一端硫代磷酸酯结构的嵌段型核酸，在硫代修饰位点进行药物修饰；因胶束型球形核酸的亲水外壳保持了其碱基互补配对的性质，可通过此性质赋予胶束型球形核酸靶向、成像的功能；
载药核酸水凝胶。
优选地，上述用于制备精确组装的DNA纳米结构的药物分子可尽量选择疏水性弱、分子量小的分子，且可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可选择疏水性强的分子，且可增加核酸骨架硫代修饰的数量。
在又一方面，本发明提供了一种上述的基于核酸硫代磷酸酯修饰的核酸-药物结合物的制备方法，主要包括如下步骤：
第一步、制备硫代修饰的核酸分子；优选地，通过固相合成方法制备硫代修饰的核酸分子，能够实现硫代修饰的位点和数量可调；
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；在优选的实施方式中，所述药物分子还含有可断裂的响应型化学键，更优选地，所述可断裂的响应型化学键为二硫键，所述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，可以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羟基的酯化反应得到苄溴结构修饰的紫杉醇药物分子；
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。反应完的处理过程可以是：加入一定体积的水溶液(水溶液的体积可通过有限次实验确定，此处不详述)，然后通过乙酸乙酯萃取的方法或乙醇沉淀的方法除去多余的小分子，水相干燥后即可得到核酸-药物结合物。
优选地，上述制备方法中，通过控制核酸分子浓度、药物分子和硫代磷酸酯的比例以及反应溶液中是否含有盐溶液可以控制药物分子的接枝效率。
在一些实施例中，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。
在一些实施例中，所述反应的温度和时间是：在50℃-58℃反应过夜或更长时间，但反应温度和时间可改变，并不限于此。
在又一方面，本发明提供了上述的核酸-药物结合物药物递送系统的制备方法，选自以下方法中的一种：
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；或
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上述的方法得到的核酸-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；以喜树碱为例：将四种不同序列的核酸-喜树碱前药在1×TAE/Mg 2+溶液缓冲液中以等摩尔量混合，在90℃下放置5min后迅速冷却至4℃，即得到载喜树碱的DNA纳米四面体；或
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，按照上述的方法得到的核酸-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；具体地，以紫杉醇为例：将所得到的嵌段结构的核酸-紫杉醇结合物溶于二甲基亚砜中，加入等体积的水分后在水中透析过夜，即得载紫杉醇的胶束型球形核酸；
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。具体地，例如选择含有与此球形核酸中的磷酸二酯键嵌段互补配对部分的功能性核酸序列(靶向或荧光修饰)，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。
在又一方面，本发明提供了一种基于上述的核酸-药物结合物及药物递送 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。
在又一方面，本发明提供了一种药物，其包括上述的核酸-药物结合物或其形成的药物递送系统。
优选地，所述药物为肿瘤治疗药物。
本发明的益处在于：
1、通过在硫代磷酸酯修饰的核酸分子上接枝药物分子，得到核酸-药物结合物，并通过核酸-药物结合物的自组装得到药物递送系统，实现了利用核酸对药物进行递送的目的，成功实现了利用核酸进行药物递送，满足载体材料的生物相容、体内可降解和低免疫原性的三大要求；
2、通过固相合成的方式得到硫代修饰的寡核苷酸，无需繁琐的化学合成，并且硫代修饰的位点和数量可以根据需要调整控制，对于后期的载药量和载药位点实现了精确设计的目的；
3、对可接枝的药物限制较少，实现了核酸作为载体材料的普适性；
4、合成过程简单；
5、可以通过自主设计寡核苷酸的序列和链段种类得到所需要的DNA纳米结构，进而制备可控的药物递送系统；
6、载药体系采用生物相容性的核酸为材料，对生物体有更低的免疫原性，更低的代谢负担，无毒副作用；
7、此载药体系还可以进一步引入靶向基团和功能性核酸序列，制备多功能的核酸载药体系，实现基因治疗、化疗联用的目的；还可实现肿瘤成像为一体的DNA纳米结构；
8、实现了多功能载药体系的合理简单构建。
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为实施例1和实施例2中溴代喜树碱前药和溴代紫杉醇分子的合成路线；
图2为实施例1中前药化合物1的 1H NMR谱图；
图3为实施例1中前药化合物1的LC-MS谱图；
图4为实施例1中前药化合物2的 1H NMR谱图；
图5为实施例1中前药化合物2的LC-MS谱图；
图6为实施例1中喜树碱修饰的寡核苷酸前药的紫外–可见光分光光度谱图；
图7为实施例1中喜树碱修饰的寡核苷酸前药的变性凝胶电泳图；
图8为实施例1中喜树碱修饰的寡核苷酸前药的基质辅助激光解吸电离飞行时间质谱图；
图9为实施例2中DTDP-Bz-Br的 1H NMR谱图；
图10为实施例2中PTX-Bz-Br的 1H NMR谱图；
图11为实施例2中PTX-Bz-Br的质谱图；
图12为实施例2中DNA-b-PTX-g-DNA的 31P NMR谱图；
图13为实施例2中DNA-b-PTX-g-DNA的基质辅助激光解吸电离飞行时间质谱图；
图14为实施例3中喜树碱修饰的DNA四面体折纸结构的琼脂糖凝胶电泳图；
图15为实施例3中喜树碱修饰的DNA四面体折纸结构的流体动力学直径数据图；
图16为实施例3中喜树碱修饰的DNA四面体折纸结构的原子力显微镜照片；
图17为实施例3中喜树碱修饰的DNA四面体折纸结构对癌细胞毒性评价示意图；
图18为实施例3中喜树碱修饰的DNA四面体折纸结构的实现癌细胞凋 亡示意图；
图19为实施例4中PTX-SNA的20％变形凝胶电泳图；
图20为实施例4中PTX-SNA的1％琼脂糖凝胶电泳图和动态光散射图；
图21为实施例4中PTX-SNA的透射电镜图；
图22为实施例4中多功能球形核酸的FAM/AS1411/PTX-SNA的1％琼脂糖凝胶电泳图；
图23为实施例4中靶向的基因和化疗共存的球形核酸AS1411/Bcl-2-PTX-SNA的琼脂糖凝胶电泳图；
图24为实施例4中载紫杉醇的各种胶束型球形核酸的体外抗肿瘤和逆转肿瘤多药耐药图。
具体实施方式
本发明提供一种基于核酸硫代磷酸酯骨架修饰的核酸-药物结合物、药物递送系统、它们的制备方法和应用。
本发明属于生物医药领域，具体公开了一种基于硫代磷酸酯修饰核酸的核酸-药物结合物、药物递送系统，及它们的制备方法。所述的核酸-药物结合物是由核酸硫代磷酸酯骨架中的硫代磷酸酯通过与可与其发生反应的亲电子反应基团修饰的药物分子接枝而成，其中，可通过不同目标来选择包括功能性核酸在内的核酸序列，此外，所述的核酸-药物结合物可自组装为含药纳米载体进行药物递送。与现有技术相比，本发明所采用的硫代磷酸酯核酸骨架通过简单的固相合成技术即可达到，通过硫代磷酸酯基团进行药物接枝，可精确控制药物分子在核酸骨架上的接枝位点及其组装行为，此方法对化疗药物具有普适性。本发明采用生物相容和可体内降解的水溶性核酸大分子作为载体，可显著改善化疗药物的理化性质和体内分布性质并促进其治疗作用，还可实现基因治疗、化疗的联合治疗，并避免了复杂的合成和修饰步骤。
在本文中，由「一数值至另一数值」表示的范围，是一种避免在说明书中一一列举该范围中的所有数值的概要性表示方式。因此，某一特定数值范 围的记载，涵盖该数值范围内的任意数值以及由该数值范围内的任意数值界定出的较小数值范围，如同在说明书中明文写出该任意数值和该较小数值范围一样。
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
1.1溴代喜树碱前药的合成，步骤见图1(A)，合成分为两步：
(一)、氧化还原敏感的前药化合物1的合成：氩气保护下，将喜树碱(1g)与三光气(313mg)溶于150ml无水二氯甲烷中，缓慢滴加4-二甲氨基吡啶(DMAP，1.12g溶于20mL二氯甲烷)，室温下搅拌反应半小时，加入2,2’-二硫二乙醇(4.43g)，室温反应过夜。
反应后混合液用80mL 0.1M HCL溶液洗涤，分层，弃去上清，HCL溶液反复清洗三次后，用80mL饱和NaCl溶液洗涤，分层，弃去上清，最后用80mL蒸馏水洗涤，分层，弃去上清，用无水MgSO 4干燥，粗产物采用柱层析法梯度淋洗进行分离纯化，分离极性为甲醇:二氯甲烷＝1:100。
本步骤产物产率64.5％，产物的核磁谱图见图2，其谱峰归属为： 1H NMR(400MHz,d 6-DMSO)δ(ppm):8.67(s,1H),8.15(d,J＝8.6Hz,1H),8.11(d,J＝7.9Hz,1H),7.85(ddd,J＝8.4,6.9,1.3Hz,1H),7.70(dd,J＝11.1,3.9Hz,1H),7.03(s,1H),5.56–5.38(m,2H),5.34–5.19(m,2H),4.38–4.19(m,2H),3.62–3.46(m,2H),3.02–2.88(m,2H),2.80–2.64(m,2H),2.30–2.01(m,2H),0.98–0.84(m,3H). 13C NMR(400MHz,d 6-DMSO)δ(ppm):167.54,156.93,153.29,152.62,148.33,146.69,145.22,132.00,130.86,130.19,129.45,128.94,128.40,128.14,119.58,94.86,78.36,66.92,59.75,50.70,41.56,36.63,30.78,8.05.。LC-MS液相色谱质谱联用测得化合物1的分子量与理论值吻合，如图3，ESI-MS m/z＝(M+H +)计算值529.106,检测值m/z＝(M+H +)529.103。
(二)、溴化喜树碱前药化合物2的合成：取化合物1(330mg)与N,N-二异丙基乙胺(88.8mg)在氩气保护下溶于150mL的无水二氯甲烷中，搅拌， 缓慢滴加溴乙酰溴(126.1mg，溶于超干二氯甲烷)，室温下反应过夜。反应完成后，利用旋转蒸发仪将溶剂蒸干，柱层析法梯度淋洗进行产物的纯化。二氧化硅做填料柱层析，分离极性为甲醇:二氯甲烷＝1:200。
本步骤产物产率80.7％，产物的核磁谱图见图4，其谱峰归属为： 1H NMR(400MHz,CDCl3)δ(ppm)：8.41(d,J＝13.0Hz,1H),8.21(d,J＝8.5Hz,1H),7.91(t,J＝10.3Hz,1H),7.90–7.71(m,1H),7.66(ddd,J＝8.0,7.0,1.0Hz,1H),7.33(s,1H),5.48(dt,J＝27.5,13.8Hz,2H),5.31–5.22(m,2H),4.48–4.20(m,4H),3.87–3.72(m,2H),3.01–2.80(m,4H),2.38–2.04(m,2H),0.99(t,J＝7.5Hz,3H). 13C NMR(400MHz,CDCl3)δ(ppm)：167.27,166.98,157.24,153.44,152.18,148.71,146.40,145.57,131.35,130.82,129.51,128.50,128.25,128.18,128.01,120.24,96.06,78.06,67.06,66.48,63.76,53.50,36.66,31.85,29.93,25.66,7.67.。LC-MS液相色谱质谱联用检测化合物2的分子量为m/z(M+H +)＝649.031，与理论值m/z(M+H +)649.530一致，如图5所示。
1.2寡核苷酸-喜树碱结合物前药的合成
本实例1.2中选择四条碱基互补的寡核苷酸序列，其硫代修饰位点在骨架中间，硫代磷酸酯基团个数为TET-A，TET-C:7个；TET-B，TET-D:8个。四条寡核苷酸的序列如下：
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’；
(*表示硫代磷酸酯基团修饰位点)。
取溴化喜树碱前药化合物2溶于80μL二甲基亚砜溶液中，加入20μL硫 代修饰寡核苷酸的磷酸缓冲液，寡核苷酸浓度在350μM(硫代磷酸酯基团与化合物2的比例为1:50)，置于55℃下，震荡反应20h。反应结束后，利用乙酸乙酯多次萃取除去反应中过量的化合物2，蒸干后，重新溶于超纯水中，即得到喜树碱修饰的寡核苷酸前药。将获得的4种碱基互补喜树碱修饰的寡核苷酸前药溶液通过紫外分光光度计检测，除260nm处DNA的特征吸收外，365nm处出现喜树碱分子的特征吸收峰，如图6所示。
本步骤中每条寡核苷酸链上接入了2-6个不同数量的喜树碱分子，大多数寡核苷酸链接入了4个喜树碱分子，TET-A和TET-C接入数量少于TET-B和TET-D，如图7所示的20％变性聚丙烯酰胺(PAGE)凝胶电泳检测分析，及图8所示的基质辅助激光解吸电离飞行时间质谱检测。
实例2 核酸-紫杉醇接枝物的合成
2.1苄溴修饰的紫杉醇药物(PTX-Bz-Br)的合成，步骤见图1(B)，其合成也分为两步：
(一)将4-溴甲基苯甲醇(500mg，1当量)和二硫代二丙酸(DTDP，2.6g，5当量)溶于超干的二氯甲烷和四氢呋喃的混合溶液(1/1，v/v)；然后加入DMAP(91mg，0.3当量)，搅拌几分钟后滴加二环己基碳二亚胺(DCC，615mg，1.2当量，溶于超干的二氯甲烷中)，室温反应过夜后，利用旋转蒸发仪将溶剂蒸干，通过硅胶柱层析分离得到含有二硫键的苄溴结构(DTPA-Bz-Br)，洗脱剂为石油醚/乙酸乙酯。产物核磁图及归属见图9。
(二)将紫杉醇(500mg，1当量)和DTPA-Bz-Br(230mg,1当量)溶于二氯甲烷中，然后加入DMAP(71mg，1当量)，搅拌几分钟后滴加二环己基碳二亚胺(DCC，145mg，1.2当量，溶于超干的二氯甲烷中)，室温反应过夜后，利用旋转蒸发仪将溶剂蒸干，通过硅胶柱层析分离得到苄溴修饰的紫杉醇分子(PTX-Bz-Br)，洗脱剂为石油醚/乙酸乙酯体系。产物核磁图见图10，质谱图见图11，所得的m/z的[M+1] +为1229。
2.2寡核苷酸-紫杉醇结合物前药的合成
本实例中所采用的是嵌段型DNA核酸序列，靠近5’端为磷酸二酯键修饰的DNA(26个碱基)，靠近3’端为连续的硫代磷酸酯键修饰的DNA(19个碱基，18个硫代磷酸二酯键)。
核酸序列如下：
POT 26- PST 18-T：5’-TTTTTTTTTTTTTTTTTTTTTTTTTTT*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T-3’；(*表示硫代磷酸酯基团修饰位点)。
Bcl-2- POT 10- PST 18-T：5’-TCTCCCAGCGTGCGCCATTTTTTTTTTTT*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T-3；(*表示硫代磷酸酯基团修饰位点)。
合成方法为：将PTX-Bz-Br溶于DMSO中，加入DNA后，置于55℃下震荡反应过夜。然后加入水后，用乙酸乙酯萃取除去反应中过量的PTX-Bz-Br，浓缩蒸干后，即得到嵌段型核酸-紫杉醇分子。通过核磁共振磷谱和基质辅助激光解吸电离飞行时间质谱验证其成功接枝，见图12和13。
实例3 缀合喜树碱的DNA多面体
3.1缀合喜树碱的DNA多面体的自组装
将获得的四种碱基互补的寡核苷酸前药在1×TAE/Mg 2+(40mM三羟甲基氨基甲烷,2mM乙二胺四乙酸二钠,20mM乙酸,12.5mM四水合乙酸镁,pH＝7.4,乙酸调节pH值)缓冲液中等摩尔量混合，升温至90℃，在2min内迅速冷却至4℃。通过2％琼脂糖凝胶电泳检测，逐步加入不同种寡核苷酸前药，纳米结构不断变大，最终组装得到多面体结构，相比于未修饰的DNA多面体，尺寸有所增加，如图14所示。动态光散射检测显示，药物修饰的DNA多面体的水合粒径在24nm左右，相对无修饰的DNA多面体粒径增加了约10nm，如图15。图16为原子力显微镜拍摄的药物修饰的DNA多面体，粒径在18nm左右。
3.2缀合喜树碱的DNA多面体体外抗肿瘤作用
以上制得的通过硫代寡核苷酸进行药物修饰的DNA多面体，可以有效地 被肿瘤细胞摄取而产生与原药相近甚至更佳的癌细胞杀伤效果。
将实例3.1中制备的药物修饰多面体与HCT 116细胞共孵育72h后，通过MTT检测细胞存活率，结果如图17所示，与药物修饰的多面体共孵育的细胞随药物浓度升高，存活率逐渐降低，并且在较高药物浓度下，药物修饰的多面体导致了相对于原药更低的细胞存活率，证明将药物通过硫代修饰寡核苷酸实现药物输送的方式可以实现药物的有效输送，并且产生良好的肿瘤细胞杀伤效果。
3.3缀合喜树碱的DNA多面体体外细胞凋亡作用
本发明的通过硫代寡核苷酸进行药物修饰的DNA多面体，通过诱导癌细胞凋亡的方式实现对肿瘤细胞杀伤作用。
将实例3.1中制备的药物修饰多面体与HCT 116细胞共孵育48h后，采用Annexin V-FITC/PI方法检测肿瘤细胞凋亡，结果如图18所示，与药物修饰的多面体共孵育的细胞可以实现良好的诱导癌细胞凋亡的作用，并且造成与原药相近的细胞凋亡率。证明将药物通过硫代修饰寡核苷酸实现药物输送的方式可以实现药物的快速释放，诱导肿瘤细胞凋亡，最终达到治疗癌症的目的。
实例4 缀合紫杉醇的胶束型球形核酸
4.1普通核酸嵌段型载紫杉醇的球形核酸的制备(PTX-SNA)
选择嵌段型的polyT碱基序列( POT 26- PST 18-T),按照实例2.2中的方法得到嵌段型核酸-紫杉醇结合物( PODNA-b-( PSDNA-g-PTX))，将其溶于DMSO中然后加入等体积的水，在水溶液中透析过夜即可得到PTX-SNA。通过20％变性的聚丙烯酰胺凝胶电泳、1％的琼脂糖凝胶电泳证明其组装成功。其粒径大小和形貌通过动态光散射和透射电镜进行表征。结果见图19-图21。
4.2具有靶向和成像功能的多功能载药胶束的制备(AS1411/PTX-SNA，FAM/AS1411/PTX-SNA)
将4.1中的PTX-SNA和polyA 20-AS1411和polyA 20-FAM碱基序列按照比例混合在1×TAE/Mg 2+，然后从65℃开始退火至室温即可得到具有靶向和成像 功能的多功能载药胶束。通过1％琼脂糖凝胶表征其组装，如图22。
核酸序列如下：
PolyA 20-FAM：5’-AAAAAAAAAAAAAAAAAAAA-FAM-3’；
PolyA 20-AS1411：5’-AAAAAAAAAAAAAAAAAAAAGGTGGTGGTGGTTGTGGTGGTGGTGG-3’。
4.3具有基因和化疗共存的无靶向或靶向的载药球形核酸的制备(Bcl-2-PTX-SNA，AS1411/Bcl-2-PTX-SNA)通过将嵌段DNA中的磷酸二酯键序列改为反义核酸Bcl-2序列，可以达到逆转肿瘤多药耐药的目的。
选择功能性核酸(反义核酸)作为嵌段的Bcl-2- POT 10- PST 18-T碱基序列,按照实例2.2中的方法得到含有功能性核酸序列嵌段型核酸-紫杉醇结合物(Bcl-2-b-( PSDNA-g-PTX))，将其溶于DMSO中然后加入等体积的水，在水溶液中透析过夜即可得到非靶向的基因和化疗共存的Bcl-2-PTX-SNA。将Bcl-2-b-( PSDNA-g-PTX)和 POT 26-b-( PSDNA-g-PTX)按比例共同透析所得的SNA，与polyA 20-AS1411按照比例混合在1×TAE/Mg 2+，然后从65℃开始退火至室温即可得到具有靶向作用的基因和化疗共存的AS1411/Bcl-2-PTX-SNA。通过0.5％琼脂糖凝胶表征其组装，如图23。
4.4缀合紫杉醇的胶束型球形核酸的体外抗肿瘤和逆转肿瘤多药耐药作用
将实例4.1-4.3中制备的各种PTX-SNA和肿瘤细胞共孵育72h后，通过MTT检测细胞存活率，结果如图24所示。其中，MCF-7和HeLa细胞为敏感肿瘤细胞，L929细胞为正常细胞，用于表征靶向AS1411/PTX-SNA的抗肿瘤效果；HeLa/PTX为紫杉醇耐药细胞，用于表征AS1411/Bcl-2-PTX-SNA逆转肿瘤耐药的效果。实验结果表明，具有靶向分子修饰的AS1411/PTX-SNA对肿瘤细胞的具有较好的靶向功能，且肿瘤细胞杀伤作用好于非靶向的PTX-SNA。对于基因和化疗共存的靶向AS1411/Bcl-2-PTX-SNA来说，其可以通过下调抗凋亡Bcl-2蛋白的基因表达发挥逆转肿瘤多药耐药的目的，实现很好的基因和化疗联合应用的目的。
本发明上述实施例首先得到了载药体系DNA四面体折纸结构，其水合纳米粒径为20纳米。将极其疏水的喜树碱药物分子精确准确地接枝到寡核苷酸 链段中，改善了喜树碱药物的溶解性，形成了水溶性纳米前药，并且通过预先设计的DNA序列实现了多面体结构的组装，达到了载药体系的结构可控性。其次，本发明上述实施例还得到了载药胶束型球形核酸，其水合粒径为68纳米；将紫杉醇药物精准接枝到核酸的硫代磷酸酯基团上，利用磷酸二酯键和硫代磷酸酯键嵌段型的DNA结构和紫杉醇的疏水性质，制备以药物为内核、以磷酸二酯键的核酸嵌段为外壳的胶束结构，所制备的胶束载药量高达53％。
在本发明及上述实施例的教导下，本领域技术人员很容易预见到，本发明所列举或例举的各原料或其等同替换物、各加工方法或其等同替换物都能实现本发明，以及各原料和加工方法的参数上下限取值、区间值都能实现本发明，在此不一一列举实施例。

Claims (24)

  1. 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。
  2. 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。
  3. 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。
  4. 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。
  5. 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。
  6. 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。
  7. 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。
  8. 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。
  9. 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。
  10. 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。
  11. 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。
  12. 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。
  13. 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。
  14. 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。
  15. 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。
  16. 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。
  17. 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。
  18. 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。
  19. 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。
  20. 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。
  21. 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。
  22. 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。
  23. 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。
  24. 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。
PCT/CN2019/072790 2019-01-23 2019-01-23 核酸-药物结合物、药物递送系统及其制备方法和应用 WO2020150915A1 (zh)

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