Pegylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond graft and hepatocyte targeting nanoformulation and method thereof

Abstract

The invention discloses a PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond graft and a hepatocyte targeting nanoformulation and method thereof, and belongs to the field of synthesis of polymer drug carriers. The PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond graft provided by the invention comprises chitosan oligosaccharide with a molecular weight of 18.9 kDa, a chitosan oligosaccharide deacetylation degree of 95%, a fatty acid carbon chain length of C18, and an amino substitution degree of 1% to 30%. The PEGylated galactosyl modified chitosan oligosaccharide fatty acid graft has a highly efficient hepatocyte targeting capability, and the PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond graft drug delivery system can significantly block the expression of hepatitis B virus antigen.

Description

Technical Field

[0001] The invention belongs to the field of synthesis of polymer drug carriers, and relates to the construction of PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafts, and the synthesized grafts realize efficient encapsulation of recombinant proteins, and are applied to the treatment of chronic hepatitis B. Background Art

[0002] Chronic hepatitis B (CHB) is a viral infectious disease that seriously endangers human health. According to the latest statistics from the WHO, there are about 296 million people with chronic HBV infection worldwide, and more than 800,000 people die from liver failure, cirrhosis and liver cancer caused by chronic HBV each year. After HBV infects liver cells, covalently closed circular DNA (cccDNA) is formed. As a template for viral replication, it is highly hidden and can circulate continuously. It is the main cause of recurrence after drug withdrawal in patients with chronic viral infection and "functional cure". The clearance rate of cccDNA is also considered to be a key indicator for the radical cure of CHB. At present, the clinical treatment drugs for CHB are nucleos(t)ide analogues (NAs) and interferon-α (IFN-α), but NAs can only inhibit HBV replication and are difficult to act on cccDNA; IFN-α is only effective for a small number of CHB patients and is usually poorly tolerated. Therefore, there is an urgent need to develop new drugs and therapies targeting HBV cccDNA to significantly improve the cure rate of CHB.

[0003] Human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) is a family of cytosine deaminases, including 7 subfamilies (APOBEC3A-H, excluding E), which can serve as a natural immune factor for the body to resist multiple viral infections. Among them, APOBEC3A (A3A) is mainly expressed in peripheral blood mononuclear cells, and the expression level in hepatocytes is very low. However, recent studies have found that A3A can act on cccDNA through the viral core protein HBc, deaminating cytosine in its transcription process to uracil, which is then recognized and excised by DNA glycosylase to form an apurinic / apyrimidinic site (AP-site). AP-site can be recognized by AP endonuclease, and finally cccDNA is degraded. No damage to the host cell genome by A3A was detected, showing its specificity for cccDNA. Based on this, human A3A recombinant protein (A3A recombinant protein, A3ARP) was obtained through gene recombination technology, in order to use it as a new drug for clearing HBV cccDNA and to improve the cure rate of CHB.

[0004] However, A3ARP is a negatively charged hydrophilic macromolecule with poor in vivo stability and difficulty in transmembrane transport. Currently, the recombinant protein drugs used in clinical practice are mainly limited to the extracellular environment, and intracellular delivery still faces great challenges. It is often difficult to achieve both stable protein encapsulation and efficient intracellular release. At the same time, the targeting of A3ARP intracellular delivery cannot be ignored. Nonspecific uptake by non-target cells may lead to reduced efficacy and toxic side effects.

[0005] Chitosan oligosaccharide stearic acid grafts can form micelles in aqueous media. Through the chemical grafting of the carboxyl group of stearic acid with the active amino group on chitosan oligosaccharide, the synthesized zwitterionic cationic grafts can be complexed with negatively charged hydrophilic macromolecules. Studies have shown that the liver is the main site for the synthesis of glutathione (Glutathione-SH, GSH). Compared with the GSH content (2-20 μM) in the extracellular fluid and systemic circulation, hepatocytes contain high concentrations of GSH (1-10 mM), and the GSH concentration of hepatocytes infected with hepatitis B virus increases in response to stress. By taking advantage of the high GSH concentration unique to hepatocytes infected with hepatitis B virus, disulfide bond functional groups are introduced into the carrier structure to achieve the intracellular environment-responsive release of the loaded drug in the hepatocytes. By utilizing an efficient endocytic desialoglycoprotein receptor (ASGPR) unique to hepatocytes, galactose can be specifically recognized to release galactose from glycolipid disulfide grafts (chitosan oligosaccharide-ss-octadecylamine, CSSO). In addition, polyethylene glycol (PEG) is a water-soluble polymer with good biocompatibility and is widely used in the modification of drugs and carriers. Studies have shown that PEG can prevent the adsorption of reducing substances such as plasma proteins, reduce phagocytosis of reticuloendothelial cells, and evade interception by the immune system. At the same time, PEG helps to delay the premature release of carriers with high positive charge loads (such as chitosan oligosaccharides) before delivering drugs to targets in vivo. Summary of the invention

[0006] The purpose of the present invention is to overcome the defects in the prior art and provide a PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond graft and its hepatocyte targeting nanoformulation and method. The PEGylated galactosyl modified chitosan oligosaccharide fatty acid disulfide bond graft (Galactosylated PEGylated chitosanoligosaccharide-ss-octadecylamine, GP-CSSO) synthesized by the present invention has an outer shell unloading type disulfide bond modification, which can not only utilize the low concentration of GSH in the extracellular fluid and systemic circulation to reduce the release of drugs outside the target cells, but also respond to the high concentration of GSH in the hepatocytes, open the disulfide bond link between glycolipids, and quickly release drugs in the hepatocyte target area; galactosyl modification can actively target liver tissue and hepatocytes; PEGylation modification can reduce the influence of extracellular reducing substances in the body, reduce the phagocytosis of non-parenchymal cells in the liver, and cooperate with galactosyl modification to improve the active targeting of hepatocytes. GP-CSSO self-aggregates in aqueous media to form cationic micelles, which can electrostatically complex A3ARP to achieve active targeting of liver tissue and hepatic parenchymal cells, thereby improving the cure rate of CHB.

[0007] The specific technical solutions adopted by the present invention are as follows:

[0008] In the first aspect, the present invention provides a PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafted product, wherein the molecular weight of the chitosan oligosaccharide is 18.9 kDa, the deacetylation degree of the chitosan oligosaccharide is 95%; the carbon chain length of the fatty acid is C18; the amino substitution degree is 1% to 30%;

[0009] The general chemical structure formula of the PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond graft is as follows:

[0010]

[0011] In a second aspect, the present invention provides a method for preparing a PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond graft (such as the compound described in the first aspect), which is as follows:

[0012] S1: A two-step amide reaction method was used to cross-link hydrophilic chitosan oligosaccharide and hydrophobic stearylamine with 3,3'-dithiodipropionic acid to synthesize chitosan oligosaccharide stearylamine disulfide bond grafts;

[0013] S2: Dissolve p-carboxybenzaldehyde in N,N-dimethylformamide, add carbodiimide and N-hydroxysuccinimide to dissolve completely, react at room temperature for 2 hours, add polyethylene glycol diamine to dissolve completely, and continue to react at room temperature overnight; then reduce pressure and concentrate the reaction solution, pour it into ice ether for precipitation, collect the product by centrifugation, and then vacuum dry it to obtain NH2-PEG 2k -CHO; the NH2-PEG 2k -CHO was dissolved in N,,N-dimethylformamide, and galactose-NHS and triethylamine were added to dissolve completely. After reacting at room temperature for 4 hours, the solvent was removed under reduced pressure, and pure water was added to dissolve, followed by extraction with dichloromethane. The organic phase was collected, concentrated under reduced pressure, poured into ice ether for precipitation, and the product was collected by centrifugation and dried in vacuum to obtain Gal-PEG. 2k -CHO;

[0014] S3: Using the Gal-PEG 2k The terminal aldehyde group of -CHO and the free amino group of the chitosan oligosaccharide stearylamine disulfide bond grafted product undergo a Schiff reaction to synthesize the PEGylated galactose-modified chitosan oligosaccharide stearylamine disulfide bond grafted product.

[0015] Preferably, the step S1 is specifically as follows:

[0016] 3,3'-dithiodipropionic acid, stearylamine, dicyclohexylcarbodiimide and 4-dimethylaminopyridine are dissolved in anhydrous dimethyl sulfoxide, and reacted for 18 hours in a water bath at 60°C with stirring and nitrogen; then carbodiimide and N-hydroxysuccinimide are added, and the reaction is carried out for 30 minutes in a water bath at 60°C with stirring to obtain an organic phase reaction liquid; chitosan oligosaccharide is dissolved in water, and then the organic phase reaction liquid is added dropwise while stirring, and the reaction is carried out for 8 hours in a water bath at 60°C with stirring; after the reaction is completed, a 7kDa dialysis bag is added, and the dialysis is performed for 48 hours, and the contents of the dialysis bag are centrifuged at room temperature at a speed of 10000 rpm for 10 minutes; the supernatant obtained by centrifugation is freeze-dried, and then washed with anhydrous ethanol to remove unreacted alcohol-soluble byproducts and stearylamine, to obtain a chitosan oligosaccharide stearylamine disulfide bond grafted product.

[0017] Furthermore, the concentration of chitosan oligosaccharide was prepared into 20 mg / ml solution, and the molar ratio of stearylamine (Octadecylamine, ODA) to chitosan oligosaccharide was 30:1.

[0018] Furthermore, the molar ratio of stearylamine, 3,3'-dithiodipropionic acid, dicyclohexylcarbodiimide, 4-dimethylaminopyridine, N-hydroxysuccinimide and carbodiimide is 1:1:3:0.3:10:10.

[0019] Preferably, in step S2, 100 mg of p-carboxybenzaldehyde is dissolved in 5 ml of N,N-dimethylformamide, and NH2-PEG 2k -CHO 500 mg was dissolved in 5 ml N,N-dimethylformamide; the feeding molar ratio of carbodiimide, N-hydroxysuccinimide, polyethylene glycol diamine, galactose-NHS and triethylamine was 1.2:1.2:1.0:1.2:2.0.

[0020] Preferably, the step S3 is specifically as follows:

[0021] The chitosan oligosaccharide stearylamine disulfide bond graft was dissolved in an acetic acid aqueous solution at pH 5.5, and then Gal-PEG 2k -CHO: chitosan oligosaccharide stearylamine disulfide bond grafted product = 1: (1-10) feed molar ratio was added Gal-PEG 2k -CHO, then add sodium triacetoxyborohydride in an amount 3 times the molar amount of chitosan oligosaccharide stearylamine disulfide bond grafted product, and stir at room temperature in the dark for 24 hours; then place in a dialysis bag MWCO 7kDa, dialyze with deionized water for 24 hours, take the solution in the dialysis bag, and obtain a PEGylated galactosyl-modified chitosan oligosaccharide stearylamine disulfide bond grafted product.

[0022] In a third aspect, the present invention provides a method for constructing a hepatocyte-targeted nanoformulation, as follows:

[0023] The PEGylated galactosyl-modified chitosan oligosaccharide stearylamine disulfide grafted product as described in the first aspect or the PEGylated galactosyl-modified chitosan oligosaccharide stearylamine disulfide grafted product freeze-dried powder obtained by any preparation method described in the second aspect is dissolved in water, filtered and sterilized, and then added to the human A3A recombinant protein solution at a theoretical drug loading amount of PEGylated galactosyl-modified chitosan oligosaccharide stearylamine disulfide grafted product: human A3A recombinant protein = (100-400):1, and incubated at room temperature to obtain a hepatocyte-targeted nanoformulation for the treatment of chronic hepatitis B.

[0024] Preferably, the PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafted product is dissolved in water, ultrasonicated in a water bath for 15 minutes at room temperature, and then sterilized by filtering with a microporous filter membrane with a pore size of 0.22 μm.

[0025] Preferably, the room temperature incubation time is 30 min.

[0026] In a fourth aspect, the present invention provides a hepatocyte-targeted nanoformulation obtained by the construction method described in the third aspect.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention constructs a polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond graft with good biological stability and safety, hepatocyte targeting, effective lysosomal escape and hepatocyte microenvironment response release, which can greatly increase the uptake of drugs by hepatocytes to increase the drug concentration at the drug molecule targeting site, improve the efficacy of the drug, increase the uptake of drugs by hepatocytes, and help reduce the distribution of drugs in normal tissues or cells, and reduce the toxic and side effects of drugs. In addition, human A3ARP is obtained by gene recombination technology, and the polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond graft has an efficient encapsulation ability for A3ARP. The synthesized polyethylene glycol galactosyl modified chitosan oligosaccharide stearylamine disulfide bond graft anti-hepatitis B gene drug delivery system has the ability to target hepatocytes and inhibit antigen expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 H NMR spectra of CSSO and GP-CSSO.

[0030] Figure 2 Cell viability of HepG2-NTCP cells after incubation with GP-CSSO for 72 h (A) and drug release from its delivery system (B).

[0031] Figure 3 Transport and release of GP-CSSO / A3ARP delivery system in HepG2-NTCP cells.

[0032] Figure 4 In vivo tissue distribution of GP-CSSO / A3ARP delivery system.

[0033] Figure 5 Synthesis route of GP-CSSO. DETAILED DESCRIPTION

[0034] The present invention is further described and illustrated below in conjunction with the accompanying drawings and specific embodiments. The technical features of each embodiment of the present invention can be combined accordingly without conflicting with each other.

[0035] The present invention provides a PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond graft, which has a highly efficient hepatocyte targeting ability. Specifically, the molecular weight of chitosan oligosaccharide is 18.9 kDa, the deacetylation degree of chitosan oligosaccharide is 95%, the carbon chain length of the fatty acid is C18, and the amino substitution degree is 1% to 30%. The representative chemical structure formula of the PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond graft is as follows:

[0036]

[0037] The synthetic route of the PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafted product of the present invention is as follows: Figure 5 As shown, the following method is used for preparation:

[0038] (1) Synthesis of glycolipid disulfide grafted product (i.e. chitosan oligosaccharide stearylamine disulfide grafted product) CSSO

[0039] Chitosan oligosaccharide stearylamine disulfide bond grafted product (CSSO) was synthesized by two-step amide reaction method, in which hydrophilic chitosan oligosaccharide and hydrophobic stearylamine were cross-linked with 3,3'-dithiodipropionic acid (DTPA).

[0040] In practical application, the steps are as follows: weigh the prescribed amount of 3,3'-dithiodipropionic acid (3,3'-Dithiodipropionic acid, DTPA), octadecylamine (Octadecylamine, ODA), dicyclohexylcarbodiimide (Dicyclohexylcarbodiimide, DCC) and 4-dimethylaminopyridine (4-dimethylaminopyridine, DMAP) and dissolve them in an appropriate amount of anhydrous dimethyl sulfoxide (DMSO), stir in a water bath at 60°C under nitrogen conditions, react for 18 hours, add the prescribed amount of carbodiimide (1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)) and N-hydroxysuccinimide (N-Hydroxysuccinimide, NHS), and stir in a water bath at 60°C for 30 minutes. Subsequently, the prescribed amount of chitosan oligosaccharide (CSO) was dissolved in water, and the organic phase reaction liquid was slowly added to the CSO solution in proportion while stirring, and the reaction was stirred in a water bath at 60°C for 8 hours. After the reaction was completed, a 7kDa dialysis bag was added and dialyzed for 48 hours. The contents of the dialysis bag were centrifuged at 10000rpm for 10 minutes at room temperature. After the supernatant was freeze-dried, it was washed with anhydrous ethanol to remove unreacted alcohol-soluble byproducts and stearylamine to obtain a glycolipid disulfide bond grafted product. The feed molar ratio was:

[0041] ODA:DTPA:DCC:DMAP:NHS::EDC=1:1:3:0.3:10:10.

[0042] (2) Synthesis of Gal-PEG-CHO

[0043] Dissolve p-carboxybenzaldehyde in N,N-dimethylformamide (DMF), add carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to dissolve completely, react at room temperature for 2 hours, then add polyethylene glycol diamine (NH2-PEG 2k -NH2) was completely dissolved, and the reaction was continued at room temperature overnight; the reaction solution was then concentrated under reduced pressure, poured into ice ether for precipitation, and the product was collected by centrifugation and then dried in vacuum to obtain NH2-PEG 2k -CHO; NH2-PEG 2k -CHO was dissolved in N,N-dimethylformamide (DMF), and galactose-NHS and triethylamine were added to dissolve completely. After reacting at room temperature for 4 hours, the solvent was removed under reduced pressure, and pure water was added to dissolve, followed by extraction with dichloromethane. The organic phase was collected, concentrated under reduced pressure, poured into ice ether for precipitation, and the product was collected by centrifugation and dried in vacuum to obtain Gal-PEG. 2k -CHO.

[0044] In practical application, this step preferably adopts the following parameters:

[0045] Weigh 100 mg of p-carboxybenzaldehyde and dissolve it in 5 ml of DMF. Add EDC (1.2 eq.) and NHS (1.2 eq.) to dissolve completely. After reacting at room temperature for 2 h, add NH2-PEG 2k -NH2 (1.0 eq.) was completely dissolved, and the reaction was continued at room temperature overnight. The reaction solution was concentrated under reduced pressure, poured into a large amount of ice ether for precipitation, and the product was collected by centrifugation and dried in vacuum to obtain NH2-PEG. 2k -CHO.

[0046] Weigh NH2-PEG 2k -CHO 500mg was dissolved in 5ml DMF, galactose-NHS (1.2eq.) and triethylamine (2.0eq.) were added to dissolve completely, reacted at room temperature for 4h, the solvent was removed under reduced pressure, pure water was added to dissolve, dichloromethane was extracted 3 times, the organic phase was collected, concentrated under reduced pressure, poured into a large amount of ice ether for precipitation, the product was collected by centrifugation, and vacuum dried to obtain Gal-PEG 2k -CHO.

[0047] (3) Synthesis of PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide grafted (GP-CSSO)

[0048] Gal-PEG prepared in step (2) 2k The terminal aldehyde group of -CHO is subjected to a Schiff reaction with the free amino group of the chitosan oligosaccharide stearylamine disulfide bond graft prepared in step (1) to synthesize the PEGylated galactosyl-modified chitosan oligosaccharide stearylamine disulfide bond graft.

[0049] In practical application, the steps are as follows:

[0050] Take the prescribed amount of CSSO and dissolve it in an appropriate amount of acetic acid solution (pH 5.5) to obtain a CSSO micelle solution with a concentration of 5.0 mg / mL. Add the raw materials according to the molar ratio of Gal-PEG-CHO:CSSO of 1:1 to 1:10, then add 3 times the molar amount of sodium triacetoxyborohydride, stir at room temperature in the dark for 24 hours, place in a dialysis bag (MWCO 7kDa), dialyze with deionized water for 24 hours, take the solution in the dialysis bag, obtain GP-CSSO, and freeze-dry for storage.

[0051] Based on the above-mentioned PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond graft, the present invention also provides a hepatocyte-targeted nanoformulation for the treatment of chronic hepatitis B, namely, a PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafted A3ARP system. It is specifically achieved by the following scheme:

[0052] The GP-CSSO freeze-dried powder was weighed and dissolved in deionized water, filtered and sterilized, and then added to the human A3A recombinant protein solution at a theoretical drug loading amount of PEGylated galactosyl-modified chitosan oligosaccharide stearylamine disulfide bond grafted product: human A3A recombinant protein = (100-400): 1. After incubation at room temperature, a hepatocyte-targeted nanoformulation for the treatment of chronic hepatitis B was obtained.

[0053] In actual application, the construction method is as follows:

[0054] Weigh the GP-CSSO freeze-dried powder and dissolve it in deionized water. Ultrasonicate in a water bath for 15 minutes to prepare GP-CSSO micelle solutions of different concentrations, and filter and sterilize them with a microporous filter membrane with a pore size of 0.22 μm. Add A3ARP solution according to the theoretical drug loading amount of 100:1-400:1 of the feed ratio (w / w) to prepare GP-CSSO / A3ARP, incubate at room temperature for 30 minutes, and obtain the delivery system GP-CSSO / A3ARP (i.e., hepatocyte-targeted nanoformulation) of composite A3ARP.

[0055] The hepatocyte-targeted nanoparticle preparation prepared by the present invention can be used to treat chronic hepatitis B and can significantly block the expression of hepatitis B virus antigens.

[0056] The technical solution of the present invention and the corresponding technical effects that can be achieved are specifically described below through embodiments.

[0057] Example 1

[0058] In this example, a PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond graft was prepared, and the performance of the obtained product was verified as follows:

[0059] (1) Preparation of low molecular weight chitosan

[0060] Take 50g of chitosan with a molecular weight of 450kDa and a deacetylation degree of 95%, slowly add it to 1500ml of 1.25% (v / v) hydrochloric acid solution under stirring in a water bath at 60°C and 500rpm, stir it overnight at 500rpm to fully swell it, slowly add chitosanase with a weight ratio of 2% chitosan, stir at 400rpm to carry out enzymolysis reaction, control the degradation degree of chitosan by gel permeation chromatography, after the reaction is completed, heat up to 80°C, add 0.3% (w / v) activated carbon, dilute the reaction solution, filter it with a Buchner funnel, and freeze-dry to obtain low molecular weight chitosan (chitosan oligosaccharide, Mw 18.9kDa) powder.

[0061] (2) Synthesis of glycolipid disulfide grafted product CSSO

[0062] The 18.9 kDa chitosan oligosaccharide prepared above was dissolved in distilled water to prepare a solution with a concentration of 20 mg / ml. According to the molar ratio of stearylamine (Octadecylamine, ODA) to chitosan oligosaccharide of 30:1, stearylamine was taken, and stearylamine, 3,3'-dithiodipropionic acid (3,3'-Dithiodipropionic acid, DTPA), dicyclohexylcarbodiimide (Dicyclohexylcarbodiimide, DCC) and 4-dimethylaminopyridine (4-dimethylaminopyridine, DMAP) were weighed in a molar ratio of 1:1:3:0.3, dissolved in anhydrous DMSO, stirred in a water bath at 60°C under nitrogen conditions, reacted for 24 hours, and filtered through a Buchner funnel. Carbodiimide (1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)) and N-hydroxysuccinimide (N-Hydroxysuccinimide, NHS) and stearylamine are added to the filtrate in a molar ratio of 10:10:1, stirred in a water bath at 60°C for reaction for 30 minutes, and then slowly added to a chitosan oligosaccharide aqueous solution preheated at 60°C, stirred in a water bath at 60°C for reaction for 12 hours. After the reaction is completed, a 7kDa dialysis bag is added and dialyzed for 48 hours. After the dialysate is freeze-dried, it is washed with anhydrous ethanol to remove unreacted stearylamine to obtain a glycolipid disulfide bond graft.

[0063] (3) Synthesis of Gal-PEG-CHO

[0064] Weigh 100 mg of p-carboxybenzaldehyde and dissolve it in 5 ml of DMF. Add EDC (1.2 eq.) and NHS (1.2 eq.) to dissolve completely. After reacting at room temperature for 2 h, add NH2-PEG 2k -NH2 (1.0 eq.) was completely dissolved, and the reaction was continued at room temperature overnight. The reaction solution was concentrated under reduced pressure, poured into a large amount of ice ether for precipitation, and the product was collected by centrifugation and dried in vacuum to obtain NH2-PEG. 2k -CHO.

[0065] Weigh NH2-PEG 2k -CHO 500mg was dissolved in 5ml DMF, galactose-NHS (1.2eq.) and triethylamine (2.0eq.) were added to dissolve completely, reacted at room temperature for 4h, the solvent was removed under reduced pressure, pure water was added to dissolve, dichloromethane was extracted 3 times, the organic phase was collected, concentrated under reduced pressure, poured into a large amount of ice ether for precipitation, the product was collected by centrifugation, and vacuum dried to obtain Gal-PEG 2k -CHO.

[0066] (4) Synthesis of PEGylated Galactosyl Modified Chitosan Oligosaccharide Stearylamine Disulfide Grafted GP-CSSO

[0067] CSSO was dissolved in an appropriate amount of acetic acid solution (pH 5.5) to obtain a CSSO micelle solution with a concentration of 5.0 mg / mL. 2k -CHO: CSSO: sodium triacetoxyborohydride was added in a molar ratio of 1:10:3, stirred at room temperature in the dark for 24 h, placed in a dialysis bag (MWCO 7 kDa), dialyzed with deionized water for 24 h, and the solution in the dialysis bag was taken to obtain GP-CSSO, which was freeze-dried and stored.

[0068] The amino substitution degree of GP-CSSO was determined by the trinitrobenzene sulfonic acid method. Take 50-1000μL of chitosan oligosaccharides of different weights, dissolve them in deionized water, prepare 1.0mg / ml chitosan oligosaccharides, and then dilute to 2.0mL. Add 2.0mL of 4% sodium bicarbonate and 0.1% trinitrobenzene sulfonic acid, bathe at 37℃ for 2h, then add 2.0mL of 2mol / L hydrochloric acid, shake well, and use a spectrophotometer to measure its absorbance at a wavelength of 344nm to prepare a standard curve. Weigh 5mg of the graft, dissolve it in an appropriate amount of deionized water, prepare a 1mg / ml graft solution, and perform the same operation as above to measure the absorbance at 344nm. Finally, the amino substitution degree of GP-CSSO is calculated to be 20.51%±0.11% through the obtained standard curve.

[0069] Example 2

[0070] In this example, a PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafted product and its hepatocyte targeting nanoformulation were prepared, and the performance of the obtained product was verified as follows: (1) Preparation of low molecular weight chitosan

[0071] Take 50g of chitosan with a molecular weight of 450kDa and a deacetylation degree of 95%, slowly add it to 1500ml of 1.25% (v / v) hydrochloric acid solution under stirring in a water bath at 60°C and 500rpm, stir it overnight at 500rpm to fully swell it, slowly add chitosanase with a weight ratio of 2% chitosan, stir at 400rpm to carry out enzymolysis reaction, control the degradation degree of chitosan by gel permeation chromatography, after the reaction is completed, heat up to 80°C, add 0.3% (w / v) activated carbon, dilute the reaction solution, filter it with a Buchner funnel, and freeze-dry to obtain low molecular weight chitosan (chitosan oligosaccharide, Mw 18.9kDa) powder.

[0072] (2) Synthesis of glycolipid disulfide grafted product CSSO

[0073] The 18.9 kDa chitosan oligosaccharide prepared above was dissolved in distilled water to prepare a solution with a concentration of 20 mg / ml. According to the molar ratio of stearylamine (Octadecylamine, ODA) to chitosan oligosaccharide of 30:1, stearylamine was taken, and stearylamine, 3,3'-dithiodipropionic acid (3,3'-Dithiodipropionic acid, DTPA), dicyclohexylcarbodiimide (Dicyclohexylcarbodiimide, DCC) and 4-dimethylaminopyridine (4-dimethylaminopyridine, DMAP) were weighed in a molar ratio of 1:1:3:0.3, dissolved in anhydrous DMSO, stirred in a water bath at 60°C under nitrogen conditions, reacted for 24 hours, and filtered through a Buchner funnel. Carbodiimide (1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)) and N-hydroxysuccinimide (N-Hydroxysuccinimide, NHS) and stearylamine are added to the filtrate in a molar ratio of 10:10:1, stirred in a water bath at 60°C for reaction for 30 minutes, and then slowly added to a chitosan oligosaccharide aqueous solution preheated at 60°C, stirred in a water bath at 60°C for reaction for 12 hours. After the reaction is completed, a 7kDa dialysis bag is added and dialyzed for 48 hours. After the dialysate is freeze-dried, it is washed with anhydrous ethanol to remove unreacted stearylamine to obtain a glycolipid disulfide bond graft.

[0074] (3) Synthesis of Gal-PEG-CHO

[0075] Weigh 100 mg of p-carboxybenzaldehyde and dissolve it in 5 ml of DMF. Add EDC (1.2 eq.) and NHS (1.2 eq.) to dissolve completely. After reacting at room temperature for 2 h, add NH2-PEG 2k -NH2 (1.0 eq.) was completely dissolved, and the reaction was continued at room temperature overnight. The reaction solution was concentrated under reduced pressure, poured into a large amount of ice ether for precipitation, and the product was collected by centrifugation and dried in vacuum to obtain NH2-PEG. 2k -CHO.

[0076] Weigh NH2-PEG 2k -CHO 500mg was dissolved in 5ml DMF, galactose-NHS (1.2eq.) and triethylamine (2.0eq.) were added to dissolve completely, reacted at room temperature for 4h, the solvent was removed under reduced pressure, pure water was added to dissolve, dichloromethane was extracted 3 times, the organic phase was collected, concentrated under reduced pressure, poured into a large amount of ice ether for precipitation, the product was collected by centrifugation, and vacuum dried to obtain Gal-PEG 2k -CHO.

[0077] (4) Synthesis of PEGylated Galactosyl Modified Chitosan Oligosaccharide Stearylamine Disulfide Grafted GP-CSSO

[0078] CSSO was dissolved in an appropriate amount of acetic acid solution (pH 5.5) to obtain a CSSO micelle solution with a concentration of 5.0 mg / mL. 2k -CHO: CSSO: sodium triacetoxyborohydride were added in a molar ratio of 1:1:3, stirred at room temperature in the dark for 24 h, placed in a dialysis bag (MWCO 7 kDa), dialyzed with deionized water for 24 h, and the solution in the dialysis bag was taken to obtain GP-CSSO, which was freeze-dried for storage.

[0079] The amino substitution degree of GP-CSSO was determined by the trinitrobenzene sulfonic acid method. Take 50-1000μL of chitosan oligosaccharides of different weights, dissolve them in deionized water, prepare 1.0mg / ml chitosan oligosaccharides, and then dilute to 2.0mL. Add 2.0mL of 4% sodium bicarbonate and 0.1% trinitrobenzene sulfonic acid, bathe at 37℃ for 2h, then add 2.0mL of 2mol / L hydrochloric acid, shake well, and use a spectrophotometer to measure its absorbance at a wavelength of 344nm to prepare a standard curve. Weigh 5mg of the graft, dissolve it in an appropriate amount of deionized water, prepare a 1mg / ml graft solution, and perform the same operation as above to measure the absorbance at 344nm. Finally, the amino substitution degree of GP-CSSO is calculated as 8.56%±0.37% through the obtained standard curve.

[0080] (5) Physicochemical properties of PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafted GP-CSSO

[0081] Determination of CSO, DTPA, ODA, CSSO, Gal-PEG-CHO, GP-CSSO by H NMR spectrum. Weigh 10 mg of CSO, DTPA, ODA, CSSO, Gal-PEG-CHO, GP-CSSO respectively and dissolve them in 1 ml of D2O (the concentration is 10 mg / mL). Determine the H NMR spectrum by H NMR. The results are shown in Figure 1 . After DCC / DMAP-catalyzed amidation reaction grafted to chitosan oligosaccharide, the carboxyl hydrogen with a chemical shift of 12ppm by DTPA disappeared, and the amino hydrogen with a chemical shift of 1.3ppm by ODA still existed, indicating that the hydrophobic end had been successfully grafted to chitosan oligosaccharide and CSSO was successfully synthesized. After the terminal aldehyde group of Gal-PEG-CHO reacted with the free amino group of CSSO by Schiff reaction, the aldehyde hydrogen with a chemical shift of 10ppm by Gal-PEG-CHO disappeared, and the methyleneoxy hydrogen with a chemical shift of 3.6ppm still existed, indicating that Gal-PEG-CHO had been grafted to CSSO and GP-CSSO was successfully synthesized.

[0082] The critical micelle concentration of GP-CSSO was determined by pyrene fluorescence spectrophotometry. The details are as follows:

[0083] Weigh 12 mg of pyrene, dissolve it in an appropriate amount of acetone to prepare a 0.0012 mg / mL pyrene acetone solution, transfer 0.5 mL of the pyrene acetone solution to a 10 mL test tube, and evaporate the acetone at 50°C. Prepare a 1.0 mg / mL concentration of GP-CSSO mother solution. Use deionized water to dilute the mother solution to obtain GP-CSSO solutions of different concentrations (0.005, 0.01, 0.02, 0.05, 0.1, 0.3, 0.5, 0.7 and 1.0 mg / mL). Transfer 5.0 mL of each graft solution of different dilution concentrations to the pyrene-containing test tube and sonicate in a water bath for 30 minutes. Use a fluorescence spectrophotometer to scan the emission spectrum of GP-CSSO (emission wavelength range is 360-450 nm, emission light slit is 2.5 nm, excitation wavelength is 337 nm, and excitation light slit is 10 nm), and record the first peak (I l The ratio of the first peak (I1=374nm) to the third peak (I3=385nm) (I1 / I3). Finally, the critical micelle concentration of GP-CSSO was calculated to be 52.44±1.02μg / mL.

[0084] The GP-CSSO graft was weighed and dissolved in distilled water to prepare a 1.0 mg / ml GP-CSSO micelle solution. The particle size and surface potential analyzer was used to measure the GP-CSSO particle size to be 107.41±4.55 nm and the Zeta potential to be 15.12±0.73 mV.

[0085] (6) Cytotoxicity of GP-CSSO, a PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond graft

[0086] HepG2-NTCP cells in the logarithmic growth phase were taken and 1×10 4 The cells were inoculated in a 96-well cell culture plate and cultured in an incubator containing 5% CO2 at 37°C until the cells adhered to the wall and grew. Then, 100-2000 μg / mL GP-CSSO micelles were added and incubated for 72 h. The in vitro cytotoxicity of GP-CSSO was measured by CCK-8 method. The results are shown in the figure. Figure 2 A. The results showed that the cell survival rate was greater than 90% when the cells were co-incubated with 2000 μg / mL GP-CSSO micelles (higher than the in vitro administration dose) for 72 hours. GP-CSSO, as a low-toxic carrier with good biocompatibility, has a good application prospect.

[0087] (7) Preparation of PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond graft delivery system (i.e., hepatocyte-targeted nanoformulation)

[0088] Weigh the GP-CSSO freeze-dried powder and dissolve it in deionized water. Ultrasonicate in a water bath for 15 minutes to prepare a 1.5 mg / ml GP-CSSO micelle solution, and filter and sterilize it with a microporous filter membrane with a pore size of 0.22 μm. Add the A3ARP solution according to the theoretical drug loading of the carrier / drug feed ratio (w / w) of 100:1 to prepare GP-CSSO / A3ARP, incubate at room temperature for 30 minutes, and obtain the delivery system GP-CSSO / A3ARP of the composite A3ARP.

[0089] Using the mechanism and method of immunofluorescence staining, DDK is a Flag-Tag fused to the C-terminus of A3ARP protein. The Flag-Tag can be specifically identified by the DDK immunofluorescence primary antibody, and the detection signal can be amplified by immunofluorescence secondary antibody staining. GP-CSSO and A3ARP were incubated at different feed ratios and fixed on a 96-well black bottom ELISA plate at the end of incubation. A3ARP encapsulated by GP-CSSO will not bind to the DDK immunofluorescence primary antibody, but the unencapsulated free A3ARP is dissolved in the incubation solution and can be recognized by the DDK immunofluorescence primary antibody. The signal is amplified by immunofluorescence secondary antibody staining, and the GE Amersham Typhoon NIR / 5 infrared imaging system is used to scan the well plate fluorescence image; the fluorescence intensity is analyzed by ImageQuant TL software, and the encapsulation rate of GP-CSSO / A3ARP prepared with a feed ratio of 100:1 is 34.32%.

[0090] (8) Physicochemical properties of PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafted delivery system

[0091] Weigh the GP-CSSO freeze-dried powder and dissolve it in deionized water, sonicate in a water bath for 15 minutes to prepare a 1.5 mg / ml GP-CSSO micelle solution, and filter and sterilize it with a microporous filter membrane with a pore size of 0.22 μm. Add the A3ARP solution according to the theoretical drug loading of the carrier / drug feed ratio (w / w) of 100:1, and prepare the GP-CSSO / A3ARP, incubate at room temperature for 30 minutes to obtain the composite A3ARP delivery system GP-CSSO / A3ARP. The particle size and surface potential analyzer measured the GP-CSSO / A3ARP particle size of 158.43±7.78nm and the Zeta potential of 13.1±2.5mV.

[0092] Example 3

[0093] In this example, a PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafted product and its hepatocyte targeting nanoformulation were prepared, and the performance of the obtained product was verified as follows: (1) Preparation of low molecular weight chitosan

[0094] Take 50g of chitosan with a molecular weight of 450kDa and a deacetylation degree of 95%, slowly add it to 1500ml of 1.25% (v / v) hydrochloric acid solution under stirring in a water bath at 60°C and 500rpm, stir it overnight at 500rpm to fully swell it, slowly add chitosanase with a weight ratio of 2% chitosan, stir at 400rpm to carry out enzymolysis reaction, control the degradation degree of chitosan by gel permeation chromatography, after the reaction is completed, heat up to 80°C, add 0.3% (w / v) activated carbon, dilute the reaction solution, filter it with a Buchner funnel, and freeze-dry to obtain low molecular weight chitosan (chitosan oligosaccharide, Mw 18.9kDa) powder.

[0095] (2) Synthesis of glycolipid disulfide grafted product CSSO

[0096] The 18.9 kDa chitosan oligosaccharide prepared above was dissolved in distilled water to prepare a solution with a concentration of 20 mg / ml. The molar ratio of stearylamine (Octadecylamine, ODA) to chitosan oligosaccharide was 30:1. Stearylamine, 3,3'-dithiodipropionic acid (3,3'-Dithiodipropionic acid, DTPA), dicyclohexylcarbodiimide (Dicyclohexylcarbodiimide, DCC) and 4-dimethylaminopyridine (4-dimethylaminopyridine, DMAP) were weighed in a molar ratio of 1:1:3:0.3, dissolved in anhydrous DMSO, stirred in a water bath at 60°C under nitrogen conditions, reacted for 24 hours, and filtered through a Buchner funnel. Carbodiimide (1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)) and N-hydroxysuccinimide (N-Hydroxysuccinimide, NHS) and stearylamine are added to the filtrate in a molar ratio of 10:10:1, stirred in a water bath at 60°C for reaction for 30 minutes, and then slowly added to a chitosan oligosaccharide aqueous solution preheated at 60°C, stirred in a water bath at 60°C for reaction for 12 hours. After the reaction is completed, a 7kDa dialysis bag is added and dialyzed for 48 hours. After the dialysate is freeze-dried, it is washed with anhydrous ethanol to remove unreacted stearylamine to obtain a glycolipid disulfide bond graft.

[0097] (3) Synthesis of Gal-PEG-CHO

[0098] Weigh 100 mg of p-carboxybenzaldehyde and dissolve it in 5 ml of DMF. Add EDC (1.2 eq.) and NHS (1.2 eq.) to dissolve completely. After reacting at room temperature for 2 h, add NH2-PEG 2k-NH2 (1.0 eq.) was completely dissolved, and the reaction was continued at room temperature overnight. The reaction solution was concentrated under reduced pressure, poured into a large amount of ice ether for precipitation, and the product was collected by centrifugation and dried in vacuum to obtain NH2-PEG. 2k -CHO.

[0099] Weigh NH2-PEG 2k -CHO 500mg was dissolved in 5ml DMF, galactose-NHS (1.2eq.) and triethylamine (2.0eq.) were added to dissolve completely, reacted at room temperature for 4h, the solvent was removed under reduced pressure, pure water was added to dissolve, dichloromethane was extracted 3 times, the organic phase was collected, concentrated under reduced pressure, poured into a large amount of ice ether for precipitation, the product was collected by centrifugation, and vacuum dried to obtain Gal-PEG 2k -CHO.

[0100] (4) Synthesis of PEGylated Galactosyl Modified Chitosan Oligosaccharide Stearylamine Disulfide Grafted GP-CSSO

[0101] CSSO was dissolved in an appropriate amount of acetic acid solution (pH 5.5) to obtain a CSSO micelle solution with a concentration of 5.0 mg / mL. The raw materials were added according to the molar ratio of Gal-PEG-CHO: CSSO: sodium triacetoxyborohydride of 1:1:3, stirred at room temperature in the dark for 24 hours, placed in a dialysis bag (MWCO 7kDa), dialyzed with deionized water for 24 hours, and the solution in the dialysis bag was taken to obtain GP-CSSO, which was freeze-dried for storage.

[0102] The amino substitution degree of GP-CSSO was determined by the trinitrobenzene sulfonic acid method. Take 50-1000μL of chitosan oligosaccharides of different weights, dissolve them in deionized water, prepare 1.0mg / ml chitosan oligosaccharides, and then dilute to 2.0mL. Add 2.0mL of 4% sodium bicarbonate and 0.1% trinitrobenzene sulfonic acid, bathe at 37℃ for 2h, then add 2.0mL of 2mol / L hydrochloric acid, shake well, and use a spectrophotometer to measure its absorbance at a wavelength of 344nm to prepare a standard curve. Weigh 5mg of the graft, dissolve it in an appropriate amount of deionized water, prepare a 1mg / ml graft solution, and perform the same operation as above to measure the absorbance at 344nm. Finally, the amino substitution degree of GP-CSSO is calculated as 8.56%±0.37% through the obtained standard curve.

[0103] (5) Physicochemical properties of PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafted GP-CSSO

[0104] Determination of CSO, DTPA, ODA, CSSO, Gal-PEG-CHO, GP-CSSO by H NMR spectrum. Weigh 10 mg of CSO, DTPA, ODA, CSSO, Gal-PEG-CHO, GP-CSSO respectively and dissolve them in 1 ml of D2O (the concentration is 10 mg / mL). Determine the H NMR spectrum by H NMR. The results are shown in Figure 1 . After DCC / DMAP-catalyzed amidation reaction grafted to chitosan oligosaccharide, the carboxyl hydrogen with a chemical shift of 12ppm by DTPA disappeared, and the amino hydrogen with a chemical shift of 1.3ppm by ODA still existed, indicating that the hydrophobic end had been successfully grafted to chitosan oligosaccharide and CSSO was successfully synthesized. After the terminal aldehyde group of Gal-PEG-CHO reacted with the free amino group of CSSO by Schiff reaction, the aldehyde hydrogen with a chemical shift of 10ppm by Gal-PEG-CHO disappeared, and the methyleneoxy hydrogen with a chemical shift of 3.6ppm still existed, indicating that Gal-PEG-CHO had been grafted to CSSO and GP-CSSO was successfully synthesized.

[0105] The critical micelle concentration of GP-CSSO was determined by pyrene fluorescence spectrophotometry. The details are as follows:

[0106] Weigh 12 mg of pyrene, dissolve it in an appropriate amount of acetone to prepare a 0.0012 mg / mL pyrene acetone solution, transfer 0.5 mL of the pyrene acetone solution to a 10 mL test tube, and evaporate the acetone at 50°C. Prepare a 1.0 mg / mL concentration of GP-CSSO mother solution. Use deionized water to dilute the mother solution to obtain GP-CSSO solutions of different concentrations (0.005, 0.01, 0.02, 0.05, 0.1, 0.3, 0.5, 0.7 and 1.0 mg / mL). Transfer 5.0 mL of each graft solution of different dilution concentrations to the pyrene-containing test tube and sonicate in a water bath for 30 minutes. Use a fluorescence spectrophotometer to scan the emission spectrum of GP-CSSO (emission wavelength range is 360-450 nm, emission light slit is 2.5 nm, excitation wavelength is 337 nm, and excitation light slit is 10 nm), and record the first peak (I l The ratio of the first peak (I1=374nm) to the third peak (I3=385nm) (I1 / I3). Finally, the critical micelle concentration of GP-CSSO was calculated to be 52.44±1.02μg / mL.

[0107] The GP-CSSO graft was weighed and dissolved in distilled water to prepare a 1.0 mg / ml GP-CSSO micelle solution. The particle size and surface potential analyzer was used to measure the GP-CSSO particle size to be 107.41±4.55 nm and the Zeta potential to be 15.12±0.73 mV.

[0108] (6) Cytotoxicity of GP-CSSO, a PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond graft

[0109] HepG2-NTCP cells in the logarithmic growth phase were taken and 1×10 4 The cells were inoculated in a 96-well cell culture plate and cultured in an incubator containing 5% CO2 at 37°C until the cells adhered to the wall and grew. Then, 100-2000 μg / mL GP-CSSO micelles were added and incubated for 72 h. The in vitro cytotoxicity of GP-CSSO was measured by CCK-8 method. The results are shown in the figure. Figure 2 A. The results showed that the cell survival rate was greater than 90% when the cells were co-incubated with 2000 μg / mL GP-CSSO micelles (higher than the in vitro administration dose) for 72 hours. GP-CSSO, as a low-toxic carrier with good biocompatibility, has a good application prospect.

[0110] (7) Preparation of PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond graft delivery system (i.e., hepatocyte-targeted nanoformulation)

[0111] Weigh the GP-CSSO freeze-dried powder and dissolve it in deionized water. Ultrasonicate in a water bath for 15 minutes to prepare a 5 mg / ml GP-CSSO micelle solution, and filter and sterilize it with a microporous filter membrane with a pore size of 0.22 μm. Add the A3ARP solution according to the theoretical drug loading of the carrier / drug feed ratio (w / w) of 400:1 to prepare GP-CSSO / A3ARP, incubate at room temperature for 30 minutes, and obtain the delivery system GP-CSSO / A3ARP of the composite A3ARP.

[0112] Using the mechanism and method of immunofluorescence staining, DDK is the Flag-Tag fused to the C-terminus of A3ARP protein. The DDK immunofluorescence primary antibody can specifically identify the Flag-Tag, and the immunofluorescence secondary antibody staining can amplify the detection signal. GP-CSSO and A3ARP are incubated at different feed ratios and fixed on a 96-well black bottom ELISA plate at the end of incubation. A3ARP encapsulated by GP-CSSO will not bind to the DDK immunofluorescence primary antibody, but the unencapsulated free A3ARP is dissolved in the incubation solution and can be recognized by the DDK immunofluorescence primary antibody. The signal is amplified by immunofluorescence secondary antibody staining, and the GE Amersham Typhoon NIR / 5 infrared imaging system is used to scan the well plate fluorescence image; the fluorescence intensity is analyzed by ImageQuant TL software, and the encapsulation rate of GP-CSSO / A3ARP prepared with a feed ratio of 400:1 is 80.43%.

[0113] (8) Physicochemical properties of PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafted delivery system

[0114] Weigh the GP-CSSO freeze-dried powder and dissolve it in deionized water, sonicate in a water bath for 15 minutes to prepare a 5 mg / ml GP-CSSO micelle solution, and filter and sterilize it with a microporous filter membrane with a pore size of 0.22 μm. Add the A3ARP solution according to the theoretical drug loading of the carrier / drug feed ratio (w / w) of 400:1, and prepare the GP-CSSO / A3ARP, incubate at room temperature for 30 minutes to obtain the composite A3ARP delivery system GP-CSSO / A3ARP. The particle size and surface potential analyzer measured the GP-CSSO / A3ARP particle size of 239.57±9.36nm and the Zeta potential of 18.86±0.92mV.

[0115] Example 4

[0116] In this example, a PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafted product and its hepatocyte targeting nanoformulation were prepared, and the performance of the obtained product was verified as follows: (1) Preparation of low molecular weight chitosan

[0117] Take 50g of chitosan with a molecular weight of 450kDa and a deacetylation degree of 95%, slowly add it to 1500ml of 1.25% (v / v) hydrochloric acid solution under stirring in a water bath at 60°C and 500rpm, stir it overnight at 500rpm to fully swell it, slowly add chitosanase with a weight ratio of 2% chitosan, stir at 400rpm to carry out enzymolysis reaction, control the degradation degree of chitosan by gel permeation chromatography, after the reaction is completed, heat up to 80°C, add 0.3% (w / v) activated carbon, dilute the reaction solution, filter it with a Buchner funnel, and freeze-dry to obtain low molecular weight chitosan (chitosan oligosaccharide, Mw 18.9kDa) powder.

[0118] (2) Synthesis of glycolipid disulfide grafted product CSSO

[0119] The 18.9 kDa chitosan oligosaccharide prepared above was dissolved in distilled water to prepare a solution with a concentration of 20 mg / ml. The molar ratio of stearylamine (Octadecylamine, ODA) to chitosan oligosaccharide was 30:1. Stearylamine, 3,3'-dithiodipropionic acid (3,3'-Dithiodipropionic acid, DTPA), dicyclohexylcarbodiimide (Dicyclohexylcarbodiimide, DCC) and 4-dimethylaminopyridine (4-dimethylaminopyridine, DMAP) were weighed in a molar ratio of 1:1:3:0.3, dissolved in anhydrous DMSO, stirred in a water bath at 60°C under nitrogen conditions, reacted for 24 hours, and filtered through a Buchner funnel. Carbodiimide (1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)) and N-hydroxysuccinimide (N-Hydroxysuccinimide, NHS) and stearylamine are added to the filtrate in a molar ratio of 10:10:1, stirred in a water bath at 60°C for reaction for 30 minutes, and then slowly added to a chitosan oligosaccharide aqueous solution preheated at 60°C, stirred in a water bath at 60°C for reaction for 12 hours. After the reaction is completed, a 7kDa dialysis bag is added and dialyzed for 48 hours. After the dialysate is freeze-dried, it is washed with anhydrous ethanol to remove unreacted stearylamine to obtain a glycolipid disulfide bond graft.

[0120] (3) Synthesis of Gal-PEG-CHO

[0121] Weigh 100 mg of p-carboxybenzaldehyde and dissolve it in 5 ml of DMF. Add EDC (1.2 eq.) and NHS (1.2 eq.) to dissolve completely. After reacting at room temperature for 2 h, add NH2-PEG 2k -NH2 (1.0 eq.) was completely dissolved, and the reaction was continued at room temperature overnight. The reaction solution was concentrated under reduced pressure, poured into a large amount of ice ether for precipitation, and the product was collected by centrifugation and dried in vacuum to obtain NH2-PEG. 2k -CHO.

[0122] Weigh NH2-PEG 2k -CHO 500mg was dissolved in 5ml DMF, galactose-NHS (1.2eq.) and triethylamine (2.0eq.) were added to dissolve completely, reacted at room temperature for 4h, the solvent was removed under reduced pressure, pure water was added to dissolve, dichloromethane was extracted 3 times, the organic phase was collected, concentrated under reduced pressure, poured into a large amount of ice ether for precipitation, the product was collected by centrifugation, and vacuum dried to obtain Gal-PEG 2k -CHO.

[0123] (4) Synthesis of PEGylated Galactosyl Modified Chitosan Oligosaccharide Stearylamine Disulfide Grafted GP-CSSO

[0124] CSSO was dissolved in an appropriate amount of acetic acid solution (pH 5.5) to obtain a CSSO micelle solution with a concentration of 5.0 mg / mL. The raw materials were added according to the molar ratio of Gal-PEG-CHO: CSSO: sodium triacetoxyborohydride of 1:1:3, stirred at room temperature in the dark for 24 hours, placed in a dialysis bag (MWCO 7kDa), dialyzed with deionized water for 24 hours, and the solution in the dialysis bag was taken to obtain GP-CSSO, which was freeze-dried for storage.

[0125] The amino substitution degree of GP-CSSO was determined by the trinitrobenzene sulfonic acid method. Take 50-1000 μL of chitosan oligosaccharides of different weights, dissolve them in deionized water, prepare 1.0 mg / ml chitosan oligosaccharides, and then dilute to 2.0 mL. Add 2.0 mL of 4% sodium bicarbonate and 0.1% trinitrobenzene sulfonic acid, bathe at 37°C for 2 hours, then add 2.0 mL of 2 mol / L hydrochloric acid, shake well, and use a spectrophotometer to measure its absorbance at a wavelength of 344 nm to prepare a standard curve. Weigh 5 mg of the graft, dissolve it in an appropriate amount of deionized water, and prepare a 1 mg / ml graft solution. After the same operation as above, measure the absorbance at 344 nm. Finally, the amino substitution degree of GP-CSSO is calculated to be 8.56% ± 0.37% through the obtained standard curve.

[0126] (5) Physicochemical properties of PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafted GP-CSSO

[0127] Determination of CSO, DTPA, ODA, CSSO, Gal-PEG-CHO, GP-CSSO by H NMR spectrum. Weigh 10 mg of CSO, DTPA, ODA, CSSO, Gal-PEG-CHO, GP-CSSO respectively and dissolve them in 1 ml of D2O (the concentration is 10 mg / mL). Determine the H NMR spectrum by H NMR. The results are shown in Figure 1 . After DCC / DMAP-catalyzed amidation reaction grafted to chitosan oligosaccharide, the carboxyl hydrogen with a chemical shift of 12ppm by DTPA disappeared, and the amino hydrogen with a chemical shift of 1.3ppm by ODA still existed, indicating that the hydrophobic end had been successfully grafted to chitosan oligosaccharide and CSSO was successfully synthesized. After the terminal aldehyde group of Gal-PEG-CHO reacted with the free amino group of CSSO by Schiff reaction, the aldehyde hydrogen with a chemical shift of 10ppm by Gal-PEG-CHO disappeared, and the methyleneoxy hydrogen with a chemical shift of 3.6ppm still existed, indicating that Gal-PEG-CHO had been grafted to CSSO and GP-CSSO was successfully synthesized.

[0128] The critical micelle concentration of GP-CSSO was determined by pyrene fluorescence spectrophotometry. Weigh 12 mg of pyrene, dissolve it in an appropriate amount of acetone, and prepare a 0.0012 mg / mL pyrene acetone solution. Pipette 0.5 mL of pyrene acetone solution into 10 mL test tubes respectively, and evaporate the acetone at 50 ° C. Prepare a 1.0 mg / mL concentration of GP-CSSO mother solution. Use deionized water to dilute the mother solution to obtain GP-CSSO solutions of different concentrations (0.005, 0.01, 0.02, 0.05, 0.1, 0.3, 0.5, 0.7 and 1.0 mg / mL). Pipette 5.0 mL of each graft solution of different dilution concentrations, add it to the test tube containing pyrene, and ultrasonicate in a water bath for 30 minutes. Use a fluorescence spectrophotometer to scan the emission spectrum of GP-CSSO (emission wavelength range: 360-450nm, emission light slit: 2.5nm, excitation wavelength: 337nm, excitation light slit: 10nm), and record the first peak (I l The ratio of the first peak (I1=374nm) to the third peak (I3=385nm) (I1 / I3). Finally, the critical micelle concentration of GP-CSSO was calculated to be 52.44±1.02μg / mL.

[0129] The GP-CSSO graft was weighed and dissolved in distilled water to prepare a 1.0 mg / ml GP-CSSO micelle solution. The particle size and surface potential analyzer was used to measure the GP-CSSO particle size to be 107.41±4.55 nm and the Zeta potential to be 15.12±0.73 mV.

[0130] (6) Cytotoxicity of GP-CSSO, a PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond graft

[0131] HepG2-NTCP cells in the logarithmic growth phase were taken and 1×10 4 The cells were inoculated in a 96-well cell culture plate and cultured in an incubator containing 5% CO2 at 37°C until the cells adhered to the wall and grew. Then, 100-2000 μg / mL GP-CSSO micelles were added and incubated for 72 h. The in vitro cytotoxicity of GP-CSSO was measured by CCK-8 method. The results are shown in the figure. Figure 2 A. The results showed that the cell survival rate was greater than 90% when the cells were co-incubated with 2000 μg / mL GP-CSSO micelles (higher than the in vitro administration dose) for 72 hours. GP-CSSO, as a low-toxic carrier with good biocompatibility, has a good application prospect.

[0132] (7) Preparation of PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond graft delivery system (i.e., hepatocyte-targeted nanoformulation)

[0133] Weigh the GP-CSSO freeze-dried powder and dissolve it in deionized water. Ultrasonicate in a water bath for 15 minutes to prepare a 3 mg / ml GP-CSSO micelle solution, and filter and sterilize it with a microporous filter membrane with a pore size of 0.22 μm. Add the A3ARP solution according to the theoretical drug loading of the carrier / drug feed ratio (w / w) of 200:1 to prepare GP-CSSO / A3ARP, incubate at room temperature for 30 minutes, and obtain the delivery system GP-CSSO / A3ARP of the composite A3ARP.

[0134] Using the mechanism and method of immunofluorescence staining, DDK is the Flag-Tag fused to the C-terminus of A3ARP protein. The Flag-Tag can be specifically identified by the DDK immunofluorescence primary antibody, and the detection signal can be amplified by immunofluorescence secondary antibody staining. GP-CSSO and A3ARP were incubated at different feed ratios and fixed on a 96-well black bottom ELISA plate at the end of incubation. A3ARP encapsulated by GP-CSSO will not bind to the DDK immunofluorescence primary antibody, but the unencapsulated free A3ARP is dissolved in the incubation solution and can be recognized by the DDK immunofluorescence primary antibody. The signal is amplified by immunofluorescence secondary antibody staining, and the GE Amersham Typhoon NIR / 5 infrared imaging system is used to scan the well plate fluorescence image; the fluorescence intensity is analyzed by ImageQuant TL software, and the encapsulation rate of GP-CSSO / A3ARP prepared with a feed ratio of 200:1 is 74.40%.

[0135] (8) Physicochemical properties of PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafted delivery system

[0136] Weigh the GP-CSSO freeze-dried powder and dissolve it in deionized water, sonicate in a water bath for 15 minutes to prepare a 3 mg / ml GP-CSSO micelle solution, and filter and sterilize it with a microporous filter membrane with a pore size of 0.22 μm. Add the A3ARP solution according to the theoretical drug loading of the carrier / drug feed ratio (w / w) of 200:1, and prepare the GP-CSSO / A3ARP, incubate at room temperature for 30 minutes to obtain the composite A3ARP delivery system GP-CSSO / A3ARP. The particle size and surface potential analyzer measured the GP-CSSO / A3ARP particle size of 161.12±9.81nm and the Zeta potential of 15.17±0.63mV.

[0137] (9) In vitro release study of PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafted delivery system

[0138] Prepare GP-CSSO / A3ARP, centrifuge at 4°C, 13000rpm for 10min, discard the supernatant, add 1mL of Tris hydrochloric acid buffer (pH7.2) containing 0, 0.1, 1 and 10mM GSH, disperse the precipitate, shake at 37°C, 60rpm, and take samples regularly. Centrifuge again at 4°C, 13000rpm for 10min, take the supernatant, use BCA protein quantification kit to determine the A3ARP content, and analyze the GSH concentration-responsive release. The measured GP-CSSO / A3ARP release is as follows Figure 2 B. As can be seen from the figure, the drug release of the delivery system is accelerated with the increase of the concentration of GSH in the release medium. Compared with 0mM GSH release medium, the drug release rate is slightly increased under the action of 0.1mM GSH release medium, while the drug release of the delivery system is significantly accelerated under the action of 1mM and 10mM GSH release medium. This should be related to the response of the disulfide bonds of the functional groups of the GP-CSSO structure to high GSH concentrations, resulting in the depolymerization of disulfide bonds between glycolipids.

[0139] (10) Intracellular transport and release of PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafted delivery system

[0140] Take GP-CSSO, Gal-CSSO (control vector) and FITC, and dissolve them in a certain volume of 50% ethanol solution at a molar ratio of 1:4. Add a certain concentration of blank micelle carrier solution to the FITC solution under ice bath, light protection and magnetic stirring (250r / min), and react for 24h under the same conditions. Put the reaction solution in a dialysis bag (MWCO 3.5kDa) and dialyze with deionized water for 72h to remove by-products. The dialysate was freeze-dried to obtain FITC-GP-CSSO, FITC-CSSO (control vector), and FITC-Gal-CSSO (control vector) labeled with FITC.

[0141] A3ARP was labeled with red fluorescent reagent Cy5, and FITC-GP-CSSO and FITC-Gal-CSSO (control vector) were used as vectors. According to the delivery system preparation method, fluorescently labeled gene delivery systems FITC-GP-CSSO / Cy5-A3ARP and FITC-Gal-CSSO / Cy5-A3ARP were prepared.

[0142] HepG2-NTCP was inoculated into a cell culture plate and cultured in a 5% CO2 cell culture incubator at 37°C. After the cells adhered to the wall and grew, the culture medium in the cell plate was aspirated and discarded. After washing with PBS twice, Cy5-A3ARP, FITC-GP-CSSO / Cy5-A3ARP, and FITC-Gal-CSSO / Cy5-A3ARP solutions (the final concentration of Cy5-A3ARP was 1.0ug / mL) were added respectively, and incubated for 1, 4, and 12 hours respectively. The blue fluorescent reagent LysoTracker was added to label the lysosomes, the culture medium was aspirated, and the cells were rinsed with PBS for 3 times to remove the carrier adsorbed on the cell surface. The coverslip was fixed with 4% paraformaldehyde in the dark, taken out after 20 minutes, embedded on a slide with glycerol, and sealed for later use. Laser confocal scanning microscopy was used to observe the cellular uptake, lysosomal escape and gene release of Cy5-A3ARP, FITC-GP-CSSO / Cy5-A3ARP and FITC-Gal-CSSO / Cy5-A3ARP. Figure 3 As can be seen from the figure, LysoTracker labels lysosomes (blue), FITC labels Gal-CSSO and GP-CSSO (green), Cy5 labels A3ARP (red), blue and red channels are superimposed (Merge 1) and blue, green and red channels are superimposed (Merge 2). Since A3ARP itself is difficult to penetrate the cell barrier, in this group of photos, the FITC channel and the Cy5 channel are not colored, and the Merge 1 and Merge 2 channels only show the blue color of the lysosomal marker. In the fluorescently labeled Gal-CSSO / A3ARP and GP-CSSO / A3ARP groups, some red dots appear in the cytoplasm, indicating that both Gal-CSSO / A3ARP and GP-CSSO / A3ARP nanoparticles can achieve lysosomal escape. In the Merge 1 figure, the number of blue-purple (red and blue overlap) fluorescent spots in the GP-CSSO / A3ARP group is less than that in the Gal-CSSO / A3ARP group, indicating that its lysosomal escape ability is stronger than that of Gal-CSSO / A3ARP. In the Merge 2 figure, the number of free red spots in the GP-CSSO / A3ARP group is also more than that of the Gal-CSSO / A3ARP group, indicating that GP-CSSO can release more A3ARP into the cytoplasm.

[0143] (11) In vivo tissue distribution of PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond graft delivery system

[0144] A3ARP was labeled with Cy5, and Hu-URG humanized mice were used as model animals. Cy5-A3ARP, CSSO / Cy5-A3ARP, and GP-CSSO / Cy5-A3ARP containing 1.0 μM A3ARP were injected into the tail vein. The animals were taken at 6h and 12h, and the small animal in vivo fluorescence imaging system was used to take pictures to investigate the tissue distribution of A3ARP in vivo; the mice were humanely killed at 12h, and the livers were collected and sliced. Hepatocytes were immunofluorescently labeled with specific antibodies Anti-ASGPR1 and BV421 (green), and Kupffer cells were fluorescently labeled with PE / Cy7anti-mouse F4 / 80 (green). The in vivo hepatocyte targeting was qualitatively observed by confocal microscopy. The results are shown in Figure 2. Figure 4 . As can be seen from the figure, the liver tissue showed different degrees of fluorescence enrichment, and the intensity was GP-CSSO / Cy5-A3ARP>Gal-CSSO / Cy5-A3ARP>CSSO / Cy5-A3ARP. This shows that CSSO / Cy5-A3ARP passively enters the liver tissue through the reticuloendothelial system phagocytosis, but the distribution amount is limited. Galactosyl-modified Gal-CSSO / Cy5-A3ARP can significantly promote the targeted distribution of liver tissue. PEG modification can prevent the adsorption of reducing substances such as plasma proteins, reduce reticuloendothelial cell phagocytosis, and escape the interception of the immune system. GP-CSSO was prepared by PEGylation of Gal-CSSO, and its liver tissue targeting was further enhanced, indicating that it can target and deliver more A3ARP to liver tissue.

[0145] (12) Efficacy of PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafted delivery system

[0146] HBV-HepG2-NTCP was inoculated into a 24-well cell culture plate and cultured in a 5% CO2 cell culture incubator at 37°C until the cell confluence reached 80%. Before adding samples, the culture medium in the cell plate prepared the day before was aspirated and discarded, washed with serum-free culture medium, and 0.5 mL of OPTI-MEMI and GP-CSSO / A3ARP (containing 1.0 μM A3ARP) were added for co-incubation. A blank control group was set up, and the culture medium containing the same drug concentration or no drug was used every 2 days. When cultured to the 10th day, the supernatant or cells were collected, and the samples were processed according to the instructions of the enzyme-linked immunosorbent assay kit, and the expression of HBeAg and HBsAg in the cell culture supernatant was determined by an enzyme-linked detector. The following formula was used to calculate the inhibition rate of GGP-CSSO / A3ARP on HBsAg, HBeAg, HBV DNA, and HBVcccDNA. The results are shown in Table 1. The results showed that after A3ARP was delivered by GP-CSSO, it had a certain inhibitory effect on the expression of HBsAg and HBeAg in the cell supernatant, but had a lower inhibitory effect on HBV DNA and HBV cccDNA.

[0147] Inhibition rate (%) = (control well X-sample well X) / (control well X) × 100%

[0148]

[0149] Table 1 Virological index inhibition rate of different drug delivery systems of GP-CSSO / A3ARP

[0150] Through experimental verification, it can be seen that the product prepared under the parameters of the method in this embodiment has the best performance. The GP-CSSO prepared under this parameter has low cytotoxicity, good GP-CSSO / A3ARP encapsulation rate, and the delivery system can target more A3ARP to liver tissue and significantly block the expression of hepatitis B virus antigen.

[0151] The above-described embodiment is only a preferred solution of the present invention, but it is not intended to limit the present invention. A person skilled in the relevant technical field may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafted product, characterized in that: The molecular weight of chitosan oligosaccharide in the graft is 18.9 kDa, the deacetylation degree of chitosan oligosaccharide is 95%, the carbon chain length of the fatty acid is C18, and the amino substitution degree is 1% to 30%. The preparation method of the PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond graft is as follows: S1: A two-step amide reaction method was used to cross-link hydrophilic chitosan oligosaccharide and hydrophobic stearylamine with 3,3'-dithiodipropionic acid to synthesize chitosan oligosaccharide stearylamine disulfide bond grafts; S2: Dissolve p-carboxybenzaldehyde in N,N-dimethylformamide, add carbodiimide and N-hydroxysuccinimide to dissolve completely, react at room temperature for 2 hours, add polyethylene glycol diamine to dissolve completely, and continue to react at room temperature overnight; then reduce pressure and concentrate the reaction solution, pour it into ice ether for precipitation, collect the product by centrifugation, and then vacuum dry it to obtain NH2-PEG 2k -CHO; the NH2-PEG 2k -CHO was dissolved in N,N-dimethylformamide, and galactose-NHS and triethylamine were added to dissolve completely. After reacting at room temperature for 4 hours, the solvent was removed under reduced pressure, and pure water was added to dissolve, followed by extraction with dichloromethane. The organic phase was collected, concentrated under reduced pressure, poured into ice ether for precipitation, and the product was collected by centrifugation and dried in vacuum to obtain Gal-PEG. 2k -CHO; S3: Using the Gal-PEG 2k -Schiff reaction between the terminal aldehyde group of CHO and the free amino group of the chitosan oligosaccharide stearylamine disulfide bond grafted product to synthesize the PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafted product; The general chemical structure formula of the PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond graft obtained by the above preparation method is as follows: 。 2. A method for preparing the PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafted product according to claim 1, characterized in that: The details are as follows: S1: A two-step amide reaction method was used to cross-link hydrophilic chitosan oligosaccharide and hydrophobic stearylamine with 3,3'-dithiodipropionic acid to synthesize chitosan oligosaccharide stearylamine disulfide bond grafts; S2: Dissolve p-carboxybenzaldehyde in N,N-dimethylformamide, add carbodiimide and N-hydroxysuccinimide to dissolve completely, react at room temperature for 2 hours, add polyethylene glycol diamine to dissolve completely, and continue to react at room temperature overnight; then reduce pressure and concentrate the reaction solution, pour it into ice ether for precipitation, collect the product by centrifugation, and then vacuum dry it to obtain NH2-PEG 2k -CHO; the NH2-PEG 2k -CHO was dissolved in N,N-dimethylformamide, and galactose-NHS and triethylamine were added to dissolve completely. After reacting at room temperature for 4 hours, the solvent was removed under reduced pressure, and pure water was added to dissolve, followed by extraction with dichloromethane. The organic phase was collected, concentrated under reduced pressure, poured into ice ether for precipitation, and the product was collected by centrifugation and dried in vacuum to obtain Gal-PEG. 2k -CHO; S3: Using the Gal-PEG 2k The terminal aldehyde group of -CHO and the free amino group of the chitosan oligosaccharide stearylamine disulfide bond grafted product undergo a Schiff reaction to synthesize the PEGylated galactose-modified chitosan oligosaccharide stearylamine disulfide bond grafted product.

3. The method for preparing a PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond graft according to claim 2, characterized in that: The step S1 is specifically as follows: 3,3'-dithiodipropionic acid, stearylamine, dicyclohexylcarbodiimide and 4-dimethylaminopyridine are dissolved in anhydrous dimethyl sulfoxide, and reacted for 18 hours in a water bath at 60°C with stirring and nitrogen; then carbodiimide and N-hydroxysuccinimide are added, and the reaction is carried out for 30 minutes in a water bath at 60°C with stirring to obtain an organic phase reaction liquid; chitosan oligosaccharide is dissolved in water, and then the organic phase reaction liquid is added dropwise while stirring, and the reaction is carried out for 8 hours in a water bath at 60°C with stirring; after the reaction is completed, a 7kDa dialysis bag is added, and the dialysis is performed for 48 hours, and the contents of the dialysis bag are centrifuged at room temperature at a speed of 10000 rpm for 10 minutes; the supernatant obtained by centrifugation is freeze-dried, and then washed with anhydrous ethanol to remove unreacted alcohol-soluble byproducts and stearylamine, to obtain a chitosan oligosaccharide stearylamine disulfide bond grafted product.

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

10.

5. The method for preparing a PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond graft according to claim 2, characterized in that: In step S2, 100 mg of p-carboxybenzaldehyde was dissolved in 5 ml of N,N-dimethylformamide, and NH2-PEG 2k -CHO 500 mg was dissolved in 5 ml N,N-dimethylformamide; the feeding molar ratio of carbodiimide, N-hydroxysuccinimide, polyethylene glycol diamine, galactose-NHS and triethylamine was 1.2:1.2:1.0:1.2:2.

0.

6. The method for preparing a PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond graft according to claim 2, characterized in that: The step S3 is specifically as follows: The chitosan oligosaccharide stearylamine disulfide bond graft was dissolved in an acetic acid aqueous solution at pH 5.5, and then the Gal-PEG 2k -CHO: chitosan oligosaccharide stearylamine disulfide bond grafted product = 1: (1-10) feed molar ratio was added Gal-PEG 2k -CHO, then add sodium triacetoxyborohydride in an amount 3 times the molar amount of chitosan oligosaccharide stearylamine disulfide bond grafted product, and stir at room temperature in the dark for 24 hours; then place in a dialysis bag MWCO 7kDa, dialyze with deionized water for 24 hours, take the solution in the dialysis bag, and obtain a PEGylated galactosyl-modified chitosan oligosaccharide stearylamine disulfide bond grafted product.

7. A method for constructing a hepatocyte-targeted nanoformulation, characterized in that: The details are as follows: The PEGylated galactosyl-modified chitosan oligosaccharide stearylamine disulfide grafted product as described in claim 1 or the PEGylated galactosyl-modified chitosan oligosaccharide stearylamine disulfide grafted product freeze-dried powder obtained by the preparation method of any one of claims 2 to 6 is dissolved in water, filtered and sterilized, and then added to the human A3A recombinant protein solution at a theoretical drug loading amount of PEGylated galactosyl-modified chitosan oligosaccharide stearylamine disulfide grafted product: human A3A recombinant protein = (100-400): 1, and incubated at room temperature to obtain a hepatocyte-targeted nanoformulation for the treatment of chronic hepatitis B.

8. The method for constructing a hepatocyte-targeted nanoformulation according to claim 7, characterized in that: The PEGylated galactosyl modified chitosan oligosaccharide stearylamine disulfide bond grafted product was dissolved in water, ultrasonicated in a water bath for 15 minutes at room temperature, and then filtered and sterilized with a microporous filter membrane with a pore size of 0.22 μm.

9. The method for constructing a hepatocyte-targeted nanoformulation according to claim 7, characterized in that: The room temperature incubation time is 30 min.

10. A hepatocyte-targeted nanoformulation obtained by the construction method of claim 7.

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