CN117126411A

CN117126411A - PEGylated galactosyl-modified chitosan oligosaccharide stearylamine disulfide graft and its liver parenchymal cell-targeted nanoformulation and method

Info

Publication number: CN117126411A
Application number: CN202310839382.6A
Authority: CN
Inventors: 缪静; 高鹏
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2023-07-10
Filing date: 2023-07-10
Publication date: 2023-11-28
Anticipated expiration: 2043-07-10
Also published as: CN117126411B

Abstract

The invention discloses a PEGylated galactosyl-modified chitosan oligosaccharide stearylamine disulfide graft and its liver parenchymal cell-targeted nano preparation and method, and belongs to the field of synthesis of polymer drug carriers. In the PEGylated galactosyl-modified chitosan oligosaccharide stearylamine disulfide graft graft provided by the invention, the chitosan oligosaccharide has a molecular weight of 18.9 kDa, a deacetylation degree of the chitosan oligosaccharide is 95%, and the carbon chain length of the fatty acid is C18; degree of amino substitution is 1% to 30%. The PEGylated galactosyl-modified chitosan oligosaccharide fatty acid graft has efficient liver parenchymal cell targeting ability, and the PEGylated galactosyl-modified chitosan oligosaccharide stearylamine disulfide graft drug delivery system can significantly Blocks hepatitis B virus antigen expression.

Description

Polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting material, liver parenchymal cell targeting nano preparation and method

Technical Field

The invention belongs to the field of synthesis of high molecular medicine carriers, and relates to construction of a polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting material, and efficient entrapment of recombinant proteins is realized by the synthesized grafting material, and the grafting material is applied to treatment of chronic hepatitis B.

Background

Chronic hepatitis b (chronic hepatitis B, CHB) is a viral infectious disease that severely jeopardizes human health, and according to WHO's latest statistics, there are about 2.96 hundred million people worldwide WHO are HBV chronic infectious agents, and over 80 ten thousand people each year die from liver failure, cirrhosis and liver cancer caused by chronic hepatitis b. After HBV infection of hepatocytes, covalently closed circular DNA (covalently closed circular DNA, cccDNA) is formed, which serves as a template for viral replication, is highly latent and sustainable in circulation, and is the most important cause of chronic viral infection and recurrence of patients after drug withdrawal of "functional cure", and cccDNA clearance is also considered as a key indicator for radical treatment of CHB. Currently, clinical treatment CHB drugs are nucleoside (t) analogs (NAs) and interferons (IFN- α), but nass can only inhibit HBV replication, and are difficult to act on cccDNA; IFN- α is effective only in a small proportion of patients with CHB and is generally poorly tolerated. Therefore, there is a need to develop new drugs and therapies for HBV cccDNA to significantly improve CHB cure rate.

Human apolipoprotein B mRNA editing enzyme catalyzes polypeptide-like protein 3 (apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3, APOBEC 3) a family of cytosine deaminase enzymes, including 7 subfamilies (APOBEC 3A-H, excluding E), as a natural immune factor in the body against a variety of viral infections. Among them, apodec 3A (a 3A) is mainly expressed in peripheral blood mononuclear cells, but the expression level in hepatocytes is low, but in recent years, studies have found that a3A can act on cccDNA mediated by the viral core protein HBc to deaminate cytosine to uracil during transcription, and then is recognized by DNA glycosylase and excised to form apurinic/apyrimidinic sites (apurinic/apyrimidinic site, AP-site). AP-site can be recognized by AP endonuclease, cccDNA degradation is finally realized, and the damage of A3A to the host cell genome is not detected, so that the specificity of the effect on cccDNA is shown. Based on this, human A3A recombinant protein (A3A recombinant protein, A3 ARP) was obtained by gene recombination technique, in hope of being a novel drug for clearing HBV cccDNA, for improving cure rate of CHB.

However, A3ARP is a negatively charged hydrophilic macromolecular substance, and has problems such as poor in vivo stability and difficulty in transmembrane transport. At present, the recombinant protein drugs applied clinically are mainly limited to extracellular environments, intracellular delivery still faces great challenges, and stable protein entrapment and efficient intracellular release are often difficult to combine. At the same time, the targeting of intracellular delivery of A3ARP is not neglected. Nonspecific uptake by non-target cells may lead to problems such as reduced efficacy and toxic or side effects.

The chitosan oligosaccharide stearic acid grafting material can form micelle in water medium, and the synthesized amphoteric cation grafting material can be compounded with hydrophilic macromolecular substance with negative electricity through the chemical grafting of carboxyl of stearic acid and active amino on chitosan oligosaccharide. Studies show that liver is the main site for synthesizing Glutathione-SH (GSH), and compared with the content of GSH (2-20 mu M) in extracellular fluid and systemic circulation, the liver cell contains high concentration GSH (1-10 mM), and the GSH concentration of liver parenchymal cells infected by hepatitis B virus is increased in a stress manner. The specific high GSH concentration of hepatitis B virus infected liver cells is utilized, disulfide bond functional groups are introduced into a carrier structure, and the response release of the environment in the liver cells for encapsulating the medicine can be realized. Galactose modification of glycolipid disulfide grafts (CSSO) was performed using a highly potent endocytosed asialoglycoprotein receptor (Asialoglycoprotein receptor, ASGPR) specific for liver parenchymal cells, capable of specifically recognizing galactosyl groups. In addition, polyethylene glycol (Polyethylene glycol, PEG) is a water-soluble polymer with good biocompatibility, and is widely used for modification of drugs and carriers. Research shows that PEG can prevent the adsorption of reducing matters such as plasma protein, reduce reticuloendothelial cell phagocytosis and avoid interception of immune system. Meanwhile, PEG helps to delay premature release of carriers with higher positive charge load (such as chitosan oligosaccharide and the like) before in vivo drug delivery to a target.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting material, and a liver parenchymal cell targeting nano preparation and a method thereof. The synthesized polyethylene glycol galactosyl modified chitosan oligosaccharide fatty acid disulfide bond grafting (Galactosylated PEGylated chitosan oligosaccharide-ss-octadecylamine, GP-CSSO) has the advantages that the outer shell unloading disulfide bond modification can be realized, so that extracellular fluid and low-concentration GSH in the systemic circulation can be utilized, the release of target extracellular medicines can be reduced, and the disulfide bond linkage between glycolipids can be opened in response to high-concentration GSH in liver cells, so that medicines can be quickly released in the target area of the liver cells; galactosyl modification can actively target liver tissues and hepatic parenchymal cells; PEGylation modification can reduce the influence of in-vivo extracellular reducing substances, reduce the phagocytosis of non-parenchymal cells in the liver, and improve the active targeting to the parenchymal cells of the liver in cooperation with galactosyl modification. GP-CSSO forms cationic micelle by self aggregation in aqueous medium, and A3ARP can be electrostatically compounded, thereby realizing active targeting of liver tissue and hepatic parenchymal cells and improving the cure rate of CHB.

The specific technical scheme adopted by the invention is as follows:

In a first aspect, the invention provides a polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting material, wherein the molecular weight of the chitosan oligosaccharide is 18.9kDa, and the deacetylation degree of the chitosan oligosaccharide is 95%; the carbon chain length of the fatty acid is C18; the substitution degree of amino is 1% -30%;

the chemical structural general formula of the polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting material is specifically as follows:

in a second aspect, the invention provides a preparation method of a polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting (such as the compound in the first aspect), which specifically comprises the following steps:

s1: respectively crosslinking hydrophilic chitosan oligosaccharide and hydrophobic stearylamine by 3,3' -dithiodipropionic acid by adopting a two-step amide reaction method to synthesize a chitosan oligosaccharide stearylamine disulfide grafting material;

s2: dissolving p-carboxybenzaldehyde in N, N-diAdding carbodiimide and N-hydroxysuccinimide into methyl formamide, completely dissolving, reacting at room temperature for 2 hours, adding polyethylene glycol diamine, completely dissolving, and continuously reacting at room temperature overnight; concentrating the reaction solution under reduced pressure, precipitating with glacial ethyl ether, centrifuging to collect the product, and vacuum drying to obtain NH ₂ -PEG _2k -CHO; NH is subjected to ₂ -PEG _2k dissolving-CHO in N, N-dimethylformamide, adding galactose-NHS and triethylamine, reacting at room temperature for 4 hr, removing solvent under reduced pressure, adding pure water for dissolving, extracting with dichloromethane, collecting organic phase, concentrating under reduced pressure, precipitating with glacial diethyl ether, centrifuging, collecting the product, and vacuum drying to obtain Gal-PEG _2k -CHO；

S3: using the Gal-PEG _2k And (3) synthesizing the polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting by Schiff reaction between the terminal aldehyde group of CHO and the free amino group of the chitosan oligosaccharide stearylamine disulfide bond grafting.

Preferably, the step S1 specifically includes the following steps:

dissolving 3,3' -dithiodipropionic acid, stearylamine, dicyclohexylcarbodiimide and 4-dimethylaminopyridine in anhydrous dimethyl sulfoxide, and reacting for 18h under the condition of stirring in a water bath at 60 ℃; then adding carbodiimide and N-hydroxysuccinimide, and reacting for 30min under the condition of water bath stirring at 60 ℃ to obtain organic phase reaction liquid; dissolving chitosan oligosaccharide in water, then dropwise adding the organic phase reaction solution while stirring, and reacting for 8 hours under the water bath stirring condition at 60 ℃; after the reaction is finished, adding a 7kDa dialysis bag, dialyzing for 48 hours, and taking the content of the dialysis bag to centrifuge for 10 minutes at normal temperature under the rotation speed of 10000 rpm; and (3) after the supernatant obtained by centrifugation is freeze-dried, washing with absolute ethyl alcohol to remove unreacted alcohol-soluble byproducts and stearylamine, and obtaining the chitosan oligosaccharide stearylamine disulfide bond grafting.

Further, the concentration of the chitosan oligosaccharide was prepared as a 20mg/ml solution, and the molar ratio of stearylamine (ODA) to chitosan oligosaccharide was 30:1, feeding.

Further, the molar ratio of stearylamine, 3' -dithiodipropionic acid, dicyclohexylcarbodiimide, 4-dimethylaminopyridine, N-hydroxysuccinimide and carbodiimide was 1:1:3:0.3:10:10.

Preferably, in the step S2, 100mg of p-carboxybenzaldehyde is dissolved in 5ml of N, N-dimethylformamide, and NH is added ₂ -PEG _2k -CHO 500mg in 5ml of n, n-dimethylformamide; the molar ratio of the carbodiimide to the N-hydroxysuccinimide to the polyethylene glycol diamine to the galactose-NHS to the triethylamine is 1.2:1.2:1.0:1.2:2.0.

Preferably, the step S3 specifically includes the following steps:

the chitosan oligosaccharide stearylamine disulfide bond grafting is dissolved in acetic acid water solution with pH of 5.5, and then Gal-PEG is adopted _2k Chitosan oligosaccharide stearylamine disulfide bond grafting=1 (1-10) molar ratio of addition Gal-PEG _2k CHO, followed by addition of 3-fold molar amounts of sodium triacetoxyborohydride to the chitosan oligosaccharide stearylamine disulfide graft, stirring for 24h at room temperature in the absence of light; then placing the chitosan oligosaccharide into a dialysis bag MWCO 7kDa, dialyzing with deionized water for 24 hours, and taking the solution in the dialysis bag to obtain the polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting.

In a third aspect, the invention provides a method for constructing a hepatic parenchymal cell targeting nano-preparation, which comprises the following steps:

dissolving the polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting material according to the first aspect or the polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting material obtained by the preparation method according to any one of the second aspect in water, filtering and sterilizing, and then adding the polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting material into a human A3A recombinant protein solution according to the theoretical drug loading rate of the polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting material of human A3A recombinant protein= (100-400): 1 according to the mass ratio, and incubating at room temperature to obtain the liver parenchymal cell targeting nano preparation for treating chronic hepatitis B.

Preferably, after the polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting is dissolved in water, the grafting is subjected to water bath ultrasonic treatment at room temperature for 15min, and then filtration sterilization is carried out by using a microporous filter membrane with the pore diameter of 0.22 mu m.

Preferably, the room temperature incubation time is 30min.

In a fourth aspect, the present invention provides a liver parenchymal cell targeting nano-preparation obtained by the construction method of the third aspect.

Compared with the prior art, the invention has the following beneficial effects:

The invention constructs the polyethylene glycol galactosyl modified chitosan oligosaccharide stearamine disulfide bond grafting material with good biostability and safety, liver parenchymal cell targeting, effective escape of lysosomes and micro-environment response release in liver parenchymal cells, can greatly increase the uptake of liver cells to medicines so as to improve the medicine concentration of medicine molecular targeting parts, improve the curative effect of the medicines, increase the uptake of the medicines by the liver parenchymal cells, be favorable for reducing the distribution of the medicines in normal tissues or cells and reduce the toxic and side effects of the medicines. In addition, the humanized A3ARP is obtained by a gene recombination technology, the polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting material has high-efficiency encapsulation capacity on the A3ARP, and the synthesized polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting material has the anti-hepatitis B gene drug-carrying system, and has the targeting capacity of hepatic parenchymal cells and the antigen expression inhibiting function.

Drawings

FIG. 1 shows hydrogen nuclear magnetic resonance spectra of CSSO and GP-CSSO.

FIG. 2GP-CSSO is a 72h incubation of cells at HepG2-NTCP (A) with their delivery system drug release profile (B).

FIG. 3 intracellular delivery of GP-CSSO/A3ARP delivery system in HepG 2-NTCP.

FIG. 4 in vivo tissue distribution of GP-CSSO/A3ARP delivery system.

FIG. 5 synthetic route pattern of GP-CSSO.

Detailed Description

The invention is further illustrated and described below with reference to the drawings and detailed description. The technical features of the embodiments of the invention can be combined correspondingly on the premise of no mutual conflict.

The invention provides a polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting material which has high-efficiency liver parenchymal cell targeting capability. Specifically, the molecular weight of the chitosan oligosaccharide is 18.9kDa, and the deacetylation degree of the chitosan oligosaccharide is 95%; the carbon chain length of the fatty acid is C18; the substitution degree of the amino is 1-30%. Representative chemical structural general formula of the polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting is specifically as follows:

the synthetic route of the polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting material is shown in fig. 5, and the preparation method specifically comprises the following steps:

(1) Synthesis of glycolipid disulfide graft (Chitosan oligosaccharide stearylamine disulfide graft) CSSO

The chitosan oligosaccharide stearylamine disulfide bond grafting (CSSO) is synthesized by respectively crosslinking hydrophilic chitosan oligosaccharide and hydrophobic stearylamine with 3,3' -dithiodipropionic acid (DTPA) by adopting a two-step amide reaction method.

In practical application, the method specifically comprises the following steps: the prescribed amounts of 3,3'-dithiodipropionic acid (3, 3' -Dithiodipropionic acid, DTPA), stearylamine (ODA), dicyclohexylcarbodiimide (DCC) and 4-Dimethylaminopyridine (DMAP) were weighed, dissolved in an appropriate amount of anhydrous dimethyl sulfoxide (DMSO), reacted for 18 hours under the condition of stirring nitrogen in a water bath at 60 ℃, and the prescribed amounts of carbodiimide (1- (3-dimethylminopropyl) -3-ethylcarbodiimide hydrochloride (EDC)) and N-Hydroxysuccinimide (N-Hydroxysuccinimide, NHS) were added and stirred in a water bath at 60 ℃ for 30 minutes. Then, after the prescription amount of chitosan oligosaccharide (CSO) is dissolved in water, the organic phase reaction solution is slowly dripped into the CSO solution while stirring for reaction for 8 hours in a water bath at 60 ℃, after the reaction is finished, a 7kDa dialysis bag is added for dialysis for 48 hours, the dialysis bag content is centrifuged at 10000rpm for 10 minutes at normal temperature, and after the supernatant is freeze-dried, unreacted alcohol-soluble byproducts and stearylamine are removed by washing with absolute ethyl alcohol, so that the glycolipid disulfide graft is obtained, wherein the feeding mole ratio is as follows:

ODA:DTPA:DCC:DMAP:NHS::EDC＝1:1:3:0.3:10:10。

(2) Synthesis of Gal-PEG-CHO

Dissolving p-carboxybenzaldehyde in N, N-Dimethylformamide (DMF), adding carbodiimide (EDC) and N-hydroxysuccinimide (NHS), dissolving completely, reacting at room temperature for 2 hr, adding polyethylene glycol diamine (NH) ₂ -PEG _2k -NH ₂ ) Complete dissolution and continuing the reaction at room temperature overnight; concentrating the reaction solution under reduced pressure, precipitating with glacial ethyl ether, centrifuging to collect the product, and vacuum drying to obtain NH ₂ -PEG _2k -CHO; NH is added to ₂ -PEG _2k dissolving-CHO in N, N-Dimethylformamide (DMF), adding galactose-NHS and triethylamine, reacting at room temperature for 4 hr, removing solvent under reduced pressure, adding pure water for dissolving, extracting with dichloromethane, collecting organic phase, concentrating under reduced pressure, precipitating with glacial diethyl ether, centrifuging, collecting the product, and vacuum drying to obtain Gal-PEG _2k -CHO。

In practical applications, the following parameters are preferably used in this step:

100mg of p-carboxybenzaldehyde is weighed and dissolved in 5ml of DMF, EDC (1.2 eq.) and NHS (1.2 eq.) are added and dissolved completely, and NH is added after 2h of reaction at room temperature ₂ -PEG _2k -NH ₂ (1.0 eq.) is completely dissolved, the reaction is continued at room temperature for overnight, the reaction solution is concentrated under reduced pressure, poured into a large amount of glacial ethyl ether for precipitation, the product is collected by centrifugation, and the NH is obtained by vacuum drying ₂ -PEG _2k -CHO。

Weighing NH ₂ -PEG _2k dissolving-CHO 500mg in 5ml DMF, adding galactose-NHS (1.2 eq.) and triethylamine (2.0 eq.) to dissolve completely, reacting at room temperature for 4h, removing solvent under reduced pressure, adding pure water to dissolve, extracting with dichloromethane for 3 times, collecting organic phase, concentrating under reduced pressure, pouring into a large amount of glacial diethyl ether for precipitation, centrifuging to collect the product, and vacuum drying to obtain Gal-PEG _2k -CHO。

(3) Synthesis of polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting (GP-CSSO)

Gal-PEG prepared by step (2) _2k Schiff reaction between terminal aldehyde group of CHO and free amino of chitosan oligosaccharide stearylamine disulfide bond grafting material prepared in the step (1),synthesizing polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting.

In practical application, the method specifically comprises the following steps:

the prescribed amount of CSSO was dissolved in an appropriate amount of acetic acid solution (pH 5.5) to give a CSSO micelle solution having a concentration of 5.0 mg/mL. According to Gal-PEG-CHO: the CSSO feeding mole ratio is 1:1 to 1:10, adding the raw materials, then adding 3 times of sodium triacetoxyborohydride, stirring at room temperature in a dark place for 24 hours, placing in a dialysis bag (MWCO 7 kDa), dialyzing with deionized water for 24 hours, taking the solution in the dialysis bag to obtain GP-CSSO, and freeze-drying for preservation.

Based on the polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting, the invention also provides a liver parenchymal cell targeting nano-preparation for treating chronic hepatitis B, namely an A3 ARP-loaded system of the polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting. The method is realized by the following scheme:

And (3) weighing GP-CSSO freeze-dried powder, dissolving in deionized water, filtering for sterilization, and then adding the theoretical drug loading rate of the humanized A3A recombinant protein= (100-400): 1 into the humanized A3A recombinant protein solution according to the mass ratio of the polyethylene glycol galactosyl modified chitosan stearylamine disulfide bond grafting material, and incubating at room temperature to obtain the liver parenchymal cell targeting nano-preparation for treating chronic hepatitis B.

In practical application, the construction method specifically comprises the following steps:

the GP-CSSO freeze-dried powder is weighed and dissolved in deionized water, the solution is treated by water bath ultrasonic treatment for 15min, so that GP-CSSO micelle solutions with different concentrations are prepared, and the solution is filtered and sterilized by a microporous filter membrane with the pore diameter of 0.22 mu m. Adding A3ARP solution according to the theoretical drug loading rate of 100:1-400:1 of the feed ratio (w/w) to prepare GP-CSSO/A3ARP, and incubating for 30min at room temperature to obtain the composite A3ARP delivery system GP-CSSO/A3ARP (namely the hepatic parenchymal cell targeting nano-preparation).

The liver parenchymal cell targeting nano preparation prepared by the invention can be applied to treating chronic hepatitis B and can obviously block the expression of hepatitis B virus antigens.

The technical scheme of the invention and corresponding technical effects which can be achieved are specifically described below through embodiments.

Example 1

The polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting material is prepared in the embodiment, and the performance of the obtained product is verified, specifically as follows:

(1) Preparation of low molecular weight chitosan

Taking 50g of chitosan with the molecular weight of 450kDa and the deacetylation degree of 95%, slowly adding the chitosan into 1500ml of 1.25% (v/v) hydrochloric acid solution under the condition of stirring in a water bath at 60 ℃ and 500rpm, stirring at 500rpm overnight to fully swell the chitosan, slowly adding chitosan enzyme with the weight ratio of 2% of chitosan, stirring at 400rpm to carry out enzymolysis reaction, controlling the degradation degree of the chitosan by using a gel permeation chromatography, heating to 80 ℃ after the reaction is finished, adding 0.3% (w/v) active carbon, diluting the reaction solution, filtering by a Buchner funnel, and freeze-drying to obtain low molecular weight chitosan (chitosan oligosaccharide, mw 18.9 kDa) powder.

(2) Synthesis of glycolipid disulfide bond grafting material CSSO

The prepared 18.9kDa chitosan oligosaccharide is taken and dissolved in distilled water to prepare a solution with the concentration of 20 mg/ml. The molar ratio of stearylamine (Octadecylamine, ODA) to chitosan oligosaccharide is 30:1 taking stearylamine, and mixing stearylamine, 3'-dithiodipropionic acid (3, 3' -Dithiodipropionic acid, DTPA), dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (4-dimethylaminopyridine, DMAP) according to a molar ratio of 1:1:3: weighing 0.3 weight, dissolving in anhydrous DMSO, reacting for 24 hours under the condition of stirring nitrogen in a water bath at 60 ℃, and filtering through a Buchner funnel. Carbodiimide (1- (3-dimethylmineopyl) -3-ethylcarbodiimide hydrochloride (EDC)) and N-Hydroxysuccinimide (NHS) to stearylamine molar ratio of 10:10:1, adding the mixture into filtrate, stirring the mixture in a water bath at 60 ℃ for reaction for 30min, slowly adding the mixture into a chitosan oligosaccharide aqueous solution preheated at 60 ℃, stirring the mixture in the water bath at 60 ℃ for reaction for 12h, adding a 7kDa dialysis bag after the reaction is finished, dialyzing the dialysis solution for 48h, and washing the dialysis solution with absolute ethyl alcohol to remove unreacted stearylamine to obtain the glycolipid disulfide bond grafting material.

(3) Synthesis of Gal-PEG-CHO

(4) Synthesis of polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting GP-CSSO

CSSO was dissolved in an appropriate amount of acetic acid solution (pH 5.5) to give a CSSO micelle solution at a concentration of 5.0 mg/mL. According to Gal-PEG _2k -CHO: CSSO: sodium triacetoxyborohydride molar ratio is 1:10:3 adding the raw materials, stirring for 24 hours at room temperature in a dark place, placing in a dialysis bag (MWCO 7 kDa), dialyzing with deionized water for 24 hours, taking the solution in the dialysis bag to obtain GP-CSSO, and freeze-drying and preserving.

The amino substitution degree of GP-CSSO was determined by the trinitrobenzenesulfonic acid method. Taking 50-1000 mu L of chitosan oligosaccharide with different weights, dissolving in deionized water, preparing 1.0mg/mL chitosan oligosaccharide, and then fixing the volume to 2.0mL. Adding 2.0mL of sodium bicarbonate with the mass fraction of 4% and 2.1% of trinitrobenzenesulfonic acid with the mass fraction of 0.1% respectively, carrying out water bath at 37 ℃ for 2 hours, adding 2mol/L hydrochloric acid to 2.0mL, shaking uniformly, measuring the absorbance at the wavelength of 344nm by using a spectrophotometer, and preparing a standard curve. 5mg of grafting material is weighed and dissolved in a proper amount of deionized water to prepare a grafting material solution with the concentration of 1mg/ml, the absorbance at 344nm is measured through the same operation, and finally, the amino substitution degree of GP-CSSO is calculated to be 20.51% +/-0.11% through the obtained standard curve.

Example 2

The polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting material and the liver parenchymal cell targeting nano preparation are prepared in the embodiment, and the performance of the obtained product is verified, specifically as follows: (1) Preparation of low molecular weight chitosan

(2) Synthesis of glycolipid disulfide bond grafting material CSSO

The prepared 18.9kDa chitosan oligosaccharide is taken and dissolved in distilled water to prepare a solution with the concentration of 20 mg/ml. The molar ratio of stearylamine (Octadecylamine, ODA) to chitosan oligosaccharide is 30: stearylamine is taken, and the stearylamine, 3'-dithiodipropionic acid (3, 3' -Dithiodipropionic acid, DTPA), dicyclohexylcarbodiimide (DCC) and 4-Dimethylaminopyridine (DMAP) are weighed according to the molar ratio of 1:1:3:0.3, dissolved in anhydrous DMSO, and reacted for 24 hours under the condition of stirring nitrogen in a water bath at 60 ℃ and filtered by a Buchner funnel. Carbodiimide (1- (3-dimethylmineopyl) -3-ethylcarbodiimide hydrochloride (EDC)) and N-Hydroxysuccinimide (NHS) to stearylamine molar ratio of 10:10:1, adding the mixture into filtrate, stirring the mixture in a water bath at 60 ℃ for reaction for 30min, slowly adding the mixture into a chitosan oligosaccharide aqueous solution preheated at 60 ℃, stirring the mixture in the water bath at 60 ℃ for reaction for 12h, adding a 7kDa dialysis bag after the reaction is finished, dialyzing the dialysis solution for 48h, and washing the dialysis solution with absolute ethyl alcohol to remove unreacted stearylamine to obtain the glycolipid disulfide bond grafting material.

(3) Synthesis of Gal-PEG-CHO

CSSO was dissolved in an appropriate amount of acetic acid solution (pH 5.5) to give a CSSO micelle solution at a concentration of 5.0 mg/mL. According to Gal-PEG _2k -CHO: CSSO: sodium triacetoxyborohydride molar ratio is 1:1:3 adding the raw materials, stirring for 24 hours at room temperature in a dark place, placing in a dialysis bag (MWCO 7 kDa), dialyzing with deionized water for 24 hours, taking the solution in the dialysis bag to obtain GP-CSSO, and freeze-drying and preserving.

The amino substitution degree of GP-CSSO was determined by the trinitrobenzenesulfonic acid method. Taking 50-1000 mu L of chitosan oligosaccharide with different weights, dissolving in deionized water, preparing 1.0mg/mL chitosan oligosaccharide, and then fixing the volume to 2.0mL. Adding 2.0mL of sodium bicarbonate with the mass fraction of 4% and 2.1% of trinitrobenzenesulfonic acid with the mass fraction of 0.1% respectively, carrying out water bath at 37 ℃ for 2 hours, adding 2mol/L hydrochloric acid to 2.0mL, shaking uniformly, measuring the absorbance at the wavelength of 344nm by using a spectrophotometer, and preparing a standard curve. 5mg of grafting material is weighed and dissolved in a proper amount of deionized water to prepare a grafting material solution with the concentration of 1mg/ml, the absorbance at 344nm is measured through the same operation, and finally, the amino substitution degree of 8.56% +/-0.37% of GP-CSSO is calculated through the obtained standard curve.

(5) Physical and chemical properties of polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting GP-CSSO

Nuclear magnetic resonance hydrogen spectrometry CSO, DTPA, ODA, CSSO, gal-PEG-CHO, GP-CSSO. 10mg of CSO, DTPA, ODA, CSSO and Gal-PEG are weighed respectivelyCHO, GP-CSSO are respectively dissolved in 1ml D ₂ In O (preparation concentration: 10 mg/mL), the nuclear magnetic resonance hydrogen spectrum was measured by a nuclear magnetic resonance spectrometer, and the results are shown in FIG. 1. After grafting chitosan oligosaccharide by carrying out amidation reaction under the catalysis of DCC/DMAP, the carboxyl hydrogen with the chemical shift of 12ppm of DTPA disappears, and the amino hydrogen with the chemical shift of 1.3ppm of ODA still exists, which indicates that the hydrophobic end has been successfully grafted to chitosan oligosaccharide and CSSO is successfully synthesized. After Schiff reaction of terminal aldehyde group of Gal-PEG-CHO and CSSO free amino, chemical shift of Gal-PEG-CHO is carried out for 10ppm of aldehyde hydrogen to disappear, and chemical shift of 3.6ppm of methylenedioxy hydrogen still exists, which indicates that Gal-PEG-CHO has been grafted to CSSO and GP-CSSO is successfully synthesized.

And (3) determining the critical micelle concentration of the GP-CSSO by adopting a pyrene fluorescence spectrophotometry. The method comprises the following steps:

weighing 12mg of pyrene, dissolving in proper amount of acetone to prepare 0.0012mg/mL pyrene acetone solution, respectively transferring 0.5mL pyrene acetone solution into 10mL test tubes, and volatilizing the acetone at 50 ℃. GP-CSSO mother liquor with the concentration of 1.0mg/mL is prepared. The mother liquor was diluted with deionized water to give GP-CSSO solutions of varying concentrations (0.005, 0.01, 0.02, 0.05, 0.1, 0.3, 0.5, 0.7 and 1.0 mg/mL). 5.0mL of each grafting solution with different dilution concentrations is removed and added into a pyrene-containing test tube, and the grafting solution is subjected to water bath ultrasonic treatment for 30min. The emission spectrum of GP-CSSO was scanned using a fluorescence spectrophotometer (emission wavelength range 360-450 nm, emission slit 2.5nm, excitation wavelength 337nm, excitation slit 10 nm), and the first peak (I _l =374 nm) and a third peak (I ₃ Ratio of =385 nm (I ₁ /I ₃ ). Finally, the GP-CSSO critical micelle concentration is calculated to be 52.44 +/-1.02 mug/mL.

The GP-CSSO grafting material is weighed and dissolved in distilled water to prepare a GP-CSSO micelle solution with the concentration of 1.0mg/ml, and the particle size and the surface potential analyzer are used for measuring the GP-CSSO particle size of 107.41 +/-4.55 nm and the Zeta potential of 15.12+/-0.73 mV.

(6) Polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting GP-CSSO cytotoxicity

Taking HepG2-NTCP cells in logarithmic growth phase at 1×10 per well ⁴ Inoculating into 96-well cell culture plate at 37deg.C under conditions containing5％CO ₂ After culturing in incubator until cell adhesion growth, adding 100-2000 μg/mL GP-CSSO micelle to incubate for 72h, and measuring in vitro cytotoxicity of GP-CSSO by CCK-8 method, and the result is shown in figure 2A. The results show that the cell is incubated with GP-CSSO micelle with the concentration of 2000 mug/mL (higher than the in vitro administration dosage) for 72 hours, and the cell survival rate is more than 90 percent. GP-CSSO has good application prospect as a low-toxicity carrier with good biocompatibility.

(7) Preparation of polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting delivery system (namely liver parenchymal cell targeting nano preparation)

The GP-CSSO freeze-dried powder is weighed and dissolved in deionized water, the solution is treated by water bath ultrasonic treatment for 15min to prepare 1.5mg/ml GP-CSSO micelle solution, and the solution is filtered and sterilized by a microporous filter membrane with the pore diameter of 0.22 mu m. Adding A3ARP solution according to the theoretical drug loading rate of 100:1 of carrier/drug feed ratio (w/w), and incubating the prepared GP-CSSO/A3ARP at room temperature for 30min to obtain a composite A3ARP delivery system GP-CSSO/A3ARP.

By utilizing the mechanism and method of immunofluorescence staining, DDK is Flag-Tag of A3ARP protein C end fusion mark, the Flag-Tag can be specifically identified by adopting DDK immunofluorescence primary antibody, and the immunofluorescence secondary antibody staining can amplify detection signals. And incubating GP-CSSO and A3ARP according to different feeding ratios, and fixing the incubation on a 96-hole black bottom permeable ELISA plate. The A3ARP encapsulated by GP-CSSO can not be combined with the DDK immunofluorescence primary antibody, but the free A3ARP which is not encapsulated is dissolved in the incubation liquid, can be recognized by the DDK immunofluorescence primary antibody, signals are amplified through immunofluorescence secondary antibody staining, and a GE Amersham Typhoon NIR/5 infrared imaging system is adopted to scan the fluorescence diagram of the pore plate; fluorescence intensity was analyzed by ImageQuant TL software, resulting in a feed ratio of 100:1 prepared GP-CSSO/A3ARP encapsulation efficiency of 34.32%.

(8) Physical and chemical properties of polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting delivery system

The GP-CSSO freeze-dried powder is weighed and dissolved in deionized water, the solution is treated by water bath ultrasonic treatment for 15min to prepare 1.5mg/ml GP-CSSO micelle solution, and the solution is filtered and sterilized by a microporous filter membrane with the pore diameter of 0.22 mu m. Adding A3ARP solution according to the theoretical drug loading ratio (w/w) of 100:1, and incubating the prepared GP-CSSO/A3ARP at room temperature for 30min to obtain the composite A3ARP delivery system GP-CSSO/A3ARP, wherein the particle size and the surface potential analyzer are used for measuring the particle size of the GP-CSSO/A3ARP to be 158.43 +/-7.78 nm and the Zeta potential to be 13.1+/-2.5 mV.

Example 3

(2) Synthesis of glycolipid disulfide bond grafting material CSSO

The prepared 18.9kDa chitosan oligosaccharide is taken and dissolved in distilled water to prepare a solution with the concentration of 20 mg/ml. The molar ratio of stearylamine (Octadecylamine, ODA) to chitosan oligosaccharide was 30: stearylamine is taken, and stearylamine, 3'-dithiodipropionic acid (3, 3' -Dithiodipropionic acid, DTPA), dicyclohexylcarbodiimide (DCC) and 4-Dimethylaminopyridine (DMAP) are weighed according to the molar ratio of 1:1:3:0.3, dissolved in anhydrous DMSO, and reacted for 24 hours under the condition of stirring nitrogen in a water bath at 60 ℃ and filtered by a Buchner funnel. Carbodiimide (1- (3-dimethylmineopyl) -3-ethylcarbodiimide hydrochloride (EDC)) and N-Hydroxysuccinimide (NHS) to stearylamine molar ratio of 10:10:1, adding the mixture into filtrate, stirring the mixture in a water bath at 60 ℃ for reaction for 30min, slowly adding the mixture into a chitosan oligosaccharide aqueous solution preheated at 60 ℃, stirring the mixture in the water bath at 60 ℃ for reaction for 12h, adding a 7kDa dialysis bag after the reaction is finished, dialyzing the dialysis solution for 48h, and washing the dialysis solution with absolute ethyl alcohol to remove unreacted stearylamine to obtain the glycolipid disulfide bond grafting material.

(3) Synthesis of Gal-PEG-CHO

CSSO was dissolved in an appropriate amount of acetic acid solution (pH 5.5) to give a CSSO micelle solution at a concentration of 5.0 mg/mL. According to Gal-PEG-CHO: CSSO: sodium triacetoxyborohydride molar ratio is 1:1:3 adding the raw materials, stirring for 24 hours at room temperature in a dark place, placing in a dialysis bag (MWCO 7 kDa), dialyzing with deionized water for 24 hours, taking the solution in the dialysis bag to obtain GP-CSSO, and freeze-drying and preserving.

Nuclear magnetic resonance hydrogen spectrometry CSO, DTPA, ODA, CSSO, gal-PEG-CHO, GP-CSSO. Respectively weighing 10mg of CSO, DTPA, ODA, CSSO, gal-PEG-CHO, GP-CSSO, respectively dissolving in 1ml of D ₂ In O (preparation concentration: 10 mg/mL), the nuclear magnetic resonance hydrogen spectrum was measured by a nuclear magnetic resonance spectrometer, and the results are shown in FIG. 1. After grafting chitosan oligosaccharide by carrying out amidation reaction under the catalysis of DCC/DMAP, the carboxyl hydrogen with the chemical shift of 12ppm of DTPA disappears, and the amino hydrogen with the chemical shift of 1.3ppm of ODA still exists, which indicates that the hydrophobic end has been successfully grafted to chitosan oligosaccharide and CSSO is successfully synthesized. After Schiff reaction of terminal aldehyde group of Gal-PEG-CHO and CSSO free amino, chemical shift of Gal-PEG-CHO is carried out for 10ppm of aldehyde hydrogen to disappear, and chemical shift of 3.6ppm of methylenedioxy hydrogen still exists, which indicates that Gal-PEG-CHO has been grafted to CSSO and GP-CSSO is successfully synthesized.

Taking HepG2-NTCP cells in logarithmic growth phase at 1×10 per well ⁴ Inoculating into 96-well cell culture plate at 37deg.C under conditions containing 5% CO ₂ After culturing in incubator until cell adhesion growth, adding 100-2000 μg/mL GP-CSSO micelle to incubate for 72h, and measuring in vitro cytotoxicity of GP-CSSO by CCK-8 method, and the result is shown in figure 2A. The results show that the cell is incubated with GP-CSSO micelle with the concentration of 2000 mug/mL (higher than the in vitro administration dosage) for 72 hours, and the cell survival rate is more than 90 percent. GP-CSSO has good application prospect as a low-toxicity carrier with good biocompatibility.

The GP-CSSO freeze-dried powder is weighed and dissolved in deionized water, the solution is treated by water bath ultrasonic treatment for 15min to prepare 5mg/ml GP-CSSO micelle solution, and the solution is filtered and sterilized by a microporous filter membrane with the pore diameter of 0.22 mu m. Adding A3ARP solution according to the carrier/drug feeding ratio (w/w) 400:1 theory drug loading, and incubating the prepared GP-CSSO/A3ARP at room temperature for 30min to obtain a composite A3ARP delivery system GP-CSSO/A3ARP.

By utilizing the mechanism and method of immunofluorescence staining, DDK is Flag-Tag of A3ARP protein C end fusion mark, the Flag-Tag can be specifically identified by adopting DDK immunofluorescence primary antibody, and the immunofluorescence secondary antibody staining can amplify detection signals. And incubating GP-CSSO and A3ARP according to different feeding ratios, and fixing the incubation on a 96-hole black bottom permeable ELISA plate. The A3ARP encapsulated by GP-CSSO can not be combined with the DDK immunofluorescence primary antibody, but the free A3ARP which is not encapsulated is dissolved in the incubation liquid, can be recognized by the DDK immunofluorescence primary antibody, signals are amplified through immunofluorescence secondary antibody staining, and a GE Amersham Typhoon NIR/5 infrared imaging system is adopted to scan the fluorescence diagram of the pore plate; fluorescence intensity was analyzed by ImageQuant TL software, resulting in a feed ratio of 400:1 prepared GP-CSSO/A3ARP encapsulation efficiency of 80.43%.

The GP-CSSO freeze-dried powder is weighed and dissolved in deionized water, the solution is treated by water bath ultrasonic treatment for 15min to prepare 5mg/ml GP-CSSO micelle solution, and the solution is filtered and sterilized by a microporous filter membrane with the pore diameter of 0.22 mu m. Adding A3ARP solution according to the carrier/drug loading ratio (w/w) 400:1 theory, incubating the prepared GP-CSSO/A3ARP at room temperature for 30min to obtain a composite A3ARP delivery system GP-CSSO/A3ARP, and measuring the particle size of the GP-CSSO/A3ARP to be 239.57 +/-9.36 nm and the Zeta potential to be 18.86+/-0.92 mV by using a particle size and surface potential analyzer.

Example 4

(2) Synthesis of glycolipid disulfide bond grafting material CSSO

(3) Synthesis of Gal-PEG-CHO

The amino substitution degree of GP-CSSO was determined by the trinitrobenzenesulfonic acid method. Taking 50-1000 mu L of chitosan oligosaccharide with different weights, dissolving in deionized water, preparing 1.0mg/mL chitosan oligosaccharide, and then fixing the volume to 2.0mL. After adding 2.0mL of each of 4% sodium bicarbonate and 0.1% trinitrobenzenesulfonic acid in a water bath at 37℃for 2 hours, 2.0mL of hydrochloric acid was added thereto and shaken well, and the absorbance was measured at 344nm using a spectrophotometer to prepare a standard curve. 5mg of grafting material is weighed and dissolved in a proper amount of deionized water to prepare a grafting material solution with the concentration of 1mg/ml, the absorbance at 344nm is measured through the same operation, and finally, the amino substitution degree of 8.56% +/-0.37% of GP-CSSO is calculated through the obtained standard curve.

And (3) determining the critical micelle concentration of the GP-CSSO by adopting a pyrene fluorescence spectrophotometry. Weighing 12mg of pyrene, dissolving in proper amount of acetone to prepare 0.0012mg/mL pyrene acetone solution, respectively transferring 0.5mL pyrene acetone solution into 10mL test tubes, and volatilizing the acetone at 50 ℃. GP-CSSO mother liquor with the concentration of 1.0mg/mL is prepared. The mother liquor was diluted with deionized water to give GP-CSSO solutions of varying concentrations (0.005, 0.01, 0.02, 0.05, 0.1, 0.3, 0.5, 0.7 and 1.0 mg/mL). 5.0mL of each grafting solution with different dilution concentrations is removed and added into a pyrene-containing test tube, and the grafting solution is subjected to water bath ultrasonic treatment for 30min. The emission spectrum of GP-CSSO was scanned using a fluorescence spectrophotometer (emission wavelength range 360-450 nm, emission slit 2.5nm, excitation wavelength 337nm, excitation slit 10 nm), and the first peak (I _l =374 nm) and a third peak (I ₃ Ratio of =385 nm (I ₁ /I ₃ ). Finally, the GP-CSSO critical micelle concentration is calculated to be 52.44 +/-1.02 mug/mL.

The GP-CSSO freeze-dried powder is weighed and dissolved in deionized water, the solution is treated by water bath ultrasonic treatment for 15min to prepare 3mg/ml GP-CSSO micelle solution, and the solution is filtered and sterilized by a microporous filter membrane with the pore diameter of 0.22 mu m. Adding A3ARP solution according to the theoretical drug loading rate of 200:1 of carrier/drug feed ratio (w/w), and incubating the prepared GP-CSSO/A3ARP at room temperature for 30min to obtain a composite A3ARP delivery system GP-CSSO/A3ARP.

By utilizing the mechanism and method of immunofluorescence staining, DDK is Flag-Tag of A3ARP protein C end fusion mark, the Flag-Tag can be specifically identified by adopting DDK immunofluorescence primary antibody, and the immunofluorescence secondary antibody staining can amplify detection signals. And incubating GP-CSSO and A3ARP according to different feeding ratios, and fixing the incubation on a 96-hole black bottom permeable ELISA plate. The A3ARP encapsulated by GP-CSSO can not be combined with the DDK immunofluorescence primary antibody, but the free A3ARP which is not encapsulated is dissolved in the incubation liquid, can be recognized by the DDK immunofluorescence primary antibody, signals are amplified through immunofluorescence secondary antibody staining, and a GE Amersham Typhoon NIR/5 infrared imaging system is adopted to scan the fluorescence diagram of the pore plate; fluorescence intensity was analyzed by ImageQuant TL software, resulting in a feed ratio of 200:1 prepared GP-CSSO/A3ARP encapsulation efficiency of 74.40%.

The GP-CSSO freeze-dried powder is weighed and dissolved in deionized water, the solution is treated by water bath ultrasonic treatment for 15min to prepare 3mg/ml GP-CSSO micelle solution, and the solution is filtered and sterilized by a microporous filter membrane with the pore diameter of 0.22 mu m. Adding A3ARP solution according to the theoretical drug loading ratio (w/w) of 200:1, and incubating the prepared GP-CSSO/A3ARP at room temperature for 30min to obtain the composite A3ARP delivery system GP-CSSO/A3ARP, wherein the particle size and the surface potential analyzer are used for measuring the particle size of the GP-CSSO/A3ARP to be 161.12 +/-9.81 nm and the Zeta potential to be 15.17+/-0.63 mV.

(9) In vitro release study of polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide graft delivery system

GP-CSSO/A3ARP was prepared, centrifuged at 13000rpm at 4℃for 10min, the supernatant was discarded, 1mL of Tris-HCl buffer (pH 7.2) containing 0, 0.1, 1 and 10mM GSH was added, the precipitate was dispersed, and the mixture was shaken at a constant temperature of 37℃at 60rpm to sample at regular time. And centrifuging at 13000rpm at 4 ℃ for 10min again, taking supernatant, measuring the A3ARP content by using a BCA protein quantitative kit, and analyzing GSH concentration responsive release condition. GP-CSSO/A3ARP release was measured as shown in FIG. 2B. As can be seen from the figure, the drug release of the delivery system all showed an increase in GSH concentration with increasing release medium. The drug release rate was slightly increased with 0.1mM GSH release medium compared to 0mM GSH release medium, whereas drug release was significantly faster with both 1mM and 10mM GSH release medium, which should be related to the disaggregation of the disulfide bonds between glycolipids in response to high GSH concentrations of functional groups of the GP-CSSO structure.

(10) Intracellular delivery conditions of a polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide graft delivery system

GP-CSSO, gal-CSSO (control vector) and FITC are taken according to the mole ratio of 1:4 are respectively dissolved in a certain volume of 50% ethanol solution. And (3) dropwise adding a blank micelle carrier solution with a certain concentration into the FITC solution under the conditions of ice bath, light shielding and magnetic stirring (250 r/min), and reacting for 24 hours under the same conditions. The reaction solution was placed in a dialysis bag (MWCO 3.5 kDa), dialyzed against deionized water for 72 hours, and the by-product was removed. The dialyzate was freeze-dried to obtain FITC-labeled FITC-GP-CSSO, FITC-CSSO (control vector), FITC-Gal-CSSO (control vector).

A red fluorescent reagent Cy5 is used for marking A3ARP, FITC-GP-CSSO and FITC-Gal-CSSO (control vector) are used as carriers, and a fluorescent marked gene delivery system FITC-GP-CSSO/Cy5-A3ARP and FITC-Gal-CSSO/Cy5-A3ARP are prepared according to a preparation method of a delivery system.

Inoculating HepG2-NTCP into cell culture plate, and placing in 5% CO at 37deg.C ₂ Culturing in a cell culture box, sucking and discarding culture solution in a cell plate after cells are grown on the wall, washing for 2 times by using PBS, respectively adding Cy5-A3ARP, FITC-GP-CSSO/Cy5-A3ARP, FITC-Gal-CSSO/Cy5-A3ARP solution (the final concentration of Cy5-A3ARP is 1.0 ug/mL), respectively incubating for 1, 4 and 12 hours, adding a blue fluorescent reagent LysoTracker-labeled lysosome, sucking and discarding the culture medium, and washing the cells for 3 times by using PBS to remove carriers adsorbed on the surface of the cells. Fixing the cover glass with 4% paraformaldehyde in dark place, taking out after 20min, embedding glycerol on the glass slide, and sealing for use. Cy5-A3ARP, FITC-GP-CSSO/Cy5-A3ARP, cell uptake, lysosome escape and gene release of FITC-Gal-CSSO/Cy5-A3ARP were observed by laser confocal scanning microscopy. The results are shown in FIG. 3, where Lysozagrel labeled lysosomes (blue), FITC labeled Gal-CSSO and GP-CSSO (green), cy5 labeled A3ARP (red), blue-red two channel stack (Merge 1) and blue-green-red two channel stack (Merge 2) are seen. Since A3ARP itself is difficult to penetrate the cell barrier, neither FITC channel nor Cy5 channel is developed in this group of photographs, nor the mere 1 and mere 2 channels are only blue in appearance of lysosome markers. And fluorescence marked Gal-CSSO/A3ARP and GP-CSSO/A3ARP groups, and partial red spots appear in cytoplasm, which indicates that Gal-CSSO/A3ARP and GP-CSSO/A3ARP nanoparticles can realize lysosome escape. In the Merge 1 graph, the GP-CSSO/A3ARP group is less than the Gal-CSSO/A3ARP group in the blue-violet (red-blue overlapping color) fluorescent spot, and the lysosome escape performance of the fluorescent spot is more than the Gal-CSSO/A3 ARP. In the Merge 2 plot, the free red spots, the GP-CSSO/A3ARP group, also was more than the Gal-CSSO/A3ARP group, indicating that GP-CSSO could release more A3ARP to the cytosol.

(11) In vivo tissue distribution of polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting delivery system

Cy 5-labeled A3ARP and Hu-URG humanized mice were used as model animals, and Cy5-A3ARP, CSSO/Cy5-A3ARP and GP-CSSO/Cy5-A3ARP containing 1.0 mu M A ARP were administered by tail vein injection, respectively. Taking animals at 6h and 12h respectively, taking pictures by adopting a small animal living body fluorescence imaging system, and observing the in-vivo tissue distribution rule of A3 ARP; mice were sacrificed at 12h humane, livers were harvested and tissue sections were immunofluorescence (green) labeled with specific antibodies Anti-ASGPR1 and BV421, respectively, hepatoblasts, PE/Cy7Anti-mouse F4/80 fluorescence (green) labeled Kupffer cells, and in vivo hepatocyte targeting was qualitatively observed by confocal microscopy, as shown in fig. 4. As can be seen from the figure, liver tissues showed different degrees of fluorescence enrichment with intensities GP-CSSO/Cy5-A3ARP > Gal-CSSO/Cy5-A3ARP > CSSO/Cy5-A3ARP, respectively. It was shown that CSSO/Cy5-A3ARP was passively incorporated into liver tissue by reticuloendothelial system phagocytosis, but the distribution was limited. Galactosyl modified Gal-CSSO/Cy5-A3ARP can obviously promote liver tissue targeting distribution. PEG modification can reduce reticuloendothelial cell phagocytosis by preventing the adsorption of reducing substances such as plasma proteins and the like, and avoid the interception of an immune system. PEGylation of Gal-CSSO yields GP-CSSO with further enhanced liver tissue targeting, which characterizes it as targetable delivery of more A3ARP to liver tissue.

(12) Drug effect of polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting delivery system

Inoculating HBV-HepG2-NTCP into 24-well cell culture plate, and placing into 5% CO at 37deg.C ₂ Culturing in a cell culture box until the cell fusion degree reaches 80%. Before sample addition, the culture solution in the cell plate paved on the previous day is sucked and discarded, the cell plate is washed by a culture medium without serum, 0.5mL of OPTI-MEMI and GP-CSSO/A3ARP (containing 1.0 mu M A ARP) are added for co-incubation, a blank control group is arranged, the culture medium with the same drug concentration or without drug is replaced every 2 days, when the cell plate is cultured to 10 days, supernatant fluid or cells are collected, samples are processed according to the operation guideline of an enzyme-linked immunosorbent assay kit, and the expression quantity of HBeAg and HBsAg of the cell culture supernatant fluid is measured by an enzyme-linked detector. By usingThe inhibition rate of GGP-CSSO/A3ARP on HBsAg, HBeAg, HBV DNA and HBV cccDNA is calculated according to the following formula, and the results are shown in table 1, and the results show that after A3ARP is delivered through GP-CSSO, the inhibition effect on the expression of cell supernatant HBsAg and HBeAg is certain, but the inhibition effect on HBV DNA and HBV cccDNA is lower.

Inhibition (%) = (control well X-loading well X)/(control well X) ×100%

TABLE 1 virologic index inhibition ratio of GP-CSSO/A3ARP different drug delivery systems

The experimental verification proves that the product prepared under the method parameters of the embodiment has the best performance. The GP-CSSO prepared under the parameters has low cytotoxicity, the GP-CSSO/A3ARP encapsulation rate is good, the delivery system can target more A3 ARPs to liver tissues, and the expression of hepatitis B virus antigens can be obviously blocked.

The above embodiment is only a preferred embodiment of the present invention, but it is not intended to limit the present invention. Various changes and modifications may be made by one of ordinary skill in the pertinent art without departing from the spirit and scope of the present invention. Therefore, all the technical schemes obtained by adopting the equivalent substitution or equivalent transformation are within the protection scope of the invention.

Claims

1. The polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting material is characterized in that the molecular weight of chitosan oligosaccharide in the grafting material is 18.9kDa, and the deacetylation degree of the chitosan oligosaccharide is 95%; the carbon chain length of the fatty acid is C18; the substitution degree of amino is 1% -30%;

2. the preparation method of the polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting material is characterized by comprising the following steps of:

s2: dissolving p-carboxybenzaldehyde in N, N-dimethylformamide, adding carbodiimide and N-hydroxysuccinimide to dissolve completely, reacting at room temperature for 2h, adding polyethylene glycol diamine to dissolve completely, and continuing to react at room temperature overnight; concentrating the reaction solution under reduced pressure, precipitating with glacial ethyl ether, centrifuging to collect the product, and vacuum drying to obtain NH ₂ -PEG _2k -CHO; NH is subjected to ₂ -PEG _2k dissolving-CHO in N, N-dimethylformamide, adding galactose-NHS and triethylamine, reacting at room temperature for 4 hr, removing solvent under reduced pressure, adding pure water for dissolving, extracting with dichloromethane, collecting organic phase, concentrating under reduced pressure, precipitating with glacial diethyl ether, centrifuging, collecting the product, and vacuum drying to obtain Gal-PEG _2k -CHO；

3. The method for preparing the polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond graft according to claim 2, wherein the step S1 is specifically as follows:

4. The method for preparing the polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond graft according to claim 3, wherein the molar ratio of stearylamine, 3' -dithiodipropionic acid, dicyclohexylcarbodiimide, 4-dimethylaminopyridine, N-hydroxysuccinimide and carbodiimide is 1:1:3:0.3:10:10.

5. The method for preparing a polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond graft according to claim 2, wherein in the step S2, 100mg of p-carboxybenzaldehyde is dissolved in 5ml of N, N-dimethylformamide, and NH is added ₂ -PEG _2k -CHO 500mg in 5ml of n, n-dimethylformamide; the molar ratio of the carbodiimide to the N-hydroxysuccinimide to the polyethylene glycol diamine to the galactose-NHS to the triethylamine is 1.2:1.2:1.0:1.2:2.0.

6. The method for preparing the polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond graft according to claim 2, wherein the step S3 is specifically as follows:

7. The construction method of the liver parenchymal cell targeting nano preparation is characterized by comprising the following steps of:

dissolving the polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting material according to the invention 1 or the polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting material obtained by the preparation method according to any one of claims 2-6 in water, filtering and sterilizing, and then adding the theoretical drug loading rate of the polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting material, namely human A3A recombinant protein= (100-400): 1, into a human A3A recombinant protein solution, and incubating at room temperature to obtain the liver parenchymal cell targeting nano-preparation for treating chronic hepatitis B.

8. The method for constructing a hepatic parenchymal cell targeting nano-preparation according to claim 7, wherein after the polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafting is dissolved in water, the water bath is carried out for 15min at room temperature, and then filtration sterilization is carried out by using a microporous filter membrane with the pore diameter of 0.22 μm.

9. The method for constructing a hepatocyte-targeted nano-preparation according to claim 7, wherein the incubation time at room temperature is 30min.

10. A liver parenchymal cell targeting nanomaterial formulation obtainable by the construction method of claim 7.