CN107903341B - Application of double-aliphatic-chain substituent phosphatidyl ethanolamine chitosan - Google Patents

Application of double-aliphatic-chain substituent phosphatidyl ethanolamine chitosan Download PDF

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CN107903341B
CN107903341B CN201711363440.3A CN201711363440A CN107903341B CN 107903341 B CN107903341 B CN 107903341B CN 201711363440 A CN201711363440 A CN 201711363440A CN 107903341 B CN107903341 B CN 107903341B
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chitosan
liposome
phosphatidylethanolamine
double
fatty
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CN107903341A (en
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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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Dalian Minzu University
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Dalian Nationalities University
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • 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
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/12—Macromolecular compounds
    • A61K49/126—Linear polymers, e.g. dextran, inulin, PEG
    • 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

Abstract

The application provides application of double-fatty-chain substituent phosphatidylethanolamine chitosan, and the double-fatty-chain substituent phosphatidylethanolamine chitosan is synthesized from chitosan and double-fatty-chain substituent phosphatidylethanolamine. The liposome is modified by adopting the synthesized double-fatty-chain substituent phosphatidylethanolamine chitosan through a post-insertion self-assembly method, and the double-fatty-chain substituent phosphatidylethanolamine chitosan-liposome drug carrier is formed through assembly. The drug carrier assembled by the invention has strong cell adhesion performance and antiserum capacity, and is suitable for intravenous injection. The invention also provides application of the chitosan-liposome coated superparamagnetic ferroferric oxide nano particle in drug delivery, has high drug delivery efficiency and high biocompatibility, provides a magnetic field-oriented targeting function and a nuclear magnetic resonance imaging function, and has wide application prospect.

Description

Application of double-aliphatic-chain substituent phosphatidyl ethanolamine chitosan
The application is divisional application with application number of 2017106207042, application date of 2017, 7 and 27, and invention name of 'double-fatty-chain substituent phosphatidylethanolamine chitosan and preparation method and application thereof'.
Technical Field
The invention relates to double-fatty-chain substituent phosphatidylethanolamine chitosan and a liposome drug carrier thereof, in particular to double-fatty-chain substituent phosphatidylethanolamine chitosan and a drug carrier which is constructed by the double-fatty-chain substituent phosphatidylethanolamine chitosan and the liposome and wraps superparamagnetic ferroferric oxide nano particles, and belongs to a preparation method of a novel drug carrier in the field of drug delivery.
Background
Drug carriers are systems that alter the way drugs enter the body and distribute within the body, control the rate of release of the drug, and deliver the drug to the targeted organ. The drug carrier effectively improves the utilization rate, safety and effectiveness of the drug by controlling the release. The chitosan and the liposome are common drug carriers, the chitosan has good biocompatibility and biodegradability, 2-amino and 6-hydroxyl are easy to carry out structural modification, the chitosan has biological adhesion performance, and the instantaneous permeability of the drug among cells can be improved by opening cell channels; the liposome is a microspherical particle with a one-layer or multi-layer lipid vesicle structure formed by dispersing an amphiphilic surfactant in water, can load water-soluble or fat-soluble drugs, and is widely applied to drug carriers.
The multifunctional nano carrier is a new generation nano carrier developed on the basis of a single-function nano carrier, and overcomes the defects of the single-function carrier in tumor diagnosis and treatment, such as real-time monitoring of in-vivo cell activity, special targeting of a target site or effective transmission of a drug in a target cell. Multifunctional carriers combine different functions in a single stable structure. For example, the tumor imaging agent or the diagnostic reagent is combined to realize the early diagnosis of the tumor, and the tumor treatment effect is monitored in real time. The multifunctional nano-carrier provides new opportunities for early diagnosis of tumors and individualized drug therapy.
magnetic Resonance Imaging (MRI) has good soft tissue resolution and spatial resolution, can perform accurate positioning and quantitative analysis on the imaging characteristics of soft tissues while clearly displaying anatomical structures of the tissues, and is one of the most effective methods for early diagnosis of tumors. In order to enhance the contrast between the images of diseased tissue and normal tissue to improve the clarity of the diseased tissue, a suitable contrast agent needs to be selected to display the anatomical features. The T2 contrast agent has a higher magnetic moment than the paramagnetic substance and is effective in treating adjacent tissueThe relaxation of the mesogen has obvious acceleration effect, and the detection sensitivity can be obviously improved. The commonly used superparamagnetic contrast agents are mainly microcrystalline metal particles (e.g. Fe) of different sizes3O4、Fe2O3)。
Malignant tumors are the first killer of human health. Although the survival rate of tumor patients has been improved with the improvement of detection and treatment means in recent years, the mortality rate of tumor patients remains high. At present, one of the main means of tumor treatment is chemotherapy, but the toxic reaction of the drug and the drug resistance of tumor cells cause low cure rate of the chemotherapy. On the other hand, lack of effective early diagnosis is also a major cause of low cure rate. Therefore, the search for new effective tumor early diagnosis and treatment methods is a difficult problem to be solved in clinical oncology. Gene therapy is to introduce therapeutic genes into target nuclei to repair defective genes causing diseases or to inhibit harmful genes causing diseases, thereby restoring normal functions of the body and achieving the purpose of treating diseases. Safe and efficient vectors are one of the keys to the success of gene therapy.
The stability of the drug carrier in blood is critical to the functioning of the drug carrier. The liposome structure is susceptible to destruction by components such as high density lipoproteins in serum, resulting in leakage of encapsulated drugs. The chitosan has good antiserum performance and is beneficial to improving the stability of the drug-loaded nanoparticles in serum. The liposome is modified by chitosan in a post-insertion mode, a drug carrier with a chitosan brush on the surface is constructed, nano ferroferric oxide nanoparticles (SPIO) are wrapped to form a multifunctional carrier integrating drug delivery and image diagnosis, gene transfection performance evaluation is carried out by taking genes as model drugs, treatment and diagnosis integration is realized, and a new way is opened up for tumor treatment.
Disclosure of Invention
The invention aims to overcome the defects of the traditional medicament carrier and provides double-fatty-chain substituent phosphatidylethanolamine chitosan, a liposome carrier which is constructed in a post-insertion mode and provided with a chitosan brush and SPIO, and a preparation method thereof.
the invention provides a double-aliphatic-chain substituent phosphatidylethanolamine chitosan, which has a structure shown in a formula (I):
Wherein R and R' are the same or different CxHyWherein x is 11 to 17, and y is 21 to 35.
Preferably, R and R' are the same or different C11H23、C13H27、C17H35Or C17H33。
The double-aliphatic-chain substituent phosphatidylethanolamine chitosan is realized by the following technical scheme:
Firstly, reacting one or more than two of sodium hypochlorite, sodium nitrite, periodate or hydrogen peroxide as an oxidant with chitosan under the conditions of pH 2-6 and 0-50 ℃ for 1-24 hours, dialyzing reaction liquid, and freeze-drying to prepare formyl chitosan; dispersing formyl chitosan into an alcohol solution, adding the double-fatty-chain substituent phosphatidyl ethanolamine, stirring and refluxing for 2-48 h at 20-100 ℃ under the protection of inert gas, adding sodium borohydride, continuously stirring for 3-24 h, removing an ethanol solvent through rotary evaporation, dispersing into water, dialyzing with deionized water, and freeze-drying to obtain the double-fatty-chain substituent phosphatidyl ethanolamine chitosan.
Preferably, the molar ratio of the chitosan glucosamine unit to the oxidant is 1: 3-10: 1.
Preferably, the chitosan has a weight average molecular weight of 500-10000Da and a degree of deacetylation of 65-95%.
preferably, the double-fatty-chain substituent phosphatidyl ethanolamine is one or more than two of 1, 2-dilauroyl phosphatidyl ethanolamine, 1, 2-distearoyl phosphatidyl ethanolamine, 1, 2-dimyristoyl phosphatidyl ethanolamine, 1, 2-dipalmitoyl phosphatidyl ethanolamine and 1, 2-dioleoyl phosphatidyl ethanolamine, but is not limited to the above raw materials, the dosage is 0.1-1 times, preferably 0.3-0.6 times of the molar equivalent of the formyl chitosan repeating unit, and the reaction condition is reflux for 2-48 h at 20-100 ℃, preferably reflux for 4-12h at 30-50 ℃.
The invention also aims to provide a compound (polysome @ SPIO) of the double-aliphatic-chain substituent phosphatidylethanolamine chitosan and the liposome @ SPIO.
Preferably, the cationic liposome is DOTAP, Lipofectin, LipofectaminTM2000, the particle diameter of the SPIO nano particle is 1-30 nm.
the polysome @ SPIO compound is realized by the following technical scheme: the liposome @ SPIO composite material is prepared by adopting a film ultrasonic method, and then double-fatty-chain substituent phosphatidylethanolamine chitosan is inserted into a liposome phospholipid bilayer in a post-insertion mode to obtain the polysome @ SPIO composite.
The invention also provides application of the polysome @ SPIO compound as a drug carrier, in particular application in gene transfection.
According to the invention, the chitosan is modified by the double-fatty-chain substituent phosphatidylethanolamine, and the liposome @ SPIO is modified by the post-insertion mode, so that the liposome composite material with the chitosan brush on the surface is obtained, the antiserum capability and the biocompatibility of the liposome are improved, and meanwhile, the efficient drug delivery is realized.
Compared with the prior art, the invention has the following advantages:
1. The invention adopts double-fatty-chain substituent phosphatidylethanolamine to modify chitosan, and prepares the double-fatty-chain substituent phosphatidylethanolamine chitosan.
2. The invention adopts double-fatty-chain substituent phosphatidylethanolamine chitosan to modify the liposome in a post-insertion mode, thereby improving the biocompatibility and blood stability of the liposome.
3. The liposome disclosed by the invention simultaneously encapsulates the SPIO to obtain a polysome @ SPIO composite carrier, so that the application of the liposome in drug delivery is realized, and particularly the application of the liposome in gene transfection is realized. The composite carrier has high drug delivery efficiency and high biocompatibility, provides a magnetic field-oriented targeting function and a nuclear magnetic resonance imaging function, and has wide application prospects.
Drawings
FIG. 1 is an FTIR spectrum of a double aliphatic chain substituent phosphatidylethanolamine chitosan prepared in example 2;
FIG. 2 shows the preparation of double fatty chain substituent phosphatidylethanolamine chitosan prepared in example 21HNMR spectrogram;
FIG. 3 is a TEM photograph of the double-aliphatic-chain-substituent phosphatidylethanolamine chitosan-DOTAP liposome-SPIO composite vector prepared in example 2;
FIG. 4 shows the retardation ability of the polysome @ SPIO composite vector prepared in example 2 on DNA;
FIG. 5 shows the gene transfection efficiency of the polysome @ SPIO composite vector prepared in the present invention;
FIG. 6 shows the cytotoxicity of polysome @ SPIO composite vector prepared in the present invention.
Detailed Description
the invention is described in detail below with reference to the figures and the specific examples, without limiting the scope of protection of the invention. Unless otherwise specified, the experimental methods adopted by the invention are all conventional methods, and experimental equipment, materials, reagents and the like used in the experimental method can be purchased from chemical companies.
0.8554g of low molecular weight Chitosan (CSO) was dissolved in 50mL of NaAc/HAc (pH 4.5) buffer solution and then dissolved by sonication for 30 min. 0.1093g of sodium periodate was dissolved in 50mL of NaAc/HAc (pH 4.5) buffer and sonicated for 30 min. Placing the two solutions in ice bath respectively, introducing high-purity nitrogen, degassing for 30min, mixing the two solutions, reacting for 24h at 0-4 deg.C under stirring, and adding 10mL ethylene glycol to stop the reaction. The reaction solution was transferred to a dialysis bag (MWCO ═ 3000Da), dialyzed, lyophilized, and formyl chitosan was prepared.
Weighing 0.16g of formyl chitosan, dispersing into 20mL of methanol solution, adding 0.14g of 1, 2-distearoyl phosphatidyl ethanolamine, performing stirring reaction at 60 ℃ for 42h under the protection of nitrogen, adding 0.1g of sodium borohydride, continuously stirring at room temperature for 24h, performing rotary evaporation to remove an ethanol solvent, dispersing into water, dialyzing with deionized water, and freeze-drying to obtain the 1, 2-distearoyl phosphatidyl ethanolamine chitosan.
preparing 1mg/mL 1, 2-distearoyl phosphatidyl ethanolamine chitosan aqueous solution, taking 100uL, mixing with 1mL of DOTAP cationic liposome containing SPIO in an ultrasonic mode, standing for 1h, modifying the liposome in a post-insertion self-assembly mode, and obtaining the liposome drug carrier with a chitosan brush on the surface.
example 2
Weighing 0.16g of formyl chitosan, dispersing into 20mL of methanol solution, adding 0.14g of 1, 2-dioleoyl phosphatidylethanolamine, stirring and reacting at 60 ℃ for 42h under the protection of nitrogen, adding 0.1g of sodium borohydride, continuously stirring at room temperature for 24h, removing an ethanol solvent by rotary evaporation, dispersing into water, dialyzing with deionized water, and freeze-drying to obtain the 1, 2-dioleoyl phosphatidylethanolamine chitosan.
Preparing 1mg/mL 1, 2-dioleoyl phosphatidylethanolamine aqueous solution, taking 100uL, mixing with 1mL DOTAP cationic liposome containing SPIO in an ultrasonic mode, standing for 1h, modifying the liposome in a post-insertion self-assembly mode, and obtaining the liposome drug carrier with the chitosan brush on the surface.
Example 3
Weighing 0.16g of formyl chitosan, dispersing into 20mL of methanol solution, adding 0.14g of 1, 2-dilauroyl phosphatidyl ethanolamine, stirring for reaction at 60 ℃ for 42h under the protection of nitrogen, adding 0.1g of sodium borohydride, continuously stirring for 24h at room temperature, removing an ethanol solvent by rotary evaporation, dispersing into water, dialyzing with deionized water, and freeze-drying to obtain the 1, 2-dilauroyl phosphatidyl ethanolamine chitosan.
Preparing 1mg/mL 1, 2-dilauroyl phosphatidyl ethanolamine aqueous solution, mixing 100uL with 1mL DOTAP cationic liposome containing SPIO in an ultrasonic mode, standing for 1h, and modifying the liposome in a post-insertion self-assembly mode to obtain the liposome drug carrier with the chitosan brush on the surface.
Example 4
Weighing 0.16g of formyl chitosan, dispersing into 20mL of methanol solution, adding 0.14g of 1, 2-dimyristoyl phosphatidylethanolamine, stirring for reaction at 60 ℃ for 42h under the protection of nitrogen, adding 0.1g of sodium borohydride, continuously stirring for 24h at room temperature, removing an ethanol solvent by rotary evaporation, dispersing into water, dialyzing with deionized water, and freeze-drying to obtain the 1, 2-dimyristoyl phosphatidylethanolamine chitosan.
Preparing 1mg/mL 1, 2-dimyristoyl phosphatidylethanolamine aqueous solution, taking 100uL, mixing with 1mL DOTAP cationic liposome containing SPIO in an ultrasonic mode, standing for 1h, modifying the liposome in a post-insertion self-assembly mode, and obtaining the liposome drug carrier with the chitosan brush on the surface.
Example 5
Weighing 0.16g of formyl chitosan, dispersing into 20mL of methanol solution, adding 0.14g of 1, 2-dipalmitoyl phosphatidyl ethanolamine, stirring for reaction at 60 ℃ for 42h under the protection of nitrogen, adding 0.1g of sodium borohydride, continuously stirring for 24h at room temperature, removing an ethanol solvent by rotary evaporation, dispersing into water, dialyzing with deionized water, and freeze-drying to obtain the 1, 2-dipalmitoyl phosphatidyl ethanolamine chitosan.
Preparing 1mg/mL 1, 2-dipalmitoyl phosphatidyl ethanolamine chitosan aqueous solution, taking 100uL, mixing with 1mL of DOTAP cationic liposome containing SPIO in an ultrasonic mode, standing for 1h, modifying the liposome in a post-insertion self-assembly mode, and obtaining the liposome drug carrier with the chitosan brush on the surface.
Example 6
Weighing 0.16g of formyl chitosan, dispersing into 20mL of methanol solution, adding 0.3g of 1, 2-distearoyl phosphatidyl ethanolamine, performing stirring reaction at 60 ℃ for 42h under the protection of nitrogen, adding 0.1g of sodium borohydride, continuously stirring at room temperature for 24h, performing rotary evaporation to remove an ethanol solvent, dispersing into water, dialyzing with deionized water, and freeze-drying to obtain the 1, 2-distearoyl phosphatidyl ethanolamine chitosan.
Preparing 1mg/mL 1, 2-distearoyl phosphatidyl ethanolamine chitosan aqueous solution, taking 100uL, mixing with 1mL of DOTAP cationic liposome containing SPIO in an ultrasonic mode, standing for 1h, modifying the liposome in a post-insertion self-assembly mode, and obtaining the liposome drug carrier with a chitosan brush on the surface.
Example 7
weighing 0.16g of formyl chitosan, dispersing into 20mL of methanol solution, adding 0.3g of 1, 2-dioleoyl phosphatidylethanolamine, stirring and reacting at 60 ℃ for 42h under the protection of nitrogen, adding 0.1g of sodium borohydride, continuously stirring at room temperature for 24h, removing an ethanol solvent by rotary evaporation, dispersing into water, dialyzing with deionized water, and freeze-drying to obtain the 1, 2-dioleoyl phosphatidylethanolamine chitosan.
Preparing 1mg/mL 1, 2-dioleoyl phosphatidylethanolamine chitosan aqueous solution, taking 100uL, mixing with 1mL DOTAP cationic liposome containing SPIO in an ultrasonic mode, standing for 1h, modifying the liposome in a post-insertion self-assembly mode, and obtaining the liposome drug carrier with the chitosan brush on the surface.
Example 8
Weighing 0.16g of formyl chitosan, dispersing into 20mL of methanol solution, adding 0.3g of 1, 2-dilauroyl phosphatidyl ethanolamine, stirring for reaction at 60 ℃ for 42h under the protection of nitrogen, adding 0.1g of sodium borohydride, continuously stirring for 24h at room temperature, removing an ethanol solvent by rotary evaporation, dispersing into water, dialyzing with deionized water, and freeze-drying to obtain the 1, 2-dilauroyl phosphatidyl ethanolamine chitosan.
Preparing 1mg/mL 1, 2-dilauroyl phosphatidyl ethanolamine aqueous solution, mixing 100uL with 1mL DOTAP cationic liposome containing SPIO in an ultrasonic mode, standing for 1h, and modifying the liposome in a post-insertion self-assembly mode to obtain the liposome drug carrier with the chitosan brush on the surface.
Example 9
Weighing 0.16g of formyl chitosan, dispersing into 20mL of methanol solution, adding 0.3g of 1, 2-dimyristoyl phosphatidylethanolamine, stirring for reaction at 60 ℃ for 42h under the protection of nitrogen, adding 0.1g of sodium borohydride, continuously stirring for 24h at room temperature, removing an ethanol solvent by rotary evaporation, dispersing into water, dialyzing with deionized water, and freeze-drying to obtain the 1, 2-dimyristoyl phosphatidylethanolamine chitosan.
Preparing 1mg/mL 1, 2-dimyristoyl phosphatidylethanolamine aqueous solution, taking 100uL, mixing with 1mL DOTAP cationic liposome containing SPIO in an ultrasonic mode, standing for 1h, modifying the liposome in a post-insertion self-assembly mode, and obtaining the liposome drug carrier with the chitosan brush on the surface.
Example 10
Weighing 0.16g of formyl chitosan, dispersing into 20mL of methanol solution, adding 0.3g of 1, 2-dipalmitoyl phosphatidyl ethanolamine, stirring for reaction at 60 ℃ for 42h under the protection of nitrogen, adding 0.1g of sodium borohydride, continuously stirring for 24h at room temperature, removing an ethanol solvent by rotary evaporation, dispersing into water, dialyzing with deionized water, and freeze-drying to obtain the 1, 2-dipalmitoyl phosphatidyl ethanolamine chitosan.
Preparing 1mg/mL 1, 2-dipalmitoyl phosphatidyl ethanolamine chitosan aqueous solution, taking 100uL, mixing with 1mL of DOTAP cationic liposome containing SPIO in an ultrasonic mode, standing for 1h, modifying the liposome in a post-insertion self-assembly mode, and obtaining the liposome drug carrier with the chitosan brush on the surface.
Example 11
Weighing 0.16g of formyl chitosan, dispersing into 20mL of methanol solution, adding 0.3g of 1, 2-distearoyl phosphatidyl ethanolamine, performing stirring reaction at 50 ℃ for 42h under the protection of nitrogen, adding 0.1g of sodium borohydride, continuously stirring at room temperature for 24h, performing rotary evaporation to remove an ethanol solvent, dispersing into water, dialyzing with deionized water, and freeze-drying to obtain the 1, 2-distearoyl phosphatidyl ethanolamine chitosan.
Preparing 1mg/mL 1, 2-distearoyl phosphatidyl ethanolamine chitosan aqueous solution, taking 500uL, mixing with 1mL of DOTAP cationic liposome containing SPIO in an ultrasonic mode, standing for 1h, modifying the liposome in a post-insertion self-assembly mode, and obtaining the liposome drug carrier with a chitosan brush on the surface.
Example 12
Weighing 0.16g of formyl chitosan, dispersing into 20mL of methanol solution, adding 0.3g of 1, 2-dioleoyl phosphatidylethanolamine, stirring and reacting at 50 ℃ for 42h under the protection of nitrogen, adding 0.1g of sodium borohydride, continuously stirring at room temperature for 24h, removing an ethanol solvent by rotary evaporation, dispersing into water, dialyzing with deionized water, and freeze-drying to obtain the 1, 2-dioleoyl phosphatidylethanolamine chitosan.
Preparing 1mg/mL 1, 2-dioleoyl phosphatidylethanolamine aqueous solution, taking 500uL, mixing with 1mL DOTAP cationic liposome containing SPIO in an ultrasonic mode, standing for 1h, modifying the liposome in a post-insertion self-assembly mode, and obtaining the liposome drug carrier with the chitosan brush on the surface.
Example 13
Weighing 0.16g of formyl chitosan, dispersing into 20mL of methanol solution, adding 0.3g of 1, 2-dilauroyl phosphatidyl ethanolamine, stirring at 50 ℃ for reaction for 42h under the protection of nitrogen, adding 0.1g of sodium borohydride, continuously stirring at room temperature for 24h, removing an ethanol solvent by rotary evaporation, dispersing into water, dialyzing with deionized water, and freeze-drying to obtain the 1, 2-dilauroyl phosphatidyl ethanolamine chitosan.
Preparing 1mg/mL 1, 2-dilauroyl phosphatidyl ethanolamine aqueous solution, mixing 500uL with 1mL DOTAP cationic liposome containing SPIO in an ultrasonic mode, standing for 1h, and modifying the liposome in a post-insertion self-assembly mode to obtain the liposome drug carrier with the chitosan brush on the surface.
Example 14
Weighing 0.16g of formyl chitosan, dispersing into 20mL of methanol solution, adding 0.3g of 1, 2-dimyristoyl phosphatidylethanolamine, stirring for reaction at 50 ℃ for 42h under the protection of nitrogen, adding 0.1g of sodium borohydride, continuously stirring for 24h at room temperature, removing an ethanol solvent by rotary evaporation, dispersing into water, dialyzing with deionized water, and freeze-drying to obtain the 1, 2-dimyristoyl phosphatidylethanolamine chitosan.
preparing 1mg/mL 1, 2-dimyristoyl phosphatidylethanolamine aqueous solution, taking 500uL, mixing with 1mL DOTAP cationic liposome containing SPIO in an ultrasonic mode, standing for 1h, modifying the liposome in a post-insertion self-assembly mode, and obtaining the liposome drug carrier with the chitosan brush on the surface.
Example 15
Weighing 0.16g of formyl chitosan, dispersing into 20mL of methanol solution, adding 0.3g of 1, 2-dipalmitoyl phosphatidyl ethanolamine, stirring for reaction at 50 ℃ for 42h under the protection of nitrogen, adding 0.1g of sodium borohydride, continuously stirring for 24h at room temperature, removing an ethanol solvent by rotary evaporation, dispersing into water, dialyzing with deionized water, and freeze-drying to obtain the 1, 2-dipalmitoyl phosphatidyl ethanolamine chitosan.
Preparing 1mg/mL 1, 2-dipalmitoyl phosphatidyl ethanolamine chitosan aqueous solution, taking 500uL, mixing with 1mL of DOTAP cationic liposome containing SPIO in an ultrasonic mode, standing for 1h, modifying the liposome in a post-insertion self-assembly mode, and obtaining the liposome drug carrier with the chitosan brush on the surface.
Example 16
weighing 0.16g of formyl chitosan, dispersing into 20mL of methanol solution, adding 0.3g of 1, 2-distearoyl phosphatidyl ethanolamine, carrying out stirring reaction at 80 ℃ for 42h under the protection of nitrogen, adding 0.2g of sodium borohydride, continuously stirring at room temperature for 24h, carrying out rotary evaporation to remove an ethanol solvent, dispersing into water, dialyzing with deionized water, and freeze-drying to obtain the 1, 2-distearoyl phosphatidyl ethanolamine chitosan.
Preparing 1mg/mL 1, 2-distearoyl phosphatidyl ethanolamine chitosan aqueous solution, taking 500uL, mixing with 1mL of DOTAP cationic liposome containing SPIO in an ultrasonic mode, standing for 2h, modifying the liposome in a post-insertion self-assembly mode, and obtaining the liposome drug carrier with a chitosan brush on the surface.
Example 17
Weighing 0.16g of formyl chitosan, dispersing into 20mL of methanol solution, adding 0.3g of 1, 2-dioleoyl phosphatidylethanolamine, stirring and reacting at 80 ℃ for 42h under the protection of nitrogen, adding 0.2g of sodium borohydride, continuously stirring at room temperature for 24h, removing an ethanol solvent by rotary evaporation, dispersing into water, dialyzing with deionized water, and freeze-drying to obtain the 1, 2-dioleoyl phosphatidylethanolamine chitosan.
Preparing 1mg/mL 1, 2-dioleoyl phosphatidylethanolamine aqueous solution, taking 500uL, mixing with 1mL DOTAP cationic liposome containing SPIO in an ultrasonic mode, standing for 2h, modifying the liposome in a post-insertion self-assembly mode, and obtaining the liposome drug carrier with the chitosan brush on the surface.
Example 18
Weighing 0.16g of formyl chitosan, dispersing into 20mL of methanol solution, adding 0.3g of 1, 2-dilauroyl phosphatidyl ethanolamine, stirring for reaction at 80 ℃ for 42h under the protection of nitrogen, adding 0.2g of sodium borohydride, continuously stirring for 24h at room temperature, removing an ethanol solvent by rotary evaporation, dispersing into water, dialyzing with deionized water, and freeze-drying to obtain the 1, 2-dilauroyl phosphatidyl ethanolamine chitosan.
preparing 1mg/mL 1, 2-dilauroyl phosphatidyl ethanolamine aqueous solution, mixing 500uL with 1mL DOTAP cationic liposome containing SPIO in an ultrasonic mode, standing for 2h, and modifying the liposome in a post-insertion self-assembly mode to obtain the liposome drug carrier with the chitosan brush on the surface.
Example 19
Weighing 0.16g of formyl chitosan, dispersing into 20mL of methanol solution, adding 0.3g of 1, 2-dimyristoyl phosphatidylethanolamine, stirring for reaction at 80 ℃ for 42h under the protection of nitrogen, adding 0.2g of sodium borohydride, continuously stirring for 24h at room temperature, removing an ethanol solvent by rotary evaporation, dispersing into water, dialyzing with deionized water, and freeze-drying to obtain the 1, 2-dimyristoyl phosphatidylethanolamine chitosan.
Preparing 1mg/mL 1, 2-dimyristoyl phosphatidylethanolamine aqueous solution, taking 500uL, mixing with 1mL DOTAP cationic liposome containing SPIO in an ultrasonic mode, standing for 2h, modifying the liposome in a post-insertion self-assembly mode, and obtaining the liposome drug carrier with the chitosan brush on the surface.
Example 20
Weighing 0.16g of formyl chitosan, dispersing into 20mL of methanol solution, adding 0.3g of 1, 2-dipalmitoyl phosphatidyl ethanolamine, stirring for reaction at 80 ℃ for 42h under the protection of nitrogen, adding 0.2g of sodium borohydride, continuously stirring for 24h at room temperature, removing an ethanol solvent by rotary evaporation, dispersing into water, dialyzing with deionized water, and freeze-drying to obtain the 1, 2-dipalmitoyl phosphatidyl ethanolamine chitosan.
Preparing 1mg/mL 1, 2-dipalmitoyl phosphatidyl ethanolamine chitosan aqueous solution, taking 500uL, mixing with 1mL DOTAP cationic liposome containing SPIO in an ultrasonic mode, standing for 2h, modifying the liposome in a post-insertion self-assembly mode, and obtaining the liposome drug carrier with the chitosan brush on the surface.
Liposome gene transport assay with chitosan brush
pGL3 plasmid is used as a reporter gene to evaluate the gene transfer performance of the liposome carrier with the chitosan brush, and the used cells are human non-small cell lung cancer cell A549 cell lines. The cultured cells are plated and cultured in an incubator until the cell fusion degree reaches 80%, gene transfer is carried out, when the complete culture medium is sucked and washed twice by PBS, when the cells are transferred under serum condition, 400 mu L of culture medium containing 10% of serum and polysome @ SPIO (example 3) and DNA compound (containing 1 mu g of DNA in each well) with different N/P ratios (mass ratio) are added, after 18h of culture, the culture medium is sucked out, fresh culture medium containing 10% of serum is replaced for further culture for 32h, the intensity of photons is detected on a BioTek Synergy2 multifunctional enzyme microplate reader according to the instruction provided by a luciferase kit, and the concentration of total protein is detected, so that the result is unified and standardized into RLU/mg protein (the relative photon number corresponding to each mg of protein).
cytotoxicity of liposomes with chitosan brushes
The cytotoxicity of the vector was evaluated by the MTT method. Cells were plated in 96-well cell culture plates in 3 wells in parallel, 5X 10 wells per well4Individual cells, 5% CO at 37 ℃2Culturing in a cell culture box until the cell fusion degree reaches more than 85 percent. The medium was removed, washed 2 times with PBS, and then fresh medium and the test carrier were added, and after 24 hours of incubation, 20. mu.L of 5mg/mL MTT solution was added to each well, incubation was continued at 37 ℃ for 4 hours, and the medium was removed and the incubation was terminated. Living cellReduction of MTT by succinate dehydrogenase in mitochondria resulted in formazan, solubilization was performed by addition of 150 μ L DMSO per well, and incubation was continued for 30min at 37 ℃. The absorbance of each well at a wavelength of 570nm was measured on a multifunctional microplate reader (Sunrise Tecan), the 96-well plate was shaken before detection and automatically mixed for 600s, and the microplate reader was zeroed with a cell-free medium. Cell viability was calculated according to equation 1.1:
Cell survival (%) ═ a570SMP/A570CTL×100 (1.1)
Wherein A570SMPAbsorbance of cell well plates to which test vectors or complexes are added, A570CTLAbsorbance was measured for cell well plates containing medium only.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims (1)

1. The application of the double-fatty-chain substituent phosphatidylethanolamine chitosan is characterized in that the double-fatty-chain substituent phosphatidylethanolamine chitosan-liposome prepared from the double-fatty-chain substituent phosphatidylethanolamine chitosan is used for preparing an antihuman non-small cell lung cancer cell A549 drug carrier:
0.8554g of low molecular weight chitosan is dissolved in 50mL of NaAc/HAc buffer solution with pH =4.5, and the solution is dissolved by ultrasonic treatment for 30 min; 0.1093g of sodium periodate, dissolved in 50mL of NaAc/HAc buffer solution with pH =4.5, and sonicated for 30 min; placing the two solutions in ice bath respectively, introducing high-purity nitrogen, degassing for 30min, mixing the two solutions, stirring at 0-4 deg.C for 24 hr, and adding 10mL ethylene glycol to stop reaction; transferring the reaction solution into a dialysis bag MWCO =3000 Da, dialyzing, and freeze-drying to prepare formyl chitosan;
Weighing 0.16g of formyl chitosan, dispersing into 20mL of methanol solution, adding 0.3g of 1, 2-dioleoyl phosphatidylethanolamine, stirring for reaction at 50 ℃ for 42h under the protection of nitrogen, adding 0.1g of sodium borohydride, continuously stirring for 24h at room temperature, removing an ethanol solvent by rotary evaporation, dispersing into water, dialyzing with deionized water, and freeze-drying to obtain 1, 2-dioleoyl phosphatidylethanolamine chitosan;
Preparing 1mg/mL 1, 2-dioleoyl phosphatidylethanolamine chitosan aqueous solution, taking 500 mu L, mixing with 1mL DOTAP cationic liposome containing SPIO in an ultrasonic mode, standing for 1h, modifying the liposome in a post-insertion self-assembly mode, and obtaining a liposome drug carrier with a chitosan brush on the surface;
culturing the human non-small cell lung cancer cell A549 cell line in an incubator until the cell fusion degree reaches 80%, sucking the culture medium, washing with PBS, adding the culture medium containing 10% serum and the liposome drug carrier and DNA compound with the chitosan brush on the surface when transferring under serum condition, culturing for 18h, sucking out the culture medium, and replacing with fresh culture medium containing 10% serum to continue culturing for 32 h.
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