WO2019019513A1 - 氧化还原响应壳聚糖-脂质体的制备方法和用途 - Google Patents

氧化还原响应壳聚糖-脂质体的制备方法和用途 Download PDF

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WO2019019513A1
WO2019019513A1 PCT/CN2017/114413 CN2017114413W WO2019019513A1 WO 2019019513 A1 WO2019019513 A1 WO 2019019513A1 CN 2017114413 W CN2017114413 W CN 2017114413W WO 2019019513 A1 WO2019019513 A1 WO 2019019513A1
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chitosan
redox
responsive
liposome
phosphatidylethanolamine
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French (fr)
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张树彪
陈会英
马羽
秦晓利
蓝浩铭
崔韶晖
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大连民族大学
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    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
    • C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
    • C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • 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
    • A61K47/6905—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 colloid or an emulsion
    • A61K47/6911—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 colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1806—Suspensions, emulsions, colloids, dispersions
    • A61K49/1812—Suspensions, emulsions, colloids, dispersions liposomes, polymersomes, e.g. immunoliposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
    • A61K9/0009—Galenical forms characterised by the drug release technique; Application systems commanded by energy involving or responsive to electricity, magnetism or acoustic waves; Galenical aspects of sonophoresis, iontophoresis, electroporation or electroosmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1277—Processes for preparing; Proliposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1277—Processes for preparing; Proliposomes
    • A61K9/1278—Post-loading, e.g. by ion or pH gradient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

  • the present invention relates to a redox-responsive chitosan-liposome drug carrier, and in particular to a redox-responsive double-fat chain-substituted phosphatidylethanolamine chitosan and a superparamagnetic package thereof with liposome construction
  • a pharmaceutical carrier of ferroferric oxide nanoparticles is a preparation method of a novel pharmaceutical carrier in the field of drug delivery.
  • a drug carrier refers to a system that changes the manner in which a drug enters the body and its distribution in the body, controls the release rate of the drug, and delivers the drug to the targeted organ.
  • the drug carrier effectively improves the utilization, safety and effectiveness of the drug by controlled release.
  • Chitosan and liposome are commonly used drug carriers. Chitosan has good biocompatibility and biodegradability.
  • the 2-position amino group and the 6-position hydroxyl group are easy to be structurally modified, have bioadhesive properties, and can pass.
  • the liposome is an ultrafine spherical particle having one or more layers of lipid vesicles formed by dispersing the amphiphilic surfactant in water, capable of loading Water-soluble or fat-soluble drugs are widely used in pharmaceutical carriers.
  • Multi-functional nanocarriers are a new generation of nanocarriers developed on the basis of single-function nanocarriers. They overcome some of the shortcomings of single-function carriers in tumor diagnosis and treatment, such as real-time monitoring of cell activity in vivo, for target sites. Special targeting or efficient delivery of drugs within target cells. Multifunctional nanocarriers combine different functions in a single stable structure. Such as the combination of tumor developer or diagnostic reagent to achieve early diagnosis of tumors, real-time monitoring of tumor treatment effects and so on. Multifunctional nanocarriers offer new opportunities for early diagnosis and individualized drug treatment of tumors.
  • Magnetic resonance imaging has good soft tissue resolution and spatial resolution. It can clearly display the anatomy of the tissue and accurately locate and quantify the imaging features of soft tissue. It is the most effective method for early diagnosis of tumors. One. In order to enhance the contrast between the diseased tissue and the image of normal tissue to improve the clarity of the diseased tissue, it is necessary to select a suitable contrast agent to display the anatomical features.
  • the T2 contrast agent has a higher magnetic moment than the paramagnetic substance, and has a significant acceleration effect on the relaxation of protons in adjacent tissues, which can significantly improve the detection sensitivity.
  • Commonly used superparamagnetic contrast agents are mainly microcrystalline metal particles of different sizes (such as Fe 3 O 4 , ⁇ Fe 2 O 3 ).
  • the stability of the drug carrier in the blood is critical to the function of the drug carrier. Liposomal structures are susceptible to damage by components such as high-density lipoproteins in serum, leading to leakage of encapsulated drugs. Chitosan has good antiserum properties and helps to improve the stability of drug-loaded nanoparticles in serum.
  • the chitosan was modified by post-insertion method to construct a drug carrier with a chitosan brush on the surface, and encapsulated with nano-ferric oxide nanoparticles (SPIO) to form a multifunctional drug delivery and imaging diagnosis.
  • SPIO nano-ferric oxide nanoparticles
  • the vector uses the gene as a model drug to evaluate the gene transfection performance, realize the integration of treatment and diagnosis, and open up a new way for tumor treatment.
  • the technical problem to be solved by the present invention is to overcome the defects of the existing drug carrier, to provide a redox-responsive double-fat chain-substituted phosphatidylethanolamine chitosan, and a redox-responsive chitosan brush constructed by post-insertion and Liposomal carrier of SPIO, preparation method of chitosan-liposome drug carrier.
  • a first object of the invention is to claim a redox-responsive chitosan having the structure of formula (I):
  • Another object of the present invention is to provide a method for preparing a redox-responsive chitosan, which is specifically: firstly, chitosan is dissolved in water, and if necessary, 1 to 3 glacial acetic acid is added to promote complete dissolution, and the mixture is added dropwise under stirring. After the reaction to the dithiobissuccinimidyl substituted ester in DMSO, the reaction is carried out at 20 to 60 ° C for 1 to 24 hours, and the solution of the double aliphatic chain substituted phosphatidylethanolamine alcohol is continuously added to the reaction solution, and the reaction is carried out at 20 to 60 ° C. ⁇ 24h, after the reaction solution was rotary evaporated, dialyzed and lyophilized to prepare a redox-responsive chitosan.
  • the chitosan has a weight average molecular weight of 500 to 10,000 Da and a degree of deacetylation of 65 to 95%.
  • the di-fatty chain substituent phosphatidylethanolamine is 1,2-dilauroylphosphatidylethanolamine, 1,2-distearoylphosphatidylethanolamine, 1,2-dimyristoylphosphatidylethanolamine, 1
  • 2-dipalmitoylphosphatidylethanolamine and 1,2-dioleoylphosphatidylethanolamine are 1,2-dilauroylphosphatidylethanolamine, 1,2-distearoylphosphatidylethanolamine, 1,2-dimyristoylphosphatidylethanolamine, 1
  • 2-dipalmitoylphosphatidylethanolamine and 1,2-dioleoylphosphatidylethanolamine but not limited to the above-mentioned raw materials, and the amount thereof is 0.1 to 1 times the molar equivalent of the chitosan repeating unit.
  • it is 0.3-0.6 times, and the reaction conditions are preferably 20-50 ° C, the stirring reaction is 2-48 h, and more
  • a third object of the present invention is to provide a method for preparing a redox-responsive chitosan-liposome drug carrier:
  • the liposome @SPIO composite was prepared by thin film ultrasonic method, and the cationic liposome was modified by post-insertion self-assembly.
  • the redox-responsive chitosan was inserted into the phospholipid bilayer of the liposome to obtain oxidation. The reduction is in response to chitosan-liposome, wherein the mass ratio of cationic liposome to SPIO is 30:1 to 10:1, and the mass ratio of chitosan to liposome@SPIO is 0.5:1 to 6:1.
  • the cationic liposomes as a 2000 DOTAP, Lipofectin, Lipofectamin TM.
  • the SPIO nanoparticles have a particle diameter of 1 to 30 nm.
  • the present invention simultaneously claims the use of the above-described redox-responsive chitosan-liposome as a drug carrier, especially in gene transfection.
  • the invention modifies the chitosan by redox reaction in response to the double aliphatic chain substituent phosphatidylethanolamine, and then modifies the liposome @SPIO by post-insertion method to obtain a chitosan having a surface with redox reaction.
  • Brush double-chain-substituted phosphatidylethanolamine chitosan-liposome drug carrier improve liposome anti-serum ability and biocompatibility, and improve the control and release ability of drugs by responding to redox environment
  • Magnetic field-directed targeting and magnetic resonance imaging are available to enable integrated drug delivery for therapy and diagnosis.
  • the present invention has the following advantages:
  • the present invention uses a redox environment to modify a chitosan in response to a double aliphatic chain substituted phosphatidylethanolamine to prepare a double aliphatic chain substituted phosphatidylethanolamine chitosan.
  • the present invention uses a redox environment in response to a double aliphatic chain substituent phosphatidylethanolamine chitosan to modify liposomes by post-insertion, improve lipobiocompatibility and blood stability, and through environmental responsiveness, It is suitable for intravenous injection and improves the ability to control the release of drugs.
  • the present invention uses a chitosan-liposome-encapsulated superparamagnetic ferroferric oxide nanoparticle to obtain a redox-responsive chitosan-liposome composite carrier, thereby realizing its application in drug delivery, especially in gene transfer. Application in dyeing.
  • the complex has high drug delivery efficiency and high biocompatibility, and provides magnetic field-oriented targeting function and nuclear magnetic resonance imaging function, and has broad application prospects.
  • Example 1 is a FTIR spectrum of a redox-responsive chitosan prepared in Example 1;
  • Example 2 is a 1 H NMR spectrum of the redox-responsive chitosan prepared in Example 1;
  • Example 3 is a TEM photograph of a redox-responsive chitosan-DOTAP liposome-SPIO composite carrier prepared in Example 1;
  • Figure 5 is a diagram showing the gene transfection efficiency of the redox-responsive chitosan-liposome prepared by the present invention.
  • Figure 6 is a graph showing the cytotoxicity of a redox-responsive chitosan-liposome prepared according to the present invention.
  • chitosan (CSO) with a molecular weight of 5 kDa was dissolved in 100 mL of water and sonicated for 30 min. Fully dissolved.
  • the aqueous solution of chitosan was added dropwise to a solution of dithiobissuccinimidyl propionate in DMSO under stirring, and after reacting at 30 ° C for 2 h, 0.5 g of 1,2 was continuously added to the reaction solution.
  • redox-responsive 1,2-dioleoylphosphatidylethanolamine chitosan aqueous solution take 100 uL, mix with 1 mL of DOTAP cationic liposome containing SPIO, and then let it stand for 1 h.
  • the liposomes are modified to obtain a liposome drug carrier having a redox-responsive chitosan brush on the surface.
  • chitosan having a molecular weight of 5 kDa was dissolved in 100 mL of water and sonicated for 30 minutes to dissolve it sufficiently.
  • the aqueous solution of chitosan was added dropwise to a solution of dithiobissuccinimidyl propionate in DMSO under stirring, and after reacting at 30 ° C for 2 h, 0.5 g of 1,2 was continuously added to the reaction solution.
  • redox-responsive 1,2-distearoylphosphatidylethanolamine chitosan aqueous solution take 100 uL, mix with 1 mL of DOTAP cationic liposome containing SPIO, and then let it stand for 1 h.
  • the liposome is modified to obtain a liposome drug carrier having a redox-responsive chitosan brush on its surface.
  • chitosan having a molecular weight of 5 kDa was dissolved in 100 mL of water and sonicated for 30 minutes to dissolve it sufficiently.
  • the aqueous solution of chitosan was added dropwise to a solution of dithiobissuccinimidyl propionate in DMSO under stirring, and after reacting at 30 ° C for 2 h, 0.5 g of 1,2 was continuously added to the reaction solution.
  • redox-responsive 1,2-dilauroylphosphatidylethanolamine chitosan aqueous solution take 100 uL, mix with 1 mL of DOTAP cationic liposome containing SPIO, and then let it stand for 1 h.
  • the liposomes are modified to obtain a liposome drug carrier having a redox-responsive chitosan brush on the surface.
  • chitosan having a molecular weight of 1 kDa was dissolved in 100 mL of water and sonicated for 30 minutes to dissolve it sufficiently.
  • the aqueous solution of chitosan was added dropwise to a solution of dithiobissuccinimidyl propionate in DMSO under stirring, and after reacting at 30 ° C for 2 h, 0.5 g of 1,2 was continuously added to the reaction solution.
  • chitosan having a molecular weight of 1 kDa was dissolved in 100 mL of water and sonicated for 30 minutes to dissolve it sufficiently.
  • the aqueous solution of chitosan was added dropwise to a solution of dithiobissuccinimidyl propionate in DMSO under stirring, and after reacting at 30 ° C for 2 h, 0.5 g of 1,2 was continuously added to the reaction solution.
  • chitosan having a molecular weight of 1 kDa was dissolved in 100 mL of water and sonicated for 30 minutes to dissolve it sufficiently.
  • the aqueous solution of chitosan was added dropwise to a solution of dithiobissuccinimidyl propionate in DMSO under stirring, and after reacting at 30 ° C for 2 h, 1.0 g of 1, 2 was continuously added to the reaction solution.
  • Formulated 1mg / mL redox response of 1,2-dioleoyl phosphatidyl ethanolamine aqueous solution of chitosan take 100 uL, Lipofectamin TM 2000 and cationic liposomes comprising SPIO by ultrasonically mixing 1mL, then allowed to stand IH, by the The liposome was modified by inserting a self-assembly method to obtain a liposome drug carrier having a redox-responsive chitosan brush on its surface.
  • chitosan having a molecular weight of 5 kDa was dissolved in 100 mL of water and sonicated for 30 minutes to dissolve it sufficiently.
  • the aqueous solution of chitosan was added dropwise to a solution of dithiobissuccinimidyl propionate in DMSO under stirring, and after reacting at 30 ° C for 2 h, 1.0 g of 1, 2 was continuously added to the reaction solution.
  • redox-responsive 1,2-distearoylphosphatidylethanolamine chitosan aqueous solution take 100 uL, mix with 1 mL of DOTAP cationic liposome containing SPIO, and then let it stand for 1 h.
  • the liposome is modified to obtain a liposome drug carrier having a redox-responsive chitosan brush on its surface.
  • chitosan having a molecular weight of 1 kDa was dissolved in 100 mL of water and sonicated for 30 minutes to dissolve it sufficiently.
  • the aqueous solution of chitosan was added dropwise to a solution of dithiobissuccinimidyl propionate in DMSO under stirring, and after reacting at 30 ° C for 2 h, 1.0 g of 1, 2 was continuously added to the reaction solution.
  • Formulated 1mg / mL redox response of 1,2-dilauroyl phosphatidylethanolamine chitosan solution take 100 uL, mixing ultrasonically Lipofectamin TM 2000 and comprises cationic liposomes of SPIO 1mL, and then allowed to stand IH, by the The liposome was modified by inserting a self-assembly method to obtain a liposome drug carrier having a redox-responsive chitosan brush on its surface.
  • chitosan having a molecular weight of 5 kDa was dissolved in 100 mL of water and sonicated for 30 minutes to dissolve it sufficiently.
  • the aqueous solution of chitosan was added dropwise to a solution of dithiobissuccinimidyl propionate in DMSO under stirring, and after reacting at 30 ° C for 2 h, 1.0 g of 1, 2 was continuously added to the reaction solution.
  • chitosan having a molecular weight of 1 kDa was dissolved in 100 mL of water and sonicated for 30 minutes to dissolve it sufficiently.
  • the aqueous solution of chitosan was added dropwise to a solution of dithiobissuccinimidyl propionate in DMSO under stirring, and after reacting at 30 ° C for 2 h, 1.0 g of 1, 2 was continuously added to the reaction solution.
  • chitosan having a molecular weight of 1 kDa was dissolved in 100 mL of water and sonicated for 30 minutes to dissolve it sufficiently.
  • the aqueous solution of chitosan was added dropwise to a solution of dithiobissuccinimidyl propionate in DMSO under stirring, and after reacting at 30 ° C for 4 h, 1.0 g of 1,2 was continuously added to the reaction solution.
  • chitosan having a molecular weight of 5 kDa was dissolved in 100 mL of water and sonicated for 30 minutes to dissolve it sufficiently.
  • the aqueous solution of chitosan was added dropwise to a solution of dithiobissuccinimidyl propionate in DMSO under stirring, and after reacting at 30 ° C for 4 h, 1.0 g of 1,2 was continuously added to the reaction solution.
  • redox-responsive 1,2-distearoylphosphatidylethanolamine chitosan aqueous solution take 500 uL, mix with 1 mL of DOTAP cationic liposome containing SPIO, and then let it stand for 1 h.
  • the liposome is modified to obtain a liposome drug carrier having a redox-responsive chitosan brush on its surface.
  • chitosan having a molecular weight of 5 kDa was dissolved in 100 mL of water and sonicated for 30 minutes to dissolve it sufficiently.
  • the aqueous solution of chitosan was added dropwise to a solution of dithiobissuccinimidyl propionate in DMSO under stirring, and after reacting at 30 ° C for 4 h, 1.0 g of 1,2 was continuously added to the reaction solution.
  • redox-responsive 1,2-dilauroylphosphatidylethanolamine chitosan aqueous solution take 500 uL, mix with 1 mL of DOTAP cationic liposome containing SPIO, and then let it stand for 1 h.
  • the liposomes are modified to obtain a liposome drug carrier having a redox-responsive chitosan brush on the surface.
  • chitosan having a molecular weight of 5 kDa was dissolved in 100 mL of water and sonicated for 30 minutes to dissolve it sufficiently.
  • the aqueous solution of chitosan was added dropwise to a solution of dithiobissuccinimidyl propionate in DMSO under stirring, and after reacting at 30 ° C for 4 h, 1.0 g of 1,2 was continuously added to the reaction solution.
  • chitosan having a molecular weight of 1 kDa was dissolved in 100 mL of water and sonicated for 30 minutes to dissolve it sufficiently.
  • the aqueous solution of chitosan was added dropwise to a solution of dithiobissuccinimidyl propionate in DMSO under stirring, and after reacting at 30 ° C for 4 h, 1.0 g of 1,2 was continuously added to the reaction solution.
  • an ethanol solution of dipalmitoylphosphatidylethanolamine reacted at 30 ° C for 4 h, the reaction solution was rotary evaporated, dialyzed, and lyophilized.
  • a redox-responsive 1,2-dipalmitoylphosphatidylethanolamine chitosan was prepared.
  • redox-responsive 1,2-dipalmitoylphosphatidylethanolamine chitosan aqueous solution take 500 uL, mix with 1 mL of DOTAP cationic liposome containing SPIO, and then let it stand for 1 h.
  • the liposomes are modified to obtain a liposome drug carrier having a redox-responsive chitosan brush on the surface.
  • chitosan having a molecular weight of 5 kDa was dissolved in 100 mL of water and sonicated for 30 minutes to dissolve it sufficiently.
  • the aqueous solution of chitosan was added dropwise to a solution of dithiobissuccinimidyl propionate in DMSO under stirring, and after reacting at 40 ° C for 4 h, 1.0 g of 1,2 was continuously added to the reaction solution.
  • chitosan having a molecular weight of 1 kDa was dissolved in 100 mL of water and sonicated for 30 minutes to dissolve it sufficiently.
  • the aqueous solution of chitosan was added dropwise to a solution of dithiobissuccinimidyl propionate in DMSO under stirring, and after reacting at 40 ° C for 4 h, 1.0 g of 1,2 was continuously added to the reaction solution.
  • redox-responsive 1,2-distearoylphosphatidylethanolamine chitosan aqueous solution take 500 uL, mix with 1 mL of DOTAP cationic liposome containing SPIO, and then let it stand for 2 h.
  • the liposome is modified to obtain a liposome drug carrier having a redox-responsive chitosan brush on its surface.
  • chitosan having a molecular weight of 1 kDa was dissolved in 100 mL of water and sonicated for 30 minutes to dissolve it sufficiently.
  • the aqueous solution of chitosan was added dropwise to a solution of dithiobissuccinimidyl propionate in DMSO under stirring, and after reacting at 40 ° C for 4 h, 1.0 g of 1,2 was continuously added to the reaction solution.
  • chitosan having a molecular weight of 5 kDa was dissolved in 100 mL of water and sonicated for 30 minutes to dissolve it sufficiently.
  • the aqueous solution of chitosan was added dropwise to a solution of dithiobissuccinimidyl propionate in DMSO under stirring, and after reacting at 40 ° C for 4 h, 1.0 g of 1,2 was continuously added to the reaction solution.
  • redox-responsive 1,2-dimyristoylphosphatidylethanolamine chitosan aqueous solution take 500 uL, mix with 1 mL of DOTAP cationic liposome containing SPIO, and then let it stand for 2 h.
  • the liposome is modified to obtain a liposome drug carrier having a redox-responsive chitosan brush on its surface.
  • chitosan having a molecular weight of 5 kDa was dissolved in 100 mL of water and sonicated for 30 minutes to dissolve it sufficiently.
  • the aqueous solution of chitosan was added dropwise to a solution of dithiobissuccinimidyl propionate in DMSO under stirring, and after reacting at 40 ° C for 4 h, 1.0 g of 1,2 was continuously added to the reaction solution.
  • the pGL3 plasmid was used as a reporter gene to evaluate the gene transfer performance of a liposome vector having a redox-responsive chitosan brush, and the cells used were human non-small cell lung cancer cell A549 cell line. Training The cultured cells are plated, cultured in an incubator until the cell fusion degree reaches 80%, and the gene is transported. When transporting, the complete medium is aspirated, washed twice with PBS, and transported under serum conditions, 400 ⁇ L is added.
  • the cytotoxicity of the vector was evaluated by the MTT method.
  • the cells were seeded on a 96-well cell culture plate, paralleled with 3 wells, and 5 ⁇ 10 4 cells were seeded per well, and cultured at 37 ° C in a 5% CO 2 cell incubator until the cell fusion degree reached 85% or more.
  • the medium was removed, washed twice with PBS, fresh medium and test vector were added. After 24 hours of culture, 20 ⁇ L of 5 mg/mL MTT solution was added to each well, and incubation was continued for 4 hours at 37 ° C. The medium was removed and the culture was terminated.
  • A570 SMP is the absorbance of the cell plate to which the vector or complex to be tested is added
  • A570 CTL is the absorbance of the cell plate containing only the medium.

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Abstract

本发明提供了一种氧化还原响应壳聚糖-脂质体的制备方法和用途，该方法采用二硫双琥珀酰亚胺基取代酯，合成了氧化还原响应二硫键连接的双脂肪链取代基磷脂酰乙醇胺-s-s-壳聚糖。采用合成的双脂肪链取代基磷脂酰乙醇胺壳聚糖，通过后插入自组装的方法，对脂质体进行修饰，组装形成表面具有氧化还原响应壳聚糖刷子的双脂肪链取代基磷脂酰乙醇胺壳聚糖-脂质体药物载体。本发明所组装的壳聚糖-脂质体，不仅具有强的细胞粘附性能和抗血清能力，同时还具有环境响应性能，适用于静脉注射。本发明还提供了壳聚糖-脂质体包裹超顺磁四氧化三铁纳米粒子在药物递送中的应用，兼具高的药物递送效率和高的生物相容性，具有广阔的应用前景。

Description

氧化还原响应壳聚糖-脂质体的制备方法和用途 技术领域
本发明涉及一种氧化还原响应壳聚糖-脂质体药物载体，具体地说，涉及一种氧化还原响应双脂肪链取代基磷脂酰乙醇胺壳聚糖及其与脂质体构建包裹超顺磁四氧化三铁纳米粒子的药物载体，属于药物递送领域新型药物载体的制备方法。
背景技术
药物载体是指能改变药物进入人体的方式和在体内的分布、控制药物的释放速度并将药物输送到靶向器官的体系。药物载体通过控制释放，有效提高药物的利用率、安全性和有效性。壳聚糖和脂质体是常用的药物载体，壳聚糖生物相容性和生物可降解性好，2-位氨基和6-位羟基易于进行结构修饰，具有生物粘附性能，并能通过打开细胞通道提高药物在细胞间的瞬间渗透能力；脂质体是由两亲性表面活性剂分散在水中形成的一种具有一层或多层脂质囊泡结构的超微球状粒子，能够装载水溶性或者脂溶性药物，被广泛应用于药物载体。
多功能纳米载体是在单一功能纳米载体的基础上发展起来的新一代纳米载体，它克服了单一功能载体在肿瘤诊断和治疗中存在的一些不足，如对体内细胞活动的实时监控，对于靶部位的特殊靶向或药物在靶细胞内的有效传递。多功能纳米载体在一个单一稳定的结构中结合了不同的功能。如结合肿瘤显影剂或诊断试剂实现肿瘤的早期诊断，实时监测肿瘤治疗效果等。多功能纳米载体为肿瘤的早期诊断和个体化药物治疗提供了新机遇。
磁共振成像(MRI)具有良好的软组织分辨率和空间分辨率，清晰显示组织解剖结构的同时，可以对软组织的影像学特征进行准确的定位、定量分析，是肿瘤早期诊断的最有效的方法之一。为了增强病变组织与正常组织的图像之间的对比度以提高病变组织的清晰度，需要选择合适的对比剂来显示解剖学特征。T2对比剂具有较顺磁性物质更高的磁矩，对邻近组织中质子的弛豫有明显的加速效应，能显著提高检测灵敏度。常用的超顺磁性对比剂主要为不同大小的微晶金属粒子(如Fe3O4、γFe2O3)。
恶性肿瘤是人类健康的第一杀手。尽管近年来随着检测和治疗手段的改进，肿瘤患者的生存率有所提高，但是肿瘤患者的死亡率仍居高不下。目前，肿瘤治 疗主要手段之一是化学治疗，但药物的毒性反应和肿瘤细胞耐药性导致化学治疗治愈率低。另一方面，缺乏有效的早期诊断也是导致治愈率低的主要原因。因此，寻求新的有效的肿瘤早期诊断和治疗方法是临床肿瘤学亟待解决的难题。基因治疗通过将治疗基因导入到靶细胞核内以修复导致疾病的缺陷基因或者抑制导致疾病的有害基因，从而使机体恢复正常功能，达到治疗疾病的目的。安全、高效的载体是基因治疗成功的关键之一。
药物载体在血液中的稳定性对发挥药物载体的作用十分关键。脂质体结构易受血清中高密度脂蛋白等成分的破坏，导致包封药物的泄漏。壳聚糖具有良好的抗血清性能，有助于提高载药纳米颗粒在血清中的稳定性。通过后插入方式对脂质体进行壳聚糖修饰，构建表面具有壳聚糖刷子的药物载体，并包裹纳米四氧化三铁纳米粒子(SPIO)，形成集合药物递送和影像诊断于一体的多功能载体，以基因为模型药物，进行基因转染性能评价，实现治疗和诊断一体化，为肿瘤治疗开辟新途径。
人体病变部位(如肿瘤)的细胞外和细胞内存在明显的氧化还原环境差异，细胞外倾向于氧化性环境，以保持细胞膜蛋白等二硫键的稳定性，而细胞内则是过度表达的高浓度谷胱甘肽形成的还原性环境，利用细胞外与细胞内的氧化还原物质浓度的差异，有可能实现载体对药物的控制释放，进一步提高疗效。
发明内容
本发明所要解决的技术问题在于克服现有药物载体的缺陷，提供一种氧化还原响应双脂肪链取代基磷脂酰乙醇胺壳聚糖，以及通过后插入方式构建的具有氧化还原响应壳聚糖刷子和SPIO的脂质体载体，壳聚糖-脂质体药物载体的制备方法。
本发明第一个目的是请求保护氧化还原响应壳聚糖，具有式(Ⅰ)结构：
Figure PCTCN2017114413-appb-000001
其中，L＝-CO-(CH2)a-S-S-(CH2)b-CO-，a＝1～5，b＝1～5；R和R'为相同或不相同的CxHy，其中x＝11～17，y＝21～35。
优选地，L＝-CO-(CH2)2-S-S-(CH2)2-CO-，R和R'为相同或不相同的C11H23、C13H27、C17H35或C17H33。
本发明另一个目的请求保护氧化还原响应壳聚糖的制备方法，具体为：首先将壳聚糖溶解于水，必要时加1～3冰醋酸促使其充分溶解，搅拌状态下，逐滴滴加到二硫代双琥珀酰亚胺基取代酯的DMSO溶液，20～60℃反应1～24h后，继续向反应液中滴加双脂肪链取代基磷脂酰乙醇胺醇溶液，20～60℃反应1～24h，反应液旋转蒸发后，透析、冷干，制备得到氧化还原响应壳聚糖。
优选地，所述壳聚糖的重均分子量为500-10000Da，脱乙酰度为65-95％。
优选地，所述双脂肪链取代基磷脂酰乙醇胺为1,2-二月桂酰基磷脂酰乙醇胺、1,2-二硬脂酰基磷脂酰乙醇胺、1,2-二肉豆蔻酰基磷脂酰乙醇胺、1,2-二棕榈酰基磷脂酰乙醇胺、1,2-二油酰基磷脂酰乙醇胺的一种或两种以上，但不限于上述原料，其用量为壳聚糖重复单元摩尔当量的0.1-1倍，优选为0.3-0.6倍，反应条件优选为20-50℃搅拌反应2-48h，更优选为30-50℃搅拌反应4-12h。
本发明第三个目的还在于提供一种氧化还原响应壳聚糖-脂质体药物载体的制备方法：
首先采用薄膜超声法制备脂质体@SPIO复合材料，再通过后插入自组装的方式对阳离子脂质体进行修饰，将氧化还原响应壳聚糖插入到脂质体的磷脂双分子层，得到氧化还原响应壳聚糖-脂质体，其中阳离子脂质体与SPIO的质量比为30:1～10:1，壳聚糖与脂质体@SPIO的质量比为0.5:1～6:1。
优选地，所述阳离子脂质体为DOTAP、Lipofectin、LipofectaminTM 2000中的一种。
优选地，所述SPIO纳米粒子的粒径为1～30nm。
本发明同时请求保护上述氧化还原响应壳聚糖-脂质体作为药物载体的应用，尤其是在基因转染中的应用。
本发明通过氧化还原响应双脂肪链取代基磷脂酰乙醇胺对壳聚糖进行修饰，再通过后插入方式对脂质体@SPIO进行修饰，得到表面具有氧化还原响应壳聚糖 刷子的双脂肪链取代基磷脂酰乙醇胺壳聚糖-脂质体药物载体，提高脂质体抗血清能力和生物相容性，并通过对氧化还原环境响应，提高对药物的控制释放能力，同时提供磁场导向的靶向功能和核磁共振造影功能，实现治疗与诊断一体化的药物递送。
与现有技术相比，本发明具有以下优点：
1、本发明采用氧化还原环境响应双脂肪链取代基磷脂酰乙醇胺对壳聚糖进行修饰，制备了双脂肪链取代基磷脂酰乙醇胺壳聚糖。
2、本发明采用氧化还原环境响应双脂肪链取代基磷脂酰乙醇胺壳聚糖通过后插入方式对脂质体进行修饰，改善脂质体生物相容性和血液稳定性，并通过环境响应性能，适用于静脉注射，提高对药物的控制释放能力。
3、本发明采用壳聚糖-脂质体包裹超顺磁四氧化三铁纳米粒子获得氧化还原响应壳聚糖-脂质体复合载体，实现其在药物递送中的应用，特别是在基因转染中的应用。该复合物兼具高的药物递送效率和高的生物相容性，同时提供磁场导向的靶向功能和核磁共振造影功能，具有广阔的应用前景。
附图说明
图1为实施例1所制备的氧化还原响应壳聚糖的FTIR谱图；
图2为实施例1所制备的氧化还原响应壳聚糖的1HNMR谱图；
图3为实施例1所制备的氧化还原响应壳聚糖-DOTAP脂质体-SPIO复合载体的TEM照片；
图4为实施例1所制备的氧化还原响应壳聚糖-脂质体对DNA的延滞能力考查；
图5为本发明制备的氧化还原响应壳聚糖-脂质体的基因转染效率；
图6为本发明制备的氧化还原响应壳聚糖-脂质体的细胞毒性。
具体实施方式
下面通过附图和具体实施例详述本发明，但不限制本发明的保护范围。如无特殊说明，本发明所采用的实验方法均为常规方法，所用实验器材、材料、试剂等均可从化学公司购买。
实施例1
将分子量5kDa的壳聚糖(CSO)0.5g，溶于100mL水，超声30min，使其 充分溶解。搅拌状态下，将该壳聚糖水溶液逐滴滴加到二硫代双琥珀酰亚胺基丙酸酯的DMSO溶液，30℃反应2h后，继续向反应液中滴加0.5g的1,2-二油酰基磷脂酰乙醇胺的乙醇溶液，30℃反应2h，反应液旋转蒸发后，透析、冷干，制备得到氧化还原响应1,2-二油酰基磷脂酰乙醇胺壳聚糖。
配制1mg/mL氧化还原响应1,2-二油酰基磷脂酰乙醇胺壳聚糖水溶液，取100uL，与包含SPIO的DOTAP阳离子脂质体1mL通过超声的方式混合，然后静置1h，通过后插入自组装的方式，对脂质体进行修饰，获得表面具有氧化还原响应壳聚糖刷子的脂质体药物载体。
实施例2
将分子量5kDa的壳聚糖(CSO)0.5g，溶于100mL水，超声30min，使其充分溶解。搅拌状态下，将该壳聚糖水溶液逐滴滴加到二硫代双琥珀酰亚胺基丙酸酯的DMSO溶液，30℃反应2h后，继续向反应液中滴加0.5g的1,2-二硬脂酰基磷脂酰乙醇胺的乙醇溶液，30℃反应2h，反应液旋转蒸发后，透析、冷干，制备得到氧化还原响应1,2-二硬脂酰基磷脂酰乙醇胺壳聚糖。
配制1mg/mL氧化还原响应1,2-二硬脂酰基磷脂酰乙醇胺壳聚糖水溶液，取100uL，与包含SPIO的DOTAP阳离子脂质体1mL通过超声的方式混合，然后静置1h，通过后插入自组装的方式，对脂质体进行修饰，获得表面具有氧化还原响应壳聚糖刷子的脂质体药物载体。
实施例3
将分子量5kDa的壳聚糖(CSO)0.5g，溶于100mL水，超声30min，使其充分溶解。搅拌状态下，将该壳聚糖水溶液逐滴滴加到二硫代双琥珀酰亚胺基丙酸酯的DMSO溶液，30℃反应2h后，继续向反应液中滴加0.5g的1,2-二月桂酰基磷脂酰乙醇胺的乙醇溶液，30℃反应2h，反应液旋转蒸发后，透析、冷干，制备得到氧化还原响应1,2-二月桂酰基磷脂酰乙醇胺壳聚糖。
配制1mg/mL氧化还原响应1,2-二月桂酰基磷脂酰乙醇胺壳聚糖水溶液，取100uL，与包含SPIO的DOTAP阳离子脂质体1mL通过超声的方式混合，然后静置1h，通过后插入自组装的方式，对脂质体进行修饰，获得表面具有氧化还原响应壳聚糖刷子的脂质体药物载体。
实施例4
将分子量1kDa的壳聚糖(CSO)0.5g，溶于100mL水，超声30min，使其充分溶解。搅拌状态下，将该壳聚糖水溶液逐滴滴加到二硫代双琥珀酰亚胺基丙酸酯的DMSO溶液，30℃反应2h后，继续向反应液中滴加0.5g的1,2-二肉豆蔻酰基磷脂酰乙醇胺的乙醇溶液，30℃反应2h，反应液旋转蒸发后，透析、冷干，制备得到氧化还原响应1,2-二肉豆蔻酰基磷脂酰乙醇胺壳聚糖。
配制1mg/mL氧化还原响应1,2-二肉豆蔻酰基磷脂酰乙醇胺壳聚糖水溶液，取100uL，与包含SPIO的Lipofectin阳离子脂质体1mL通过超声的方式混合，然后静置1h，通过后插入自组装的方式，对脂质体进行修饰，获得表面具有氧化还原响应壳聚糖刷子的脂质体药物载体。
实施例5
将分子量1kDa的壳聚糖(CSO)0.5g，溶于100mL水，超声30min，使其充分溶解。搅拌状态下，将该壳聚糖水溶液逐滴滴加到二硫代双琥珀酰亚胺基丙酸酯的DMSO溶液，30℃反应2h后，继续向反应液中滴加0.5g的1,2-二棕榈酰基磷脂酰乙醇胺的乙醇溶液，30℃反应2h，反应液旋转蒸发后，透析、冷干，制备得到氧化还原响应1,2-二棕榈酰基磷脂酰乙醇胺壳聚糖。
配制1mg/mL氧化还原响应1,2-二棕榈酰基磷脂酰乙醇胺壳聚糖水溶液，取100uL，与包含SPIO的Lipofectin阳离子脂质体1mL通过超声的方式混合，然后静置1h，通过后插入自组装的方式，对脂质体进行修饰，获得表面具有氧化还原响应壳聚糖刷子的脂质体药物载体。
实施例6
将分子量1kDa的壳聚糖(CSO)0.5g，溶于100mL水，超声30min，使其充分溶解。搅拌状态下，将该壳聚糖水溶液逐滴滴加到二硫代双琥珀酰亚胺基丙酸酯的DMSO溶液，30℃反应2h后，继续向反应液中滴加1.0g的1,2-二油酰基磷脂酰乙醇胺的乙醇溶液，30℃反应2h，反应液旋转蒸发后，透析、冷干，制备得到氧化还原响应1,2-二油酰基磷脂酰乙醇胺壳聚糖。
配制1mg/mL氧化还原响应1,2-二油酰基磷脂酰乙醇胺壳聚糖水溶液，取100uL，与包含SPIO的LipofectaminTM 2000阳离子脂质体1mL通过超声的方式混合，然后静置1h，通过后插入自组装的方式，对脂质体进行修饰，获得表面具有氧化还原响应壳聚糖刷子的脂质体药物载体。
实施例7
将分子量5kDa的壳聚糖(CSO)0.5g，溶于100mL水，超声30min，使其充分溶解。搅拌状态下，将该壳聚糖水溶液逐滴滴加到二硫代双琥珀酰亚胺基丙酸酯的DMSO溶液，30℃反应2h后，继续向反应液中滴加1.0g的1,2-二硬脂酰基磷脂酰乙醇胺的乙醇溶液，30℃反应2h，反应液旋转蒸发后，透析、冷干，制备得到氧化还原响应1,2-二硬脂酰基磷脂酰乙醇胺壳聚糖。
配制1mg/mL氧化还原响应1,2-二硬脂酰基磷脂酰乙醇胺壳聚糖水溶液，取100uL，与包含SPIO的DOTAP阳离子脂质体1mL通过超声的方式混合，然后静置1h，通过后插入自组装的方式，对脂质体进行修饰，获得表面具有氧化还原响应壳聚糖刷子的脂质体药物载体。
实施例8
将分子量1kDa的壳聚糖(CSO)0.5g，溶于100mL水，超声30min，使其充分溶解。搅拌状态下，将该壳聚糖水溶液逐滴滴加到二硫代双琥珀酰亚胺基丙酸酯的DMSO溶液，30℃反应2h后，继续向反应液中滴加1.0g的1,2-二月桂酰基磷脂酰乙醇胺的乙醇溶液，30℃反应2h，反应液旋转蒸发后，透析、冷干，制备得到氧化还原响应1,2-二月桂酰基磷脂酰乙醇胺壳聚糖。
配制1mg/mL氧化还原响应1,2-二月桂酰基磷脂酰乙醇胺壳聚糖水溶液，取100uL，与包含SPIO的LipofectaminTM 2000阳离子脂质体1mL通过超声的方式混合，然后静置1h，通过后插入自组装的方式，对脂质体进行修饰，获得表面具有氧化还原响应壳聚糖刷子的脂质体药物载体。
实施例9
将分子量5kDa的壳聚糖(CSO)0.5g，溶于100mL水，超声30min，使其充分溶解。搅拌状态下，将该壳聚糖水溶液逐滴滴加到二硫代双琥珀酰亚胺基丙酸酯的DMSO溶液，30℃反应2h后，继续向反应液中滴加1.0g的1,2-二肉豆蔻酰基磷脂酰乙醇胺的乙醇溶液，30℃反应2h，反应液旋转蒸发后，透析、冷干，制备得到氧化还原响应1,2-二肉豆蔻酰基磷脂酰乙醇胺壳聚糖。
配制1mg/mL氧化还原响应1,2-二肉豆蔻酰基磷脂酰乙醇胺壳聚糖水溶液，取100uL，与包含SPIO的DOTAP阳离子脂质体1mL通过超声的方式混合， 然后静置1h，通过后插入自组装的方式，对脂质体进行修饰，获得表面具有氧化还原响应壳聚糖刷子的脂质体药物载体。
实施例10
将分子量1kDa的壳聚糖(CSO)0.5g，溶于100mL水，超声30min，使其充分溶解。搅拌状态下，将该壳聚糖水溶液逐滴滴加到二硫代双琥珀酰亚胺基丙酸酯的DMSO溶液，30℃反应2h后，继续向反应液中滴加1.0g的1,2-二棕榈酰基磷脂酰乙醇胺的乙醇溶液，30℃反应2h，反应液旋转蒸发后，透析、冷干，制备得到氧化还原响应1,2-二棕榈酰基磷脂酰乙醇胺壳聚糖。
配制1mg/mL氧化还原响应1,2-二棕榈酰基磷脂酰乙醇胺壳聚糖水溶液，取100uL，与包含SPIO的DOTAP阳离子脂质体1mL通过超声的方式混合，然后静置1h，通过后插入自组装的方式，对脂质体进行修饰，获得表面具有氧化还原响应壳聚糖刷子的脂质体药物载体。
实施例11
将分子量1kDa的壳聚糖(CSO)0.5g，溶于100mL水，超声30min，使其充分溶解。搅拌状态下，将该壳聚糖水溶液逐滴滴加到二硫代双琥珀酰亚胺基丙酸酯的DMSO溶液，30℃反应4h后，继续向反应液中滴加1.0g的1,2-二油酰基磷脂酰乙醇胺的乙醇溶液，30℃反应4h，反应液旋转蒸发后，透析、冷干，制备得到氧化还原响应1,2-二油酰基磷脂酰乙醇胺壳聚糖。
配制1mg/mL氧化还原响应1,2-二油酰基磷脂酰乙醇胺壳聚糖水溶液，取500uL，与包含SPIO的DOTAP阳离子脂质体1mL通过超声的方式混合，然后静置1h，通过后插入自组装的方式，对脂质体进行修饰，获得表面具有氧化还原响应壳聚糖刷子的脂质体药物载体。
实施例12
将分子量5kDa的壳聚糖(CSO)0.5g，溶于100mL水，超声30min，使其充分溶解。搅拌状态下，将该壳聚糖水溶液逐滴滴加到二硫代双琥珀酰亚胺基丙酸酯的DMSO溶液，30℃反应4h后，继续向反应液中滴加1.0g的1,2-二硬脂酰基磷脂酰乙醇胺的乙醇溶液，30℃反应4h，反应液旋转蒸发后，透析、冷干，制备得到氧化还原响应1,2-二硬脂酰基磷脂酰乙醇胺壳聚糖。
配制1mg/mL氧化还原响应1,2-二硬脂酰基磷脂酰乙醇胺壳聚糖水溶液，取500uL，与包含SPIO的DOTAP阳离子脂质体1mL通过超声的方式混合，然后静置1h，通过后插入自组装的方式，对脂质体进行修饰，获得表面具有氧化还原响应壳聚糖刷子的脂质体药物载体。
实施例13
将分子量5kDa的壳聚糖(CSO)0.5g，溶于100mL水，超声30min，使其充分溶解。搅拌状态下，将该壳聚糖水溶液逐滴滴加到二硫代双琥珀酰亚胺基丙酸酯的DMSO溶液，30℃反应4h后，继续向反应液中滴加1.0g的1,2-二月桂酰基磷脂酰乙醇胺的乙醇溶液，30℃反应4h，反应液旋转蒸发后，透析、冷干，制备得到氧化还原响应1,2-二月桂酰基磷脂酰乙醇胺壳聚糖。
配制1mg/mL氧化还原响应1,2-二月桂酰基磷脂酰乙醇胺壳聚糖水溶液，取500uL，与包含SPIO的DOTAP阳离子脂质体1mL通过超声的方式混合，然后静置1h，通过后插入自组装的方式，对脂质体进行修饰，获得表面具有氧化还原响应壳聚糖刷子的脂质体药物载体。
实施例14
将分子量5kDa的壳聚糖(CSO)0.5g，溶于100mL水，超声30min，使其充分溶解。搅拌状态下，将该壳聚糖水溶液逐滴滴加到二硫代双琥珀酰亚胺基丙酸酯的DMSO溶液，30℃反应4h后，继续向反应液中滴加1.0g的1,2-二肉豆蔻酰基磷脂酰乙醇胺的乙醇溶液，30℃反应4h，反应液旋转蒸发后，透析、冷干，制备得到氧化还原响应1,2-二肉豆蔻酰基磷脂酰乙醇胺壳聚糖。
配制1mg/mL氧化还原响应1,2-二肉豆蔻酰基磷脂酰乙醇胺壳聚糖水溶液，取500uL，与包含SPIO的DOTAP阳离子脂质体1mL通过超声的方式混合，然后静置1h，通过后插入自组装的方式，对脂质体进行修饰，获得表面具有氧化还原响应壳聚糖刷子的脂质体药物载体。
实施例15
将分子量1kDa的壳聚糖(CSO)0.5g，溶于100mL水，超声30min，使其充分溶解。搅拌状态下，将该壳聚糖水溶液逐滴滴加到二硫代双琥珀酰亚胺基丙酸酯的DMSO溶液，30℃反应4h后，继续向反应液中滴加1.0g的1,2-二棕榈酰基磷脂酰乙醇胺的乙醇溶液，30℃反应4h，反应液旋转蒸发后，透析、冷干， 制备得到氧化还原响应1,2-二棕榈酰基磷脂酰乙醇胺壳聚糖。
配制1mg/mL氧化还原响应1,2-二棕榈酰基磷脂酰乙醇胺壳聚糖水溶液，取500uL，与包含SPIO的DOTAP阳离子脂质体1mL通过超声的方式混合，然后静置1h，通过后插入自组装的方式，对脂质体进行修饰，获得表面具有氧化还原响应壳聚糖刷子的脂质体药物载体。
实施例16
将分子量5kDa的壳聚糖(CSO)0.5g，溶于100mL水，超声30min，使其充分溶解。搅拌状态下，将该壳聚糖水溶液逐滴滴加到二硫代双琥珀酰亚胺基丙酸酯的DMSO溶液，40℃反应4h后，继续向反应液中滴加1.0g的1,2-二油酰基磷脂酰乙醇胺的乙醇溶液，40℃反应4h，反应液旋转蒸发后，透析、冷干，制备得到氧化还原响应1,2-二油酰基磷脂酰乙醇胺壳聚糖。
配制1mg/mL氧化还原响应1,2-二油酰基磷脂酰乙醇胺壳聚糖水溶液，取500uL，与包含SPIO的DOTAP阳离子脂质体1mL通过超声的方式混合，然后静置2h，通过后插入自组装的方式，对脂质体进行修饰，获得表面具有氧化还原响应壳聚糖刷子的脂质体药物载体。
实施例17
将分子量1kDa的壳聚糖(CSO)0.5g，溶于100mL水，超声30min，使其充分溶解。搅拌状态下，将该壳聚糖水溶液逐滴滴加到二硫代双琥珀酰亚胺基丙酸酯的DMSO溶液，40℃反应4h后，继续向反应液中滴加1.0g的1,2-二硬脂酰基磷脂酰乙醇胺的乙醇溶液，40℃反应4h，反应液旋转蒸发后，透析、冷干，制备得到氧化还原响应1,2-二硬脂酰基磷脂酰乙醇胺壳聚糖。
配制1mg/mL氧化还原响应1,2-二硬脂酰基磷脂酰乙醇胺壳聚糖水溶液，取500uL，与包含SPIO的DOTAP阳离子脂质体1mL通过超声的方式混合，然后静置2h，通过后插入自组装的方式，对脂质体进行修饰，获得表面具有氧化还原响应壳聚糖刷子的脂质体药物载体。
实施例18
将分子量1kDa的壳聚糖(CSO)0.5g，溶于100mL水，超声30min，使其充分溶解。搅拌状态下，将该壳聚糖水溶液逐滴滴加到二硫代双琥珀酰亚胺基丙酸酯的DMSO溶液，40℃反应4h后，继续向反应液中滴加1.0g的1,2-二月桂酰 基磷脂酰乙醇胺的乙醇溶液，40℃反应4h，反应液旋转蒸发后，透析、冷干，制备得到氧化还原响应1,2-二月桂酰基磷脂酰乙醇胺壳聚糖。
配制1mg/mL氧化还原响应1,2-二月桂酰基磷脂酰乙醇胺壳聚糖水溶液，取500uL，与包含SPIO的DOTAP阳离子脂质体1mL通过超声的方式混合，然后静置2h，通过后插入自组装的方式，对脂质体进行修饰，获得表面具有氧化还原响应壳聚糖刷子的脂质体药物载体。
实施例19
将分子量5kDa的壳聚糖(CSO)0.5g，溶于100mL水，超声30min，使其充分溶解。搅拌状态下，将该壳聚糖水溶液逐滴滴加到二硫代双琥珀酰亚胺基丙酸酯的DMSO溶液，40℃反应4h后，继续向反应液中滴加1.0g的1,2-二肉豆蔻酰基磷脂酰乙醇胺的乙醇溶液，40℃反应4h，反应液旋转蒸发后，透析、冷干，制备得到氧化还原响应1,2-二肉豆蔻酰基磷脂酰乙醇胺壳聚糖。
配制1mg/mL氧化还原响应1,2-二肉豆蔻酰基磷脂酰乙醇胺壳聚糖水溶液，取500uL，与包含SPIO的DOTAP阳离子脂质体1mL通过超声的方式混合，然后静置2h，通过后插入自组装的方式，对脂质体进行修饰，获得表面具有氧化还原响应壳聚糖刷子的脂质体药物载体。
实施例20
将分子量5kDa的壳聚糖(CSO)0.5g，溶于100mL水，超声30min，使其充分溶解。搅拌状态下，将该壳聚糖水溶液逐滴滴加到二硫代双琥珀酰亚胺基丙酸酯的DMSO溶液，40℃反应4h后，继续向反应液中滴加1.0g的1,2-二棕榈酰基磷脂酰乙醇胺的乙醇溶液，40℃反应4h，反应液旋转蒸发后，透析、冷干，制备得到氧化还原响应1,2-二棕榈酰基磷脂酰乙醇胺壳聚糖。
配制1mg/mL氧化还原响应1,2-二棕榈酰基磷脂酰乙醇胺壳聚糖水溶液，取500uL，与包含SPIO的DOTAP阳离子脂质体1mL通过超声的方式混合，然后静置2h，通过后插入自组装的方式，对脂质体进行修饰，获得表面具有氧化还原响应壳聚糖刷子的脂质体药物载体。
具有氧化还原响应壳聚糖刷子的脂质体基因转运测定
采用pGL3质粒为报告基因，对具有氧化还原响应壳聚糖刷子的脂质体载体的基因转运性能进行评价，所用细胞为人非小细胞肺癌细胞A549细胞系。将培 养好的细胞铺板，在培养箱中培养至细胞融合度达到80％后，进行基因转运，转运时，将完全培养基吸去，用PBS洗涤两次，血清条件下转运时，加入400μL含10％血清的培养基和不同N/P比(质量比)的氧化还原响应polysome@SPIO(实施例3)与DNA复合物(每孔含1μg DNA)，培养18h后，吸出培养基，换上新鲜的含10％血清的培养基继续培养32h后，按照荧光素酶试剂盒所提供的说明书在BioTek Synergy2多功能酶标仪上检测光子的强度，用BCA检测出总蛋白的浓度，从而将结果统一标准化成RLU/mg蛋白(每毫克蛋白所对应的相对光子数)。
具有氧化还原响应壳聚糖刷子的脂质体的细胞毒性
载体的细胞毒性采用MTT法进行评价。在96孔细胞培养板上种植细胞，平行3孔，每孔种植5×104个细胞，在37℃，5％CO2细胞培养箱中培养至细胞融合度达到85％以上。移去培养基，用PBS洗2次后，加入新鲜培养基和待测载体，培养24h后，每孔加入20μL 5mg/mL的MTT溶液，37℃继续培养4h，移去培养基，终止培养。活细胞线粒体中的琥珀酸脱氢酶还原MTT生成甲臢，每孔加入150μL DMSO使溶解，在37℃继续孵育30min。在多功能酶标仪(Sunrise Tecan)上测定570nm波长各孔的吸收值，检测前震动96孔板自动混匀600s，并采用无细胞的培养基对酶标仪调零。细胞存活率按公式1.1计算：
细胞存活率(％)＝A570SMP/A570CTL×100   (1.1)
其中A570SMP为加入待测载体或复合物的细胞孔板的吸光值，A570CTL为只含培养基的细胞孔板的吸光值。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 氧化还原响应壳聚糖，其特征在于，具有式(Ⅰ)结构：
    Figure PCTCN2017114413-appb-100001
    其中，L＝-CO-(CH2)a-S-S-(CH2)b-CO-，a＝1～5，b＝1～5；
    R和R'为相同或不相同的CxHy，其中x＝11～17，y＝21～35。
  2. 根据权利要求1所述氧化还原响应壳聚糖，其特征在于，L＝-CO-(CH2)2-S-S-(CH2)2-CO-，R和R'为相同或不相同的C11H23、C13H27、C17H35或C17H33。
  3. 一种如权利要求1所述氧化还原响应壳聚糖的制备方法，其特征在于，首先将壳聚糖溶解于水，充分溶解，搅拌状态下，逐滴滴加到二硫代双琥珀酰亚胺基取代酯的DMSO溶液，20～60℃反应1～24h后，继续向反应液中滴加双脂肪链取代基磷脂酰乙醇胺醇溶液，20～60℃反应1～24h，反应液旋转蒸发后，透析、冷干，制备得到氧化还原响应壳聚糖。
  4. 根据权利要求3所述氧化还原响应壳聚糖的制备方法，其特征在于，所述壳聚糖的重均分子量为500-10000Da，脱乙酰度为65-95％。
  5. 根据权利要求3所述氧化还原响应壳聚糖的制备方法，其特征在于，所述双脂肪链取代基磷脂酰乙醇胺为1,2-二月桂酰基磷脂酰乙醇胺、1,2-二硬脂酰基磷脂酰乙醇胺、1,2-二肉豆蔻酰基磷脂酰乙醇胺、1,2-二棕榈酰基磷脂酰乙醇胺、1,2-二油酰基磷脂酰乙醇胺中的一种或两种以上。
  6. 根据权利要求3所述氧化还原响应壳聚糖的制备方法，其特征在于，所述双脂肪链取代基磷脂酰乙醇胺用量为壳聚糖重复单元摩尔当量的0.1-1倍，反应条件为20-50℃搅拌反应2-48h。
  7. 根据权利要求3所述氧化还原响应壳聚糖的制备方法，其特征在于，所述双脂肪链取代基磷脂酰乙醇胺用量为壳聚糖重复单元摩尔当量的0.3-0.6倍， 反应条件为30-50℃搅拌反应4-12h。
  8. 一种氧化还原响应壳聚糖-脂质体的制备方法，其特征在于，采用权利要求1所述氧化还原响应壳聚糖通过后插入自组装的方式，对阳离子脂质体进行修饰，得到氧化还原响应壳聚糖-脂质体。
  9. 根据权利要求8所述氧化还原响应壳聚糖-脂质体的制备方法，其特征在于，阳离子脂质体为DOTAP、Lipofectin、LipofectaminTM2000中的一种，该阳离子脂质体亲水核心包裹粒径为1～30nm的超顺磁四氧化三铁纳米粒子。
  10. 一种如权利要求8所述氧化还原响应壳聚糖-脂质体在药物递送中的应用。
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