WO2020042470A1 - 一种用于抗肿瘤药物递送的聚二硫苏糖醇纳米体系及其制备方法和应用 - Google Patents

一种用于抗肿瘤药物递送的聚二硫苏糖醇纳米体系及其制备方法和应用 Download PDF

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WO2020042470A1
WO2020042470A1 PCT/CN2018/122760 CN2018122760W WO2020042470A1 WO 2020042470 A1 WO2020042470 A1 WO 2020042470A1 CN 2018122760 W CN2018122760 W CN 2018122760W WO 2020042470 A1 WO2020042470 A1 WO 2020042470A1
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polydithiothreitol
pdtt
drug
polymer
grafted
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French (fr)
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吴钧
康洋
张鑫宇
易恬琦
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中山大学
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G75/00—Macromolecular compounds obtained by reactions forming a linkage containing sulfur with or without nitrogen, oxygen, or carbon in the main chain of the macromolecule
    • C08G75/02—Polythioethers
    • C08G75/0204—Polyarylenethioethers
    • C08G75/025—Preparatory processes
    • C08G75/0268—Preparatory processes using disulfides
    • 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
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • 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
    • A61K47/69—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
    • A61K47/6931—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 the material constituting the nanoparticle being a polymer
    • A61K47/6935—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 the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention belongs to the technical field of functional polymer materials and biomedicine. More specifically, it relates to a polydithiothreitol nano system for antitumor drug delivery, and a preparation method and application thereof.
  • Stimuli-responsive polymer systems have been widely used in the field of nanomedicine because they can respond to external environments such as temperature, light, enzymes, and pH.
  • the reduction-response controlled-release method has been reported by many researchers, and is mainly used for the preparation of drug-controlled release systems targeting tumor cells.
  • Reduction responsiveness generally refers to the introduction of reduction-sensitive groups into high-molecular polymers, and the use of high-concentration glutathione in tumor cells to reduce these specific groups such as disulfide bonds in drug systems To release the drug into the cell.
  • Glutathione is an important free radical scavenger and antioxidant in the body.
  • concentration of glutathione in body fluids and extracellular matrix is 2-20 ⁇ M, and the concentration in cytoplasm is 2-10 mM.
  • concentration of glutathione is at least four times that of normal cells. This particular concentration gradient change has made the disulfide bond-containing drug release system show a broad application prospect in the field of biomedicine in recent years.
  • camptothecin is an anticancer drug screened by M.E.Wall and M.C.Wani in 1966 and is a quinoline alkaloid. Because camptothecin has low water solubility, it is difficult to prepare a more ideal formulation. At present, there are two main methods of camptothecin used in nanometer drug loading systems: physical drug loading and chemical drug loading.
  • camptothecin The former is used to encapsulate camptothecin through physical adsorption through liposomes, micelles, etc., while the latter is to graft the camptothecin to the carrier through chemical bonds.
  • Many studies have shown that the grafting of the 20-position hydroxyl group of camptothecin can increase the stability of its lactone ring and change the hydrophilic and hydrophobic properties. The most common is the grafting of camptothecin to 3 kDa to 40 kDa polyethylene glycol to form a polyethylene glycol prodrug.
  • Prothecan developed by Enzon, connects camptothecin to both ends of polyethylene glycol with a molecular weight of 4000, and has now entered the phase III clinical research stage.
  • the macromolecular carriers used for camptothecin grafting are poly-L-glutamic acid (PGA), polysaccharides, and some synthetic polymers.
  • the technical problem to be solved by the present invention is to overcome the shortcomings and deficiencies of the prior art mentioned above, and provide a polydithiothreitol nano system for antitumor drug delivery.
  • the present invention uses dithiothreitol as a monomer to quickly and simply prepare a polydithiothreitol with good biocompatibility; a hydrophobic antitumor drug grafted with polydithiothreitol to form Antitumor drug delivery system, realizes the controlled drug loading of hydrophobic drugs, improves the solubility of drugs, greatly improves the availability of hydrophobic drugs, and has the rapid reduction response of GSH, which improves the targeting of antitumor drugs Sex.
  • a first object of the present invention is to provide a polymer PDTT.
  • a second object of the present invention is to provide a method for preparing the above-mentioned polymer PDTT.
  • a third object of the present invention is to provide an application of the above-mentioned polymer PDTT as a drug delivery carrier.
  • a fourth object of the present invention is to provide a polydithiothreitol nanosystem for drug delivery with reduction responsiveness.
  • a fifth object of the present invention is to provide a method for preparing the above polydithiothreitol nano system.
  • a sixth object of the present invention is to provide an application of the above-mentioned polymer PDTT or the above-mentioned polydithiothreitol nanosystem in the preparation of anticancer drugs.
  • a polymer PDTT for drug delivery is composed of a plurality of repeating dithiothreitol structural units, and its structural formula is represented by the following formula (I):
  • n 5 to 10,000.
  • the molecular weight of the polymer PDTT is 1500-45000.
  • the invention prepares a new polymer containing disulfide bond polydithiothreitol (PDTT) with controllable composition and structure, successfully constructs a nanocarrier for targeted delivery of antitumor drugs; and grafts different antitumor drugs to Polydithiothreitol (PDTT) on the hydroxyl group (PDTT-CPT), and then through a single emulsion method, nano-precipitation method and other nano-particle preparation processes to form a uniform and stable nano-particle solution, or can also pass other
  • the preparation method is prepared into a drug carrier such as an oral sustained-release preparation or an injectable hydrogel.
  • the novel polymer polydithiothreitol of the present invention is stable in properties after grafting the drug, has good biocompatibility and degradability, and has fast response response to GSH, improves the targeted delivery efficiency of the nanocarrier, and achieves
  • the controlled and controlled release of drugs in tumor cells improves the bioavailability of hydrophobic drugs and opens up a new way for effective treatment of diseases.
  • the invention also provides a method for preparing a polymer PDTT: after disthiothreitol is dissolved with organic solvent A, a polymerization reaction is performed under vacuum conditions, so that the thiol group at the end of dithiothreitol is oxidized to disulfide bonds, thereby The polymer PDTT was obtained.
  • the temperature of the polymerization reaction is 40 to 180 ° C, and the time of the polymerization reaction is 10 min to 48 h.
  • the temperature of the polymerization reaction is 90 to 100 ° C, and the time of the polymerization reaction is 10 to 20 hours.
  • the mass-volume ratio of the dithiothreitol and the organic solvent A is 1 to 5 g: 2 to 20 mL.
  • the mass-volume ratio of the dithiothreitol and the organic solvent A is 1 to 5 g: 2 to 20 mL.
  • the mass-volume ratio of the dithiothreitol and the organic solvent A is 2 to 4 g: 5 to 11 mL.
  • the mass-volume ratio of the dithiothreitol and the organic solvent A is 2 to 4 g: 5 to 11 mL.
  • the organic solvent A is selected from the group consisting of dimethyl sulfoxide, N, N-dimethylformamide, N, N-diethylformamide, N, N-diethylacetamide, acetone, and tetrahydrofuran. One or more.
  • the invention also provides the application of the polymer PDTT as a drug delivery carrier.
  • the delivery carrier is a delivery carrier having reduction responsiveness.
  • the present invention also provides a polydithiothreitol nano system for drug delivery with reduction responsiveness, which uses the polymer PDTT as a nano-carrier to load a drug.
  • the invention also provides a method for preparing the polydithiothreitol nanometer system, comprising the following steps:
  • the above-mentioned drug-grafted polymer PDTT is prepared by a single emulsion method or a nano-precipitation method to obtain a uniform and stable nano-particle solution, which is the polydithiothreitol nano-system for drug delivery. .
  • the mass ratio of the drug and the organic basic catalyst is 1 to 5: 1 to 10.
  • the mass ratio of the drug and the organic basic catalyst is 1 to 5: 1 to 10.
  • the mass ratio of the drug and the organic basic catalyst is 1 to 2: 1 to 5.
  • the mass ratio of the drug and the organic basic catalyst is 1 to 2: 1 to 5.
  • the concentration of the polymer PDTT is 0.01 to 2.5 g / mL.
  • concentration of the polymer PDTT is 0.01 to 2.5 g / mL.
  • concentration of the polymer PDTT is 0.01 to 2.5 g / mL.
  • the concentration of the polymer PDTT is 0.01 to 1 g / mL.
  • step S1 the time of the acylation reaction is 10 to 60 min; and the time of the graft reaction is 3 to 48 h.
  • the temperature of the grafting reaction is room temperature.
  • the room temperature means 15 to 25 ° C.
  • step S1 the acylation reaction and the grafting reaction are both performed under the protection of argon gas.
  • the stirring time is 1 min to 1 h.
  • the organic solvent B is selected from methylene chloride and / or tetrahydrofuran.
  • the organic basic catalyst is selected from 4-dimethylaminopyridine (DMAP), 4-dimethylaminopyridine-p-toluenesulfonate (DPTS) or N-hydroxysuccinimide. (NHS).
  • DMAP 4-dimethylaminopyridine
  • DPTS 4-dimethylaminopyridine-p-toluenesulfonate
  • NHS N-hydroxysuccinimide.
  • the method for preparing a nanoparticle solution by a single emulsion method includes the following steps:
  • the polymer grafted with PDTT is ultrasonically dissolved in an organic solvent C which is immiscible with water and has a low boiling point to obtain an emulsion oil phase;
  • the water-immiscible organic solvent refers to an organic solvent having a solubility in water of less than 0.01 g.
  • the organic solvent having a low boiling point refers to an organic solvent having a boiling point lower than 60 ° C.
  • the organic solvent C which is immiscible with water and has a low boiling point includes, but is not limited to, chloroform, dichloromethane, acetone, or ethyl acetate.
  • the method for preparing a nanoparticle solution by a nano-precipitation method includes the following steps:
  • step S221 the oil phase is slowly dropped into the water phase, and the dropping acceleration thereof is 0.01 to 1 mL / second.
  • step S221 the drug-grafted polymer PDTT and the stabilizer are completely dissolved in the organic solvent D miscible with water at a mass ratio of 1 to 20: 1.
  • the organic solvent D miscible with water such as 1: 1, 2: 1, 5: 1, 10: 1, 20: 1, etc.
  • step S221 the drug-grafted polymer PDTT and the stabilizer are completely dissolved in the organic solvent D miscible with water at a mass ratio of 5: 1.
  • the organic solvent D miscible with water refers to an organic solvent having a solubility in water of more than 100 g.
  • the water-miscible organic solvent D includes, but is not limited to, dimethyl sulfoxide, N, N-dimethylformamide, tetrahydrofuran, dioxane, pyridine, and the like.
  • the stabilizer includes distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) type stabilizer, polyvinyl alcohol (PVA), phospholipid molecules, and the like.
  • DSPE-PEG distearoylphosphatidylethanolamine-polyethylene glycol
  • PVA polyvinyl alcohol
  • the drugs include hydrophobic or hydrophilic drugs.
  • the polymer PDTT of the present invention can not only support hydrophobic drugs, improve the solubility of hydrophobic drugs, improve its bioavailability, but also support hydrophilic drugs, and has good biocompatibility.
  • the drug includes a drug containing a -OH, -NH 2 , -COOH or halogen functional group, a protein drug or a nucleic acid drug. It was found through experiments that the polymer PDTT according to the present invention can achieve high-efficiency loading of drugs such as -OH, -NH 2 , -COOH or halogen functional groups, drugs, protein drugs, and nucleic acid drugs.
  • the protein drugs include, but are not limited to, protein drugs such as trastuzumab, ranibizumab, interferon, erythropoietin, and streptokinase.
  • the nucleic acid drugs include, but are not limited to, nucleic acid drugs such as lamivudine, adefovir dipivoxil, inosine, and coenzyme A.
  • the drugs include antitumor drugs, antibiotic drugs, and antiviral drugs.
  • the antitumor drug is selected from the group consisting of camptothecin, ursolic acid, methotrexate, 9-aminocamptothecin, trastuzumab, nitrogen mustard, cisplatin, cyclophosphamide, fluorouracil, podophyllotoxin, and the like One or more.
  • the antibiotic drugs include polymyxin, ampicillin, cephalexin, amikacin and the like.
  • the antiviral drugs include lamivudine, ganciclovir, entecavir, adefovir dipivoxil and the like.
  • the -COOH functional group-containing drugs include methotrexate, amino acid (polymyxin) drugs, protein (interferon) drugs, and the like.
  • the polydithiothreitol reacts with a drug having a carboxyl group (R-COOH) to generate a drug having an ester bond.
  • the polydithiothreitol reacts with a halogen-containing drug to form a drug having an ether bond.
  • the nanoparticle solution prepared by the above method is measured by DLS, and the particle diameter of the nanoparticle is controlled to be 10-500 nm.
  • the particle size of the nanoparticles is 10-150 nm.
  • the polymer polydithiothreitol grafted nanoparticles have fast GSH response. According to the detection results of the release of free drugs in nano-systems in PBS solutions with different GSH concentrations at 37 ° C, compared with the absence of GSH, the release of free drugs in the presence of GSH in the nanoparticles increased significantly, and with the The higher the GSH concentration, the faster the nanosystem releases free drugs. This is because the disulfide bond in the thiol attack system of GSH causes the disulfide bond to be reduced to a thiol group, and the increase of the thiol group in the environment accelerates the degradation of degradable chemical bonds (such as ester bonds). The above actions are carried out at the same time, which accelerates the degradation of the nano-system and the drug is quickly released.
  • the polydithiothreitol of the present invention has a good application prospect of a drug carrier, and is also embodied in: performing the MCF-7 / A549 / HpeG-2 with the new polydithiothreitol and the monomer dithiothreitol / CT-26 / 4T1 tumor cytotoxicity test found that polydithiothreitol did not significantly inhibit cell growth in a sufficiently large concentration range, and showed better than the monomer dithiothreitol Biocompatibility. After the grafted drug was formed into a nano-system, compared with the single-drug drug, the anti-tumor effect of polydithiothreitol was comparable, and it still showed excellent anti-tumor activity.
  • the present invention has the following beneficial effects:
  • a polymer polydithiothreitol is simply and efficiently synthesized through a polymerization reaction of a thiol group.
  • the polymer has a stable structure, good biocompatibility, can encapsulate hydrophobic antitumor drugs, and polymerizes.
  • the presence of a large number of disulfide bonds in the system makes the system have a rapid reduction response of GSH.
  • the drug according to the present invention will be grafted to polyethylene dithiothreitol side groups, such as to achieve a controlled drug camptothecin containing -OH or -NH 2 functional group such as a hydrophobic drug, improving the solubility of the drug , which greatly improves the availability of hydrophobic drugs.
  • the present invention uses polydithiothreitol as a carrier-loaded drug, which can obtain nanoparticles with a controlled particle size, a small particle size distribution range, and high permeability and high retention (EPR effect) through tumor tissue. It accumulates in tumor tissues to achieve passive targeting.
  • Polydithiothreitol nanosystems can reduce the release of drugs under normal physiological conditions to reduce the toxic and side effects of drug burst release. After being taken up by tumor cells, Under the action of glutathione in tumor cells, the disulfide bonds in the nanosystem are broken down and reduced to sulfhydryl groups. Monomers with thiol groups can promote the disintegration of the system and promote drug release. At the same time, the released small molecules act on carbonate bonds. , Accelerating the release of drugs, enhancing the therapeutic effect of cancer, has extremely high clinical application value.
  • the invention has the advantages of simple reaction process, few reaction steps, short reaction cycle, good repeatability, etc., and has good application prospects and broad development space in the field of medicine.
  • FIG. 1 is a schematic diagram of nanoparticles formed by grafting camptothecin-containing polydithiothreitol in the present invention and its release in tumor cells.
  • FIG. 2 is a nuclear magnetic hydrogen spectrum of polydithiothreitol in the present invention.
  • FIG. 3 is a nuclear magnetic hydrogen spectrum of a camptothecin-grafted polydithiothreitol in the present invention.
  • FIG. 4 is a transmission electron microscopy (TEM) result diagram of nanoparticles formed of polydithiothreitol grafted with camptothecin.
  • TEM transmission electron microscopy
  • FIG. 5 is a graph of dynamic light scattering (DLS) results of nanoparticles formed of polydithiothreitol grafted with camptothecin.
  • Figure 6 is a drug release profile of nanoparticles formed from polydithiothreitol grafted with camptothecin at different concentrations of GSH at 37 ° C.
  • FIG. 7 is a cytotoxicity chart of polydithiothreitol (PDTT) and dithiothreitol monomer (DTT) on MCF-7 breast tumor cells.
  • FIG. 8 is a cytotoxicity diagram of MCF-7 breast tumor cells formed by grafting camptothecin-containing polydithiothreitol nanoparticles (PDTT-CPT) and camptothecin monotherapy (CPT).
  • PDTT-CPT camptothecin-containing polydithiothreitol nanoparticles
  • CPT camptothecin monotherapy
  • the present invention is further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form.
  • the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.
  • the NMR spectrum of polymer PDTT is shown in Figure 2. From Figure 2, it can be seen that the signal at ⁇ 5.17 ppm is the absorption peak of solvent heavy water, and the signal at ⁇ 3.34 ppm corresponds to -CH where two hydroxyl groups of dithiothreitol are connected The proton absorption peaks on-, the signals at ⁇ 3.02ppm and ⁇ 2.72ppm correspond to the proton absorption peaks on the two methylene-CH 2 -groups connected to the two mercapto groups of dithiothreitol, and the results show that the polymer was successfully synthesized PDTT, the specific structural formula is shown in formula (I):
  • n 5 to 10,000.
  • the prepared polydithiothreitol has a stable structure, good biocompatibility, and can support hydrophobic antitumor drugs, and a large number of disulfide bonds in the polymer make the system have a rapid reduction response of GSH.
  • the drug delivery carrier has a good application prospect.
  • FIG. 3 The nuclear magnetic hydrogen spectrum of the polydithiothreitol grafted with camptothecin is shown in FIG. 3. As can be seen from FIG. 3, it is ⁇ 8.69ppm, ⁇ 8.17ppm, ⁇ 8.11ppm, ⁇ 7.86ppm, ⁇ 7.72ppm, ⁇ The signals at 7.45ppm, ⁇ 5.51ppm, and ⁇ 5.43ppm correspond to the proton absorption peaks on the aromatic ring of camptothecin.
  • ⁇ 1.74ppm, ⁇ 0.89ppm represent the absorption peaks on the methylene and ethyl groups of camptothecin
  • ⁇ 3.34ppm , ⁇ 3.05ppm and ⁇ 5.27ppm respectively represent the proton absorption peaks on the methyl, methylene and hydroxyl groups of the last polydithiothreitol.
  • Example 5 A polydithiothreitol nano system with anti-tumor drug delivery with reduction responsiveness
  • Example 3 The polydithiothreitol grafted with camptothecin in Example 3 was dissolved in methylene chloride ultrasonically to obtain an emulsion oil phase, and the concentration of the oil phase was 30 mg / mL;
  • the white mixed emulsion is transferred to room temperature, and after stirring for one day and one night, the low boiling point methylene chloride is evaporated to obtain a nanoparticle solution with a Tyndall effect.
  • the large particle impurities are removed by filtration through a 0.8 ⁇ m filter membrane to obtain a drug-loaded
  • the nanoparticle solution is the polydithiothreitol nanosystem with reduction responsiveness for antitumor drug delivery.
  • the DLS test is performed, and the particle size of the nanoparticles in this embodiment is 150 nm, and no precipitation occurs when it is placed in a refrigerator at 4 ° C.
  • the transmission electron microscopy (TEM) results and dynamic light scattering (DLS) results are shown in Figures 4 and 5, respectively.
  • the results showed that the PDTT nanoparticles with camptothecin were spherical, uniform in size and narrow in particle size distribution.
  • Nanoparticles formed by polydithiothreitol (PDTT), dithiothreitol monomer (DTT), and polydithiothreitol grafted with camptothecin in this example.
  • the camptothecin monotherapy (CPT) was used to conduct anti-MCF-7 tumor cytotoxicity experiments in vitro, and the results are shown in Figures 7 and 8. It can be seen that polydithiothreitol has good biocompatibility and does not significantly inhibit the growth of MCF-7 cells in a large range of concentration.
  • PDTT nanoparticles loaded with camptothecin maintain the excellent anticancer activity of camptothecin.
  • Example 6 A polydithiothreitol nanosystem with anti-tumor drug delivery with reduction responsiveness
  • the DLS test is performed.
  • the particle size of the nanoparticles in this embodiment is about 100 nm, and no precipitation occurs in the refrigerator at 4 ° C.
  • the transmission electron microscopy (TEM) results and dynamic light scattering (DLS) results are shown in Figures 4 and 5, respectively.
  • the results showed that the PDTT nanoparticles with camptothecin were spherical, uniform in size and narrow in particle size distribution.
  • Nanoparticles formed by polydithiothreitol (PDTT), dithiothreitol monomer (DTT), and polydithiothreitol grafted with camptothecin in this example.
  • the camptothecin monotherapy (CPT) was used to conduct anti-MCF-7 tumor cytotoxicity experiments in vitro, and the results are shown in Figures 7 and 8. It can be seen that polydithiothreitol has good biocompatibility and does not significantly inhibit the growth of MCF-7 cells in a large range of concentration.
  • PDTT nanoparticles loaded with camptothecin retain the excellent anti-cancer activity of camptothecin, have a significant anti-tumor effect, and have certain application potential in the field of cancer treatment.
  • Example 2 The other conditions are the same as those in Example 1, except that the polymerization reaction temperature is 40 ° C. and the polymerization reaction time is 48 h. A white solid polymer PDTT is prepared.
  • Example 2 The other conditions are the same as those in Example 2. The only difference is that the polymerization reaction temperature is 180 ° C. and the polymerization reaction time is 10 min. A white solid polymer PDTT is prepared.
  • the other conditions are the same as those in Example 3, except that the time for the acylation reaction is 10 min and the time for the graft reaction is 48 h.
  • a polydithiothreitol grafted with camptothecin is prepared.
  • the tumor drug delivery system realizes the controlled drug loading of hydrophobic drugs, improves the solubility of the drugs, greatly improves the availability of hydrophobic drugs, and has a fast reduction response of GSH, improving the targeting of anti-tumor drugs .
  • the other conditions were the same as those in Example 4, except that the time for the acylation reaction was 60 min and the time for the graft reaction was 3 h.
  • a polydithiothreitol grafted with camptothecin was prepared.
  • the tumor drug delivery system realizes the controlled drug loading of hydrophobic drugs, improves the solubility of the drugs, greatly improves the availability of hydrophobic drugs, and has a fast reduction response of GSH, improving the targeting of anti-tumor drugs .
  • Example 11 A polydithiothreitol nanosystem with anti-tumor drug delivery with reduction responsiveness
  • the other conditions are the same as those of Example 6, except that the mass ratio of the polydithiothreitol grafted with camptothecin and the stabilizer DSPE-PEG 3000 is 5: 1.
  • the resulting antitumor drug delivery system achieved a controlled drug loading of camptothecin, improved the solubility of camptothecin, greatly improved the availability of camptothecin, and had a fast reduction response of GSH. Improve the targeting of the drug camptothecin.
  • polydithiothreitol nanometer system of the present invention is also suitable for applications such as ursolic acid, methotrexate, 9-aminocamptothecin, trastuzumab, nitrogen mustard, cisplatin, cyclophosphamide, and fluorouracil.
  • Antitumor drugs such as podophyllotoxin, such as trastuzumab, ranibizumab, interferon, erythropoietin, streptokinase drugs, such as lamivudine, adefovir dipivoxil, inosine
  • nucleic acid drugs such as coenzyme A
  • the new polymer polydithiothreitol of the present invention is stable in nature after grafting the above-mentioned drugs, has good biocompatibility and degradability, and has fast GSH reduction responsiveness, improving The efficiency of targeted delivery of nanocarriers is achieved, the controlled release of drugs in tumor cells is achieved, the bioavailability of hydrophobic drugs is improved, and a new approach is opened for effective treatment of diseases.

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Abstract

一种基于聚二硫苏糖醇的载药纳米体体系及其制备方法和应用。所述聚二硫苏糖醇结构中包含含有二硫键的聚二硫苏糖醇并且在其羟基上接枝有抗肿瘤药物，所述聚二硫苏糖醇通过单乳法、纳米沉淀法等纳米粒制备方法制成粒径均匀且稳定的纳米粒溶液。

Description

一种用于抗肿瘤药物递送的聚二硫苏糖醇纳米体系及其制备方法和应用 技术领域
本发明属于功能高分子材料和生物医药技术领域。更具体地，涉及一种用于抗肿瘤药物递送的聚二硫苏糖醇纳米体系及其制备方法和应用。
背景技术
恶性肿瘤已成为威胁人体生命健康的主要疾病之一。尽管临床上有超过90多种抗肿瘤药物，但绝大多数抗肿瘤药物存在水溶性差、控释效果差、靶向性差和促使肿瘤产生强烈耐药性等缺点。同时，这些药物对正常组织和器官也能带来严重的毒副作用，降低人体免疫力。
刺激响应性聚合物体系因为可以对温度、光照、酶、pH等外界环境做出响应，已被广泛应用于纳米药物领域。其中还原响应控释方法已经被不少研究者们报道，主要用于靶向肿瘤细胞的药物控释体系的制备。还原响应性一般是指将具有还原敏感性的基团引入到高分子聚合物中，利用肿瘤细胞内高浓度谷胱甘肽的还原作用，将药物体系中的这些特定基团如二硫键断裂，从而将药物释放到细胞内。谷胱甘肽是体内重要的自由基清除剂和抗氧化剂，研究表明，谷胱甘肽在体液和细胞外基质中的浓度为2～20μM，细胞质内的浓度为2～10mM，而肿瘤细胞内的谷胱甘肽浓度至少是正常细胞的4倍。这一特别的浓度梯度的变化，让含二硫键的释药体系近年来在生物医学领域展现出了广泛的应用前景。
传统的化疗和靶向治疗是目前临床肿瘤治疗重要而有力的手段，但是在治疗过程中，由于药物的特异性较低，水溶性低，毒性高，导致在杀灭癌细胞的同时也会不可避免的对机体正常细胞产生损伤作用，从而影响机体健康器官的功能。例如，喜树碱是1966年由M.E.Wall和M.C.Wani筛选出来的抗癌药物，是一种喹啉类生物碱。由于喜树碱水溶性较低，难以制备较理想的制剂。目前，喜树碱用于纳米载药体系的方法主要有两种：物理载药和化学载药。前者是通过脂质体，胶束等利用物理吸附包载喜树碱，后者则是通过化学键将喜树碱接枝于载体。许多研究表明通过喜树碱20位羟基的接枝可以增加其内酯环的稳定性，改变亲疏水性能。最常见的是将喜树碱接枝到3kDa到40kDa的聚乙二醇上形成聚乙二醇前药。Enzon公司所开发的Prothecan，将喜树碱连接到分子量为4000的聚乙 二醇的两端，目前已经进入Ⅲ临床研究阶段。除此之外，用于喜树碱接枝的大分子载体还有聚L-谷氨酸(PGA)，多糖以及一些合成高分子。
发明内容
本发明要解决的技术问题是克服上述现有技术的缺陷和不足，提供一种用于抗肿瘤药物递送的聚二硫苏糖醇纳米体系。本发明以二硫苏糖醇为单体，快速而简便地制备得到具有良好的生物相容性的聚二硫苏糖醇；以聚二硫苏糖醇接枝疏水性抗肿瘤药物从而形成的抗肿瘤药物递送体系，实现了疏水药物的可控载药，改善了药物的溶解性，极大的提高了疏水药物的可利用性，并且具有GSH快速还原响应性，提高抗肿瘤药物的靶向性。
本发明的第一个目的是提供一种聚合物PDTT。
本发明的第二个目的是提供上述聚合物PDTT的制备方法。
本发明的第三个目的是提供上述聚合物PDTT在作为或制备药物传递载体方面的应用。
本发明的第四个目的是提供一种具有还原响应性的用于药物递送的聚二硫苏糖醇纳米体系。
本发明的第五个目的是提供上述聚二硫苏糖醇纳米体系的制备方法。
本发明的第六个目的是提供上述聚合物PDTT或上述聚二硫苏糖醇纳米体系在制备抗癌药物中的应用。
本发明上述目的通过以下技术方案实现：
一种用于药物递送的聚合物PDTT，所述聚合物PDTT由多个重复的二硫苏糖醇结构单元组成，其结构式如下式(I)所示：
Figure PCTCN2018122760-appb-000001
其中，n＝5～10000。
优选地，所述聚合物PDTT的分子量为1500～45000。
本发明制备组成和结构可控的含有二硫键的新聚合物聚二硫苏糖醇(PDTT)，成功构建了抗肿瘤药物靶向递送的纳米载体；并将不同的抗肿瘤药物接枝 到聚二硫苏糖醇(PDTT)上的羟基上(PDTT-CPT)，然后经过单乳法、纳米沉淀法等纳米粒制备工艺，形成粒径均一且稳定的纳米粒溶液，或者也可通过其他制剂方法制备成可口服缓控释制剂或可注射型水凝胶等药物载体。
本发明的新聚合物聚二硫苏糖醇接枝药物后性质稳定，有良好的生物相容性和可降解性，并且具有GSH快速还原响应性，提高了纳米载体的靶向递送效率，实现药物在肿瘤细胞内的定位可控释放，提高了疏水药物的生物利用度，为疾病的有效治疗开拓了一种新的途径。
本发明还提供了聚合物PDTT的制备方法：将二硫苏糖醇用有机溶剂A溶解后，在真空条件下进行聚合反应，使二硫苏糖醇末端的巯基被氧化为二硫键，从而得到聚合物PDTT。
反应式如下：
Figure PCTCN2018122760-appb-000002
优选地，所述聚合反应的温度为40～180℃，所述聚合反应的时间为10min～48h。
更优选地，所述聚合反应的温度为90～100℃，所述聚合反应的时间为10～20h。
优选地，所述二硫苏糖醇和有机溶剂A的质量体积比为1～5g：2～20mL。如1：2、1：5、1：10、1：15、1：20、3：10、4：10、5：2、5：5、5：10、5：15、5：20g/mL等。
更优选地，所述二硫苏糖醇和有机溶剂A的质量体积比为2～4g：5～11mL。如2：5、2：8、2：11、3：5、3：7、3：11、4：5、4：8、4：11g/mL等。
优选地，所述有机溶剂A选自二甲基亚砜、N,N-二甲基甲酰胺、N,N-二乙基甲酰胺、N,N-二乙基乙酰胺、丙酮、四氢呋喃中的一种或多种。
本发明还提供了聚合物PDTT在作为或制备药物传递载体方面的应用。
优选地，所述传递载体为具有还原响应性的传递载体。
本发明还提供了一种具有还原响应性的用于药物递送的聚二硫苏糖醇纳米体系，其是以所述聚合物PDTT作为纳米载体负载药物。
本发明还提供了所述聚二硫苏糖醇纳米体系的制备方法，包括以下步骤：
S1.将药物和有机碱性催化剂经有机溶剂B溶解搅拌后，加入固体光气进行 酰化反应，再逐滴加入溶解在溶剂中的所述聚合物PDTT进行接枝反应，得到接枝了药物的聚合物PDTT；
S2.将上述接枝了药物的聚合物PDTT通过单乳液法或纳米沉淀法，制备得到粒径均一、稳定的纳米粒溶液，即为所述用于药物递送的聚二硫苏糖醇纳米体系。
优选地，步骤S1中，所述药物和有机碱性催化剂的质量比为1～5：1～10。如1：1、1：2.5、1：5、1：8、1：10、1：1、1：2.5、1：5、1：8、1：10、3：5：3：9、4：1、4：9、5：1、5：8等。
更优选地，步骤S1中，所述药物和有机碱性催化剂的质量比为1～2：1～5。如1：1、1：5、1.5：1、1.5：4、2：1：2：3、2：5等。
优选地，步骤S1中，所述聚合物PDTT的浓度为0.01～2.5g/mL。如0.01、0.03、0.045、0.1、0.15、0.2、0.25g/mL等。
更优选地，步骤S1中，所述聚合物PDTT的浓度为0.01～1g/mL。
优选地，步骤S1中，所述酰化反应的时间为10～60min；所述接枝反应的时间为3～48h。
优选地，步骤S1中，所述接枝反应的温度为室温。所述室温是指15～25℃。
优选地，步骤S1中，所述酰化反应和接枝反应均是在氩气保护下进行。
优选地，步骤S1中，所述搅拌时间为1min～1h。
优选地，步骤S1中，所述有机溶剂B选自二氯甲烷和/或四氢呋喃。
对用于溶解聚合物PDTT的溶剂没有特殊要求，能溶解聚合物PDTT即可。
优选地，步骤S1中，所述有机碱性催化剂选自4-二甲氨基吡啶(DMAP)、4-二甲胺基吡啶-p-甲苯磺酸盐(DPTS)或N-羟基琥珀酰亚胺(NHS)中的任意一种。
优选地，步骤S2中，通过单乳液法制备纳米粒溶液的方法，包括以下步骤：
S21.将接枝了药物的聚合物PDTT，超声溶解于与水不互溶且沸点低的有机溶剂C中，获得乳浊液油相；
S22.向纯水中加入稳定剂作为水相，将油相与水相混合后，超声除去有机溶剂C，即可得到粒径均一、稳定的纳米粒溶液。
所述与水不互溶的有机溶剂是指在水中的溶解度小于0.01g的有机溶剂。
所述沸点低的有机溶剂是指沸点低于60℃的有机溶剂。
优选地，步骤S21中，所述与水不互溶且沸点低的有机溶剂C，包括但不限于氯仿、二氯甲烷、丙酮或乙酸乙酯等。
优选地，步骤S2中，通过纳米沉淀法制备纳米粒溶液的方法，包括以下步骤：
S221.将接枝了药物的聚合物PDTT与稳定剂，溶解于与水互溶的有机溶剂D中，形成油相；
S222.搅拌条件下，将油相滴加到水相中，即可得到粒径均一、稳定的纳米粒溶液。
优选地，步骤S221中，将油相缓慢滴加到水相中，其滴加速度为0.01～1mL/秒。
优选地，步骤S221中，将接枝了药物的聚合物PDTT与稳定剂以1～20：1的质量比，完全溶解与水互溶的有机溶剂D中。如1：1、2：1、5：1、10：1、20：1等。
更优选地，步骤S221中，将接枝了药物的聚合物PDTT与稳定剂以5：1的质量比，完全溶解与水互溶的有机溶剂D中。
所述与水互溶的有机溶剂D是指在水中的溶解度大于100g的有机溶剂。
优选地，所述与水互溶的有机溶剂D包括但不限于二甲基亚砜、N,N-二甲基甲酰胺、四氢呋喃、二氧六环、吡啶等。
优选地，步骤S22中和步骤S221中，所述稳定剂包括二硬脂酰基磷脂酰乙醇胺-聚乙二醇(DSPE-PEG)类稳定剂、聚乙烯醇(PVA)、磷脂分子等。
所述药物包括疏水性或亲水性的药物。本发明的聚合物PDTT既能负载疏水性药物，改善疏水性药物的溶解性，提高其生物利用度，也能负载亲水性药物，具有良好的生物相容性。
优选地，所述药物包括含-OH、-NH 2、-COOH或卤素官能团的药物、蛋白类药物或核酸类药物。实验发现，本发明所述聚合物PDTT能够实现含-OH、-NH 2、-COOH或卤素官能团的药物、蛋白类药物、核酸类药物等药物的高效负载。
所述蛋白类药物包括但不限于曲妥珠单抗、雷珠单抗、干扰素、促红细胞生成素、链激酶等蛋白类药物。
所述核酸类药物包括但不限于拉米夫定、阿德福韦酯、肌苷、辅酶A等核 酸类药物。
所述药物包括抗肿瘤药物、抗生素类药物、抗病毒类药物。
所述抗肿瘤药物选自喜树碱、熊果酸、甲氨蝶呤、9-氨基喜树碱、曲妥珠单抗、氮芥、顺铂、环磷酰胺、氟尿嘧啶、鬼臼毒素等中的一种或多种。
所述抗生素类药物包括多粘菌素、氨苄西林、头孢氨苄、阿米卡星等。
所述抗病毒类药物包括拉米夫定、更昔洛韦、恩替卡韦、阿德福韦酯等。
所述聚二硫苏糖醇接枝喜树碱的结构式如下式(Ⅱ)所示：
Figure PCTCN2018122760-appb-000003
所述聚二硫苏糖醇接枝熊果酸的结构式如下式(Ⅲ)所示：
Figure PCTCN2018122760-appb-000004
所述聚二硫苏糖醇接枝甲氨蝶呤的结构式如下式(Ⅳ)所示：
Figure PCTCN2018122760-appb-000005
所述聚二硫苏糖醇接枝9-氨基喜树碱的结构式如下式(Ⅴ)所示：
Figure PCTCN2018122760-appb-000006
所述含-COOH官能团的药物包括甲氨蝶呤、氨基酸类(多粘菌素)药物、蛋白类(干扰素)药物等。
所述聚二硫苏糖醇和具有羧基的药物(R-COOH)发生反应，生成具有酯键的药物。
所述聚二硫苏糖醇接枝含羧基药物的结构式如下式(Ⅵ)所示：
Figure PCTCN2018122760-appb-000007
所述含卤素的药物R-X(X＝F，Cl，Br，I)包括氮芥、顺铂、环磷酰胺、氟尿嘧啶等。
所述聚二硫苏糖醇和具有卤素的药物发生反应，生成具有醚键的药物。
所述聚二硫苏糖醇接枝含卤素药物的结构式如下式(Ⅶ)所示：
Figure PCTCN2018122760-appb-000008
通过以上方法制备的纳米粒溶液经过DLS测量，所述纳米粒的粒径控制在10～500nm。
更优选地，所述纳米粒的粒径为10～150nm。
所述聚合物聚二硫苏糖醇接枝药物后的纳米粒具有快速GSH响应性。根据37℃时在不同GSH浓度的PBS溶液中，纳米体系释放游离药物的检测结果显示：与不存在GSH时相比，该纳米粒在GSH存在的情况下，游离药物释放明显增多，而且随着GSH浓度的升高，纳米体系释放游离药物的速度越快。这是因为GSH的巯基进攻体系中的双硫键，使得双硫键还原成巯基，环境中巯基的增多加速可降解的化学键(比如酯键)的降解。以上作用同时进行，加速了纳米体系的降解，药物得以快速释放。
还原型谷胱甘肽(GSH)的结构如下式(Ⅷ)所示：
Figure PCTCN2018122760-appb-000009
本发明的聚二硫苏糖醇具有良好的药物载体应用前景，还体现在：将所述聚二硫苏糖醇新材料与单体二硫苏糖醇进行MCF-7/A549/HpeG-2/CT-26/4T1肿瘤细胞毒性检测发现，在足够大的浓度范围内，聚二硫苏糖醇对细胞生长没有明显抑制，而且与单体二硫苏糖醇相比，显示出更好的生物相容性。接枝药物形成纳米体系后，与单药药物相比，聚二硫苏糖醇的抗肿瘤效果相当，仍然呈现出优异的抗肿瘤活性。
因此，所述具有还原响应性的用于药物递送的聚二硫苏糖醇纳米体系在制备抗癌药物中的应用，也在本发明的保护范围之内。
与现有技术相比，本发明具有以下有益效果：
(1)本发明将巯基通过聚合反应，简单高效的合成了聚合物聚二硫苏糖醇，该聚合物结构稳定、其生物相容性良好，能包载疏水性的抗肿瘤药物，并且聚合物中大量存在的二硫键使得体系具有GSH快速还原响应性。
(2)本发明将药物接枝于聚二硫苏糖醇侧基，实现了如喜树碱等含-OH或-NH 2等官能团的疏水药物的可控载药，改善了药物的溶解性，极大的提高了疏水药物的可利用性。
(3)本发明以聚二硫苏糖醇作为载体负载药物，能够得到可控粒径的纳米粒，粒径分布范围小，其通过肿瘤组织的高通透性及高保留性(EPR effect)蓄积在肿瘤组织中，从而实现被动靶向的作用，聚二硫苏糖醇纳米体系能够减少药物在正常生理条件下的释放以降低药物突释带来的毒副作用；而被肿瘤细胞摄取后，在肿瘤细胞内谷胱甘肽作用下，纳米体系中的二硫键断裂，还原成巯基，具有巯基的单体能促进体系瓦解，促进药物释放；同时，游离出来的小分子作用于碳酸酯键，加速药物的释放，增强癌症的治疗作用，具有极高的临床应用价值。
(4)本发明具有反应过程简单，反应步骤少，反应周期短、重复性好等优势，在医药领域具有良好的应用前景和广阔的发展空间。
附图说明
图1是本发明接枝了喜树碱的聚二硫苏糖醇形成的纳米粒及其在肿瘤细胞中的释放示意图。
图2是本发明中聚二硫苏糖醇的核磁氢谱图。
图3是本发明中接枝了喜树碱的聚二硫苏糖醇的核磁氢谱图。
图4是接枝了喜树碱的聚二硫苏糖醇形成的纳米粒的透射电镜(TEM)结果图。
图5是接枝了喜树碱的聚二硫苏糖醇形成的纳米粒的动态光散射(DLS)结果图。
图6是接枝了喜树碱的聚二硫苏糖醇形成的纳米粒在37℃不同浓度GSH下的药物释放图。
图7是聚二硫苏糖醇(PDTT)、二硫苏糖醇单体(DTT)对MCF-7乳腺肿瘤细胞的细胞毒性图。
图8是接枝了喜树碱的聚二硫苏糖醇形成的纳米粒(PDTT-CPT)、喜树碱单药(CPT)对MCF-7乳腺肿瘤细胞的细胞毒性图。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市购。
实施例1 聚二硫苏糖醇的合成
1、通过以下方法制备作为药物载体的聚合物聚二硫苏糖醇(PDTT)，包括以下步骤：
(1)称取4.62g二硫苏糖醇，用11mL干燥后的二甲基亚砜溶解，经过液氮冷却用冻抽法使反应容器内部保持高度真空；
(2)待恢复室温后，升温至90℃，同时保持搅拌，反应10h后，抽去残余二甲基亚砜和产物水；
(3)加入乙酸乙酯，加热使固体粗产物溶解后，在室温下冷却析出固体，多次重复后，得到白色固体，即为聚二硫苏糖醇。
2、结果
聚合物PDTT的核磁氢谱图如图2所示，由图2可知～5.17ppm处的信号为 溶剂重水的吸收峰，～3.34ppm处的信号对应二硫苏糖醇两个羟基相连的-CH-上的质子吸收峰，～3.02ppm、～2.72ppm处的信号对应二硫苏糖醇两个巯基相连的两个亚甲基-CH 2-上的质子吸收峰，结果显示成功合成了聚合物PDTT，具体的结构通式如式(I)所示：
Figure PCTCN2018122760-appb-000010
其中，n＝5～10000。
实施例2 聚二硫苏糖醇的合成
1、通过以下方法制备作为药物载体的聚合物聚二硫苏糖醇(PDTT)，包括以下步骤：
(1)称取2.31g二硫苏糖醇，用5.5mL干燥后的二甲基亚砜溶解，经过液氮冷却用冻抽法使反应容器内部保持高度真空；
(2)待恢复室温后，升温至100℃，同时保持搅拌，反应20h后，抽去残余二甲基亚砜和产物水；
(3)加入乙酸乙酯，加热使固体粗产物溶解后，在室温下冷却析出固体，多次重复后，得到白色固体，即为聚二硫苏糖醇。
2、结果
制备得到的聚二硫苏糖醇结构稳定、其生物相容性良好，能包载疏水性的抗肿瘤药物，并且聚合物中大量存在的二硫键使得体系具有GSH快速还原响应性，在作为药物传递载体方面具有良好应用前景。
实施例3 接枝药物(聚二硫苏糖醇接枝喜树碱)的制备
1、通过以下方法制备接枝药物，包括以下步骤：
(1)取洁净干燥的250mL圆底烧瓶，称取喜树碱2g、4-二甲氨基吡啶(DMAP)2.11g溶解在100mL的二氯甲烷中，氩气保护下，置于磁力搅拌器上搅拌10min；
(2)称取光气0.57g，迅速加入到圆底烧瓶中，体系继续搅拌30min，进行酰化反应；
(3)将聚二硫苏糖醇0.9g溶解在30mL四氢呋喃中，置于恒压漏斗中， 在氩气保护下滴入圆底烧瓶中，室温下进行接枝反应24h；
(4)用旋蒸浓缩溶剂至30mL，然后滴入乙酸乙酯中，静置于20℃冰箱中过夜，过滤，用冰乙酸乙酯冲洗固体3遍，烘干，得到接枝了喜树碱的聚二硫苏糖醇。
接枝了喜树碱的聚二硫苏糖醇的核磁氢谱图如图3所示，由图3可知，～8.69ppm、～8.17ppm、～8.11ppm、～7.86ppm、～7.72ppm、～7.45ppm、～5.51ppm、～5.43ppm处的信号对应喜树碱芳香环上质子吸收峰，～1.74ppm、～0.89ppm代表喜树碱上亚甲基和乙基上的吸收峰，～3.34ppm、～3.05ppm、～5.27ppm分别代表聚二硫苏糖醇上次甲基，亚甲基和羟基上的质子吸收峰。以上结果说明药物喜树碱成功接枝到聚二硫苏糖醇上。
实施例4 接枝药物(聚二硫苏糖醇接枝喜树碱)的制备
1、通过以下方法制备接枝药物，包括以下步骤：
(1)取洁净干燥的100mL圆底烧瓶，称取喜树碱1g、4-二甲氨基吡啶(DMAP)1.05g溶解在50mL的四氢呋喃中，氩气保护下，置于磁力搅拌器上搅拌10min；
(2)称取光气0.57g，迅速加入到圆底烧瓶中，体系继续搅拌30min，进行酰化反应；
(3)将聚二硫苏糖醇0.45g溶解在10mL四氢呋喃中，置于恒压漏斗中，在氩气保护下滴入圆底烧瓶中，室温下进行接枝反应24h；
(4)用旋蒸浓缩溶剂至30mL，然后滴入乙酸乙酯中，静置于20℃冰箱中过夜，过滤，用冰乙酸乙酯冲洗固体3遍，烘干，得到接枝了喜树碱的聚二硫苏糖醇。经核磁氢谱分析表明，药物喜树碱成功接枝到聚二硫苏糖醇上。
实施例5 一种具有还原响应性的用于抗肿瘤药物递送的聚二硫苏糖醇纳米体系
1、通过单乳液法制备聚二硫苏糖醇纳米体系，包括以下步骤：
(1)将实施例3接枝了喜树碱的聚二硫苏糖醇超声溶解于二氯甲烷中，获得乳浊液油相，油相的浓度为30mg/mL；
(2)在10mL纯水中加入稳定剂DSPE-PEG 3000 2mg作为水相；取油相0.2mL滴入水相中，在冰浴下超声5min，获得白色混合乳液；
(3)将白色混合乳液转移至室温，搅拌一天一夜后，挥发掉低沸点的二氯 甲烷，获得具有丁达尔效应的纳米粒溶液，经0.8μm滤膜过滤除掉大颗粒杂质，得到载药的纳米粒溶液，即为所述具有还原响应性的用于抗肿瘤药物递送的聚二硫苏糖醇纳米体系。
2、结果
(1)进行DLS检测，本实施例纳米粒的粒径在150nm，且静置于4℃冰箱中无沉淀产生。其透射电镜(TEM)结果图和动态光散射(DLS)结果图分别如图4和图5所示。结果表明，载喜树碱的PDTT纳米粒呈球形，大小较均匀，粒径分布较窄。
(2)本实施例制得的纳米粒在37℃条件下，于不同浓度谷胱甘肽(0mM、2mM、10mM、20mM)的PBS溶液中进行药物释放实验，检测PBS溶液中72小时内纳米体系释放的游离喜树碱浓度。实验结果如图6所示，由此证明本发明中所述的纳米载药体系具有GSH还原响应性，且随着GSH浓度升高，还原响应性加强。
(3)将聚二硫苏糖醇(PDTT)、二硫苏糖醇单体(DTT)、本实施例接枝了喜树碱的聚二硫苏糖醇形成的纳米粒(PDTT-CPT)、喜树碱单药(CPT)分别进行体外抗MCF-7肿瘤细胞毒性实验，效果如图7和图8所示。可以看出，聚二硫苏糖醇生物相容性很好，对MCF-7细胞的生长在一个较大作用浓度的范围内均无明显抑制。载喜树碱的PDTT纳米粒，与单纯的喜树碱相比，保持了喜树碱优异的抗癌活性。
实施例6 一种具有还原响应性的用于抗肿瘤药物递送的聚二硫苏糖醇纳米体系
1、通过纳米沉淀法制备聚二硫苏糖醇纳米体系，包括以下步骤：
(1)将实施例4接枝了喜树碱的聚二硫苏糖醇和稳定剂DSPE-PEG 3000以2：1的质量比，完全溶解于二甲基亚砜中，获得聚合物浓度为20mg/mL的油相；
(2)取10mL的纯水置于磁力搅拌台上，搅拌转速为1500r/min，将0.2mL油相缓慢滴入水相中，滴加速度为0.05mL/秒，得到具有丁达尔效应的乳白色乳液，超滤3次后除去二甲基亚砜，用0.8μm有机相滤膜过滤掉大分子杂质，得到载药的纳米粒溶液，即为所述具有还原响应性的用于抗肿瘤药物递送的聚二硫苏糖醇纳米体系。
2、结果
(1)进行DLS检测，本实施例纳米粒的粒径在100nm左右，且静置于4℃冰箱中无沉淀产生。其透射电镜(TEM)结果图和动态光散射(DLS)结果图分别如图4和图5所示。结果表明，载喜树碱的PDTT纳米粒呈球形，大小较均匀，粒径分布较窄。
(2)本实施例制得的纳米粒在37℃条件下，于不同浓度谷胱甘肽(0mM、2mM、10mM、20mM)的PBS溶液中进行药物释放实验，检测PBS溶液中72小时内纳米体系释放的游离喜树碱浓度。实验结果如图6所示，由此证明本发明中所述的纳米载药体系具有GSH还原响应性，且随着GSH浓度升高，还原响应性加强。
(3)将聚二硫苏糖醇(PDTT)、二硫苏糖醇单体(DTT)、本实施例接枝了喜树碱的聚二硫苏糖醇形成的纳米粒(PDTT-CPT)、喜树碱单药(CPT)分别进行体外抗MCF-7肿瘤细胞毒性实验，效果如图7和图8所示。可以看出，聚二硫苏糖醇生物相容性很好，对MCF-7细胞的生长在一个较大作用浓度的范围内均无明显抑制。载喜树碱的PDTT纳米粒，与单纯的喜树碱相比，保持了喜树碱优异的抗癌活性，具有显著的抑瘤效果，在癌症治疗领域有一定的应用潜力。
实施例7 聚合物PDTT的制备
其他条件与实施例1相同，唯一不同之处在于，聚合反应的温度为40℃，聚合反应的时间为48h，制备得到白色固体聚合物PDTT。
实施例8 聚合物PDTT的制备
其他条件与实施例2相同，唯一不同之处在于，聚合反应的温度为180℃，聚合反应的时间为10min，制备得到白色固体聚合物PDTT。
实施例9 接枝药物(聚二硫苏糖醇接枝喜树碱)的制备
其他条件与实施例3相同，唯一不同之处在于，酰化反应的时间为10min，接枝反应的时间为48h，制备得到接枝了喜树碱的聚二硫苏糖醇，最终得到的抗肿瘤药物递送体系，实现了疏水药物的可控载药，改善了药物的溶解性，极大的提高了疏水药物的可利用性，并且具有GSH快速还原响应性，提高抗肿瘤药物的靶向性。
实施例10 接枝药物(聚二硫苏糖醇接枝喜树碱)的制备
其他条件与实施例4相同，唯一不同之处在于，酰化反应的时间为60min，接枝反应的时间为3h，制备得到接枝了喜树碱的聚二硫苏糖醇，最终得到的抗 肿瘤药物递送体系，实现了疏水药物的可控载药，改善了药物的溶解性，极大的提高了疏水药物的可利用性，并且具有GSH快速还原响应性，提高抗肿瘤药物的靶向性。
实施例11 一种具有还原响应性的用于抗肿瘤药物递送的聚二硫苏糖醇纳米体系
其他条件与实施例6相同，唯一不同之处在于，接枝了喜树碱的聚二硫苏糖醇与稳定剂DSPE-PEG 3000的质量比为5：1。最终得到的抗肿瘤药物递送体系，实现了喜树碱的可控载药，改善了喜树碱的溶解性，极大的提高了喜树碱的可利用性，并且具有GSH快速还原响应性，提高药物喜树碱的靶向性。
另外，本发明的聚二硫苏糖醇纳米体系还适用于如熊果酸、甲氨蝶呤、9-氨基喜树碱、曲妥珠单抗、氮芥、顺铂、环磷酰胺、氟尿嘧啶、鬼臼毒素等抗肿瘤药物，如曲妥珠单抗、雷珠单抗、干扰素、促红细胞生成素、链激酶蛋白类药物，如拉米夫定、阿德福韦酯、肌苷、辅酶A等核酸类药物的递送，本发明的新聚合物聚二硫苏糖醇接枝上述药物后性质稳定，有良好的生物相容性和可降解性，并且具有GSH快速还原响应性，提高了纳米载体的靶向递送效率，实现药物在肿瘤细胞内的定位可控释放，提高了疏水药物的生物利用度，为疾病的有效治疗开拓了一种新的途径。
以上具体实施方式为便于理解本发明而说明的较佳实施例，但本发明并不局限于上述实施例，即不意味着本发明必须依赖上述实施例才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明所选用原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

Claims (10)

  1. 一种用于药物递送的聚合物PDTT，其特征在于，所述聚合物PDTT由多个重复的二硫苏糖醇结构单元组成，其结构式如下式(I)所示：
    Figure PCTCN2018122760-appb-100001
    其中，n＝5～10000。
  2. 权利要求1所述聚合物PDTT的制备方法，其特征在于，将二硫苏糖醇用有机溶剂A溶解后，在真空条件下进行聚合反应，使二硫苏糖醇末端的巯基被氧化为二硫键，从而得到聚合物PDTT。
  3. 根据权利要求2所述的制备方法，其特征在于，所述聚合反应的温度为40～180℃，所述聚合反应的时间为10min～48h。
  4. 权利要求1所述聚合物PDTT在作为或制备药物传递载体方面的应用。
  5. 一种具有还原响应性的用于药物递送的聚二硫苏糖醇纳米体系，其特征在于，以权利要求1所述聚合物PDTT作为纳米载体负载药物。
  6. 根据权利要求5所述的聚二硫苏糖醇纳米体系，其特征在于，由包括以下步骤的方法制备得到：
    S1.将药物和有机碱性催化剂经有机溶剂B溶解搅拌后，加入固体光气进行酰化反应，再逐滴加入溶解在溶剂中的所述聚合物PDTT进行接枝反应，得到接枝了药物的聚合物PDTT；
    S2.将上述接枝了药物的聚合物PDTT通过单乳液法或纳米沉淀法，制备得到粒径均一、稳定的纳米粒溶液，即为所述聚二硫苏糖醇纳米体系。
  7. 根据权利要求6所述的聚二硫苏糖醇纳米体系，其特征在于，步骤S1中，所述酰化反应的时间为10～60min；所述接枝反应的时间为3～48h。
  8. 根据权利要求6所述的聚二硫苏糖醇纳米体系，其特征在于，步骤S2中，通过单乳液法制备纳米粒溶液的方法，包括以下步骤：
    S21.将接枝了药物的聚合物PDTT，超声溶解于与水不互溶且沸点低的有机溶剂C中，获得乳浊液油相；
    S22.向纯水中加入稳定剂作为水相，将油相与水相混合后，超声除去有机溶剂C，即可得到粒径均一、稳定的纳米粒溶液；
    步骤S2中，通过纳米沉淀法制备纳米粒溶液的方法，包括以下步骤：
    S221.将接枝了药物的聚合物PDTT与稳定剂，溶解于与水互溶的有机溶剂D中，形成油相；
    S222.搅拌条件下，将油相滴加到水相中，即可得到粒径均一、稳定的纳米粒溶液。
  9. 根据权利要求5所述的聚二硫苏糖醇纳米体系，其特征在于，所述药物包括含-OH、-NH 2、-COOH或卤素官能团的药物、蛋白药物或核酸药物。
  10. 权利要求1所述的聚合物PDTT或权利要求5～9任一所述的聚二硫苏糖醇纳米体系在制备抗癌药物中的应用。
PCT/CN2018/122760 2018-08-29 2018-12-21 一种用于抗肿瘤药物递送的聚二硫苏糖醇纳米体系及其制备方法和应用 WO2020042470A1 (zh)

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