Hyaluronic acid nanogel for removing aldehyde group as well as preparation method and application of hyaluronic acid nanogel

Abstract

The invention provides hyaluronic acid nanogel for removing aldehyde groups as well as a preparation method and application of the hyaluronic acid nanogel. The hyaluronic acid nanogel is combined with an oxygen amine group, acetaldehyde generated by alcohol metabolism can be removed, oxidative stress can be reduced, the ROS level can be reduced, lipid production can be reduced, and the purpose of treating the alcoholic liver can be achieved. The hyaluronic acid nanogel for removing the aldehyde group, provided by the invention, is stable in structure and uniform in particle size distribution; an in-vivo experiment shows that the uptake of the liver to the HA-Gal-Se-ON nanogel is relatively high; the HA-Gal-Se-ON nanogel can be used for removing acetaldehyde generated by alcohol metabolism in parenchymal hepatic cells, and can be used for treating acute alcoholism and chronic alcoholic hepatitis. The preparation method is simple and convenient, good in biocompatibility and good in industrial application prospect.

Description

Technical Field

[0001] The invention belongs to the field of nano drug preparations, and in particular relates to a hyaluronic acid nano gel for removing aldehyde groups, and a preparation method and application thereof. Background Art

[0002] Alcohol consumption is an important risk factor for liver damage and chronic diseases, which may be caused by long-term drinking of more than a certain amount. From alcoholic liver damage to fatty liver, liver fibrosis, cirrhosis and finally liver cancer is a gradual and irreversible damage process. About 95% of heavy drinkers will cause alcoholic liver damage and fatty liver, and about 35% of drinkers will cause more serious liver fibrosis and cirrhosis.

[0003] As for the mechanism of alcohol-induced liver damage, it is generally accepted that ethanol is metabolized by alcohol dehydrogenase and microsomal cytochrome P450 in the liver to produce acetaldehyde; acetaldehyde is extremely toxic and carcinogenic, and can bind to proteins, lipids, and DNA, leading to structural and functional changes, inducing the formation of new antigens, and generating immune responses; acetaldehyde-induced changes in mitochondrial structure lead to functional damage, including a reduction in ATP production through the respiratory chain and the production of reactive oxygen species ROS, which also lead to the formation of lipid peroxides; acetaldehyde's metabolite acetate can be converted into acetyl-CoA, which helps in fatty acid synthesis; and ultimately, through multiple mechanisms such as endoplasmic reticulum stress and apoptosis, it participates in the damage to liver cells and induces inflammatory responses, leading to fatty degeneration of the liver and the formation of alcoholic fatty liver disease. By removing acetaldehyde, the production of a series of toxic metabolites downstream can be reduced, the accumulation of ROS can be reduced, and the level of liver damage and inflammation can be alleviated.

[0004] Existing treatment strategies for alcoholic liver damage or alcoholic fatty liver disease often rely on traditional anti-inflammatory therapy or patients to reduce their alcohol intake to gradually repair liver damage. In addition, clinical emergency measures for acute alcohol poisoning include gastric lavage, sedation and detoxification, etc. There is a lack of targeted treatment methods, namely, for the toxic metabolite of alcohol - acetaldehyde. If acetaldehyde can be removed in time after drinking, it will greatly reduce liver damage, treat acute alcohol poisoning and alcoholic hepatitis, and prevent the formation of alcoholic fatty liver disease.

[0005] Acetaldehyde contains aldehyde groups and can be combined with amino compounds. This reaction can be carried out under physiological conditions, and the Schiff base formed is stable under physiological conditions and is not easy to be cleaved. Both amine groups and oxyamine groups can react with aldehyde groups to generate imines and oximes, respectively. Oxime exhibits higher hydrolysis stability due to the resonance between its carbon-nitrogen double bond and the lone pair of electrons on the oxygen atom.

[0006] Hyaluronic acid is easy to modify and has good biocompatibility, so the oxyamine group is coupled to hyaluronic acid and administered to the liver to remove excess acetaldehyde produced by ethanol metabolism. Since nanoparticles have a liver accumulation effect, hyaluronic acid needs to be made into nanogels, so diselenide bonds are selected as cross-linking groups. After reaching the liver, the diselenide bonds can be cleaved in response to ROS and can remove some ROS at the same time; and galactosamine is selected as the liver targeting group. Galactosamine can bind to the asialoglycoprotein receptors on the surface of hepatic parenchymal cells, making the nanogel have higher hepatic parenchymal cell selectivity. Summary of the invention

[0007] Therefore, the purpose of the present invention is to overcome the defects in the prior art and provide a hyaluronic acid nanogel for removing aldehyde groups, a preparation method and application thereof. The hyaluronic acid nanogel of the present invention can be delivered to liver parenchymal cells in a targeted manner, respond to ROS cleavage, effectively remove acetaldehyde produced by alcohol metabolism, repair a series of liver damage caused by acetaldehyde, and alleviate the symptoms of acute alcohol poisoning and chronic alcoholic liver disease.

[0008] Before describing the present invention, the terms used in this article are defined as follows:

[0009] The term "active oxygen species" refers to the general term for oxygen-containing free radicals and peroxides that are easily formed into free radicals and are related to oxygen metabolism in the body. Its English name is reactive oxygen species, or ROS for short.

[0010] The term "O / W emulsification method" means: oil-in-water emulsification method.

[0011] The term "TFA" means: trifluoroacetic acid.

[0012] The term "PBS" means: Phosphate Buffered Saline.

[0013] The term "HA" means: Hyaluronic acid.

[0014] The term "HA-Gal" refers to: galactosamine-conjugated hyaluronic acid.

[0015] The term "HA-ON" refers to hyaluronic acid coupled with an oxyamine group.

[0016] The term "HA-Se-Se-HA" refers to: hyaluronic acid cross-linked by diselenide bonds.

[0017] The term "HA-Gal-Se" refers to a nanogel formed by cross-linking three functionalized hyaluronic acids: HA, HA-Gal and HA-Se-Se-HA.

[0018] The term "HA-Gal-C-ON" refers to a nanogel formed by cross-linking three functionalized hyaluronic acids: HA-Gal, HA-CC-HA and HA-ON.

[0019] The term "HA-Gal-Se-NH2" refers to a nanogel formed by cross-linking three functionalized hyaluronic acids: HA-Gal, HA-Se-Se-HA and HA-NH2.

[0020] The term "HA-Gal-Se-ON" refers to a nanogel formed by cross-linking three functionalized hyaluronic acids: HA-Gal, HA-Se-Se-HA and HA-ON.

[0021] The term "HA-NT-Se-ON" refers to a nanogel formed by cross-linking three functionalized hyaluronic acids: HA, HA-Se-Se-HA and HA-ON.

[0022] The term "EDC / NHS" refers to: 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-acetylsuccinimide.

[0023] The term "ALT" refers to: alanine aminotransferase.

[0024] The term "AST" means: aspartate aminotransferase.

[0025] To achieve the above object, the first aspect of the present invention provides a hyaluronic acid nanogel for removing aldehyde groups, wherein the hyaluronic acid nanogel is prepared by an O / W emulsification method using the following raw material components:

[0026] Contains hyaluronic acid modified with aldehyde scavenging groups,

[0027] Targeting group-modified hyaluronic acid, and

[0028] Hyaluronic acid cross-linked with reactive oxygen species responsive bonds;

[0029] Preferably, the particle size of the hyaluronic acid nanogel is 100 to 200 nm, more preferably 110 to 180 nm, and further preferably 120 to 150 nm; and / or

[0030] Preferably, the potential of the hyaluronic acid nanogel is -10 mV to -20 mV, more preferably -12 mV to -18 mV, and further preferably -14 mV to -16 mV.

[0031] According to the first aspect of the present invention, the hyaluronic acid nanogel, wherein:

[0032] The ratio of the hyaluronic acid raw material components is: the mass ratio of hyaluronic acid modified with aldehyde scavenging groups: hyaluronic acid modified with targeting groups: hyaluronic acid cross-linked with active oxygen response bonds is 16:1-3:1-3, preferably 16:1-2:2-3, and most preferably 16:1:3;

[0033] The hyaluronic acid modified with the targeting group is hyaluronic acid containing galactosamine; and / or

[0034] The active oxygen responsive bond cross-linked hyaluronic acid is hyaluronic acid containing a diselenide bond or a disulfide bond.

[0035] According to the first aspect of the present invention, the hyaluronic acid nanogel, wherein:

[0036] The general structural formula of the hyaluronic acid modified with the aldehyde scavenging group is HA-X, wherein X is selected from one or more of the following: NH2, ONH2, NHNH2, preferably NH2 or ONH2, and most preferably ONH2;

[0037] The general structural formula of the hyaluronic acid modified with the targeting group is: HA-Y, wherein Y is a targeting group, preferably a liver targeting group, more preferably one or more selected from the following: galactose, N-acetylgalactose, mannose, 6-phosphomannose, N-acetylmannose, lactose, further preferably one or more selected from the following: galactose, N-acetylgalactose, mannose, further preferably galactose or N-acetylgalactose; and / or

[0038] The general structural formula of the active oxygen responsive bond cross-linked hyaluronic acid is HA-Z-HA, wherein Z is selected from one or more of the following: sulfur, selenium, tellurium, disulfide bond, diselenide bond, thioacetal, preferably selected from one or more of the following: disulfide bond, diselenide bond, thioacetal, more preferably disulfide bond and / or diselenide bond.

[0039] The second aspect of the present invention provides a method for preparing the hyaluronic acid nanogel according to the first aspect, the method comprising the following steps:

[0040] 1) preparing hyaluronic acid modified with an aldehyde scavenging group, hyaluronic acid modified with a targeting group, and hyaluronic acid cross-linked with an active oxygen responsive bond;

[0041] 2) mixing the three hyaluronic acids prepared in step 1) in proportion and dissolving them in a buffer solution to obtain a hyaluronic acid mixed solution;

[0042] 3) preparing an organic mixed solution by taking a nonionic surfactant and a diluent;

[0043] 4) The hyaluronic acid mixed solution prepared in step 2) and the organic mixed solution prepared in step 3) are mixed and ultrasonically homogeneously, and then subjected to an O / W emulsification method to obtain the hyaluronic acid nanogel.

[0044] According to the method of the second aspect of the present invention,

[0045] The hyaluronic acid modified with the aldehyde scavenging group in step 1) is prepared by coupling an oxyamine group to hyaluronic acid; and / or

[0046] The step 1) comprises:

[0047] a) dissolving sodium hyaluronate in water and placing the mixture in a dialysis bag for overnight dialysis, and freeze-drying the mixture to obtain acidified sodium hyaluronate, dissolving the acidified sodium hyaluronate in an organic solvent, and activating the hydroxyl groups to obtain a hyaluronic acid monomer;

[0048] b) adding modified molecules with different modification functions to the hyaluronic acid monomer prepared in step a), reacting overnight, dialyzing and freeze-drying to obtain the hyaluronic acid modified with the aldehyde scavenging group, the hyaluronic acid modified with the targeting group and / or the hyaluronic acid cross-linked with the active oxygen response bond, respectively;

[0049] Preferably, in step a):

[0050] The external environment of the dialysis is an acid environment, and the acid in the acid environment is preferably hydrochloric acid; and / or the concentration of the acid is preferably 0.01M to 0.1M, and most preferably 0.01M; and / or the organic solvent is selected from one or more of the following: dimethyl sulfoxide, N,N-dimethylformamide, dichloromethane, preferably dimethyl sulfoxide or N,N-dimethylformamide, and most preferably dimethyl sulfoxide; and / or the hydroxyl-activated activator is selected from one or more of the following: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, N,N,N ',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)urea hexafluorophosphate, 1,3-dicyclohexylcarbodiimide, 1,3-diisopropylcarbodiimide, 1-hydroxybenzotriazole, preferably one or more selected from the following: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)urea hexafluorophosphate, more preferably 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and / or N-hydroxysuccinimide; and / or

[0051] Preferably, in step b):

[0052] The modified molecules are selected from one or more of the following: aldehyde scavenging group modified molecules, targeting group modified molecules, and active oxygen response bond cross-linked modified molecules; wherein the molar ratio of the aldehyde scavenging group modified molecules to the hyaluronic acid monomer is 1-4:1, preferably 1-2:1, and most preferably 1:1; the molar ratio of the targeting group modified molecules to the hyaluronic acid monomer is 1-4:1, preferably 1-2:1, and most preferably 1:1; the molar ratio of the active oxygen response bond cross-linked modified molecules to the hyaluronic acid monomer is 1-2:1-2, preferably 1:1-2, and most preferably 1:2; the reaction temperature is 18-25°C, preferably 20-25°C, and most preferably 25°C; the reaction time is 12-24h, preferably 18-24h, and most preferably 24h; and / or the dialysis time is 12-24h, preferably 18-24h, and most preferably 24h; wherein:

[0053] Preferably, the aldehyde scavenging group modification molecule is selected from one or more of the following: tert-butyl 2-((2-aminoethyl)amino)-2-oxoethoxycarbamate, tert-butyl N-(5-aminopentyl)carbamate, 3-aminopropylhydrazine, more preferably tert-butyl 2-((2-aminoethyl)amino)-2-oxoethoxycarbamate or tert-butyl N-(5-aminopentyl)carbamate, most preferably tert-butyl 2-((2-aminoethyl)amino)-2-oxoethoxycarbamate;

[0054] Preferably, the targeting group modification molecule is selected from one or more of the following: galactosamine, N-acetylgalactosamine, mannosamine, 6-phosphomannosamine, N-acetylmannosamine, lactosamine, more preferably selected from one or more of the following: galactosamine, N-acetylgalactosamine, mannosamine, further preferably galactosamine or N-acetylgalactosamine; and / or

[0055] Preferably, the active oxygen responsive bond cross-linking modification molecule is selected from one or more of the following: selenocystamine hydrochloride, cystamine dihydrochloride, cysteamine hydrochloride, selenocystamine hydrochloride, tellurylcysteamine hydrochloride, tellurylcysteamine hydrochloride, acetal diamine, selenocystamine dihydrochloride, more preferably selected from one or more of the following: selenocystamine hydrochloride, cystamine dihydrochloride, cysteamine hydrochloride, more preferably selenocystamine hydrochloride or cystamine dihydrochloride.

[0056] According to the method of the second aspect of the present invention, the step 1) further comprises:

[0057] c) using a dialysis bag to remove the unreacted activator and the modified molecules from the hyaluronic acid modified with the aldehyde scavenging group, the hyaluronic acid modified with the targeting group and the hyaluronic acid cross-linked with the active oxygen responsive bond prepared in step b).

[0058] According to the method of the second aspect of the present invention, in said step 2):

[0059] The mass ratio of the hyaluronic acid modified with the aldehyde scavenging group, the hyaluronic acid modified with the targeting group and the hyaluronic acid cross-linked with the active oxygen response bond is 16:1-3:1-3, preferably 16:1-2:2-3, more preferably 16:1:3; and / or

[0060] The buffer is selected from one or more of the following: PBS, Hank's balanced salt solution, Earle's balanced salt solution, preferably PBS or Hank's balanced salt solution, most preferably PBS.

[0061] According to the method of the second aspect of the present invention, in said step 3):

[0062] The nonionic surfactant is selected from one or more of the following: Tween, Span, sucrose fatty acid ester, polyoxyethylene fatty acid ester, polyoxyethylene fatty alcohol ether, poloxamer, preferably selected from one or more of the following: Tween 80, Span 80, polyoxyethylene fatty alcohol ether, poloxamer, more preferably Tween 80 or Span 80;

[0063] The diluent is selected from one or more of the following: n-hexane, glyceride, vegetable oil, beeswax, fatty alcohol, preferably selected from one or more of the following: n-hexane, glyceride, vegetable oil, more preferably n-hexane or glyceride.

[0064] According to the method of the second aspect of the present invention, in said step 4):

[0065] The O / W emulsification method further comprises: mixing the hyaluronic acid mixed solution and the organic mixed solution uniformly and ultrasonicating them, and then sequentially performing: a first centrifugation to discard the supernatant, a first resuspension, rotary evaporation, discarding the supernatant, a second centrifugation, discarding the supernatant, and a second resuspension to obtain the hyaluronic acid nanogel;

[0066] Preferably, the mixing ultrasound is probe ultrasound.

[0067] The third aspect of the present invention provides the use of the hyaluronic acid nanogel described in the first aspect in the preparation of a drug for treating liver disease;

[0068] Preferably, the liver disease is alcoholic liver disease;

[0069] More preferably, the alcoholic liver disease is selected from one or more of the following: chronic alcoholic liver disease, acute alcoholic liver disease, acute alcohol poisoning, preferably chronic alcoholic liver disease or acute alcoholic liver disease.

[0070] According to a specific embodiment, the present invention provides a new nanogel for liver-specific acetaldehyde removal, wherein an oxyamine group is coupled to hyaluronic acid to synthesize hyaluronic acid containing a diselenide bond and galactosamine, and the three are subjected to an O / W emulsification method to obtain a nanogel with hepatocyte targeting, ROS response, and acetaldehyde removal functions.

[0071] The particle size of the hyaluronic acid nanogel of the present invention is about 130 nm, and the surface potential is -15 mV;

[0072] The present invention utilizes the carboxyl groups rich in hyaluronic acid to connect various functional groups through amide condensation, and the synthesis is simple;

[0073] The hyaluronic acid modification method of the present invention comprises the following steps:

[0074] Hyaluronic acid is dissolved in dimethyl sulfoxide, and an activator carboxyl group is added for activation for 4 hours; 2-((2-aminoethyl)amino)-2-oxoethoxycarbamic acid tert-butyl ester (or N-(5-aminopentyl)carbamic acid tert-butyl ester), selenocystamine hydrochloride, galactosamine, and diamine are added, and the reaction is carried out overnight to obtain hyaluronic acid coupled with different functional groups;

[0075] Preferably, the activator is selected from carbodiimide hydrochloride and N-hydroxysuccinimide;

[0076] Preferably, the reaction temperature is 18-25°C, more preferably 20-25°C, most preferably 25°C;

[0077] Preferably, the reaction time is 12 to 24 hours, more preferably 18 to 24 hours, most preferably 24 hours;

[0078] The steps also include: removing unreacted cross-linking agent and modified molecules from the prepared hyaluronic acid modified product using a dialysis bag;

[0079] Preferably, the dialysis bag is a dialysis bag with a molecular weight cut-off of 10KDa;

[0080] The nanogel preparation method of the present invention comprises the following steps:

[0081] Weigh 20 mg of HA raw material and dissolve it in 1 ml of PBS; take 180 mg of Tween 80, 180 mg of Span 80, and 4 ml of n-hexane, put them in a 10 ml EP tube, and mix them by ultrasonication; perform probe ultrasonication; after ultrasonication, centrifuge at 12000 rpm for 10 min, discard the supernatant, and resuspend and wash it with n-hexane; resuspend it with 15 ml of water three times, and then evaporate it for 20 min to remove the organic solvent; 15000 rpm for 45 min, discard the supernatant, wash it with water three times, and then centrifuge it at 15000 rpm for 45 min, discard the supernatant, and resuspend it in 1 ml of PBS to obtain HA-Gal-Se-ON nanogel;

[0082] The hyaluronic acid nanogel with acetaldehyde scavenging function of the present invention is used for treating acute alcohol poisoning and chronic alcoholic liver disease.

[0083] According to another specific embodiment, the first aspect of the present invention provides a hyaluronic acid nanogel, which is prepared by an O / W emulsification method from hyaluronic acid modified with an aldehyde scavenging group, hyaluronic acid modified with a liver targeting group, and hyaluronic acid cross-linked with a ROS response bond; wherein,

[0084] The nanogel can be targeted to liver parenchymal cells and cleaved in response to intracellular ROS, exposing groups that can scavenge acetaldehyde.

[0085] The hydrated particle size of the hyaluronic acid nanogel is 100-200 nm as monitored by dynamic light scattering; and the potential is -10 mV to -20 mV as detected by a Zeta potential instrument.

[0086] The structure of the hyaluronic acid modified with the aldehyde scavenging group is: HA-X-NH2, wherein X is selected from the following: C, O, N;

[0087] The liver-targeting group-modified hyaluronic acid structure is: HA-Y, where Y is a liver-targeting group, and Y is selected from the following: galactosamine, N-acetylgalactosamine, mannose, 6-phosphate mannose, N-acetylmannose, and lactose.

[0088] The ROS-responsive bond-crosslinked hyaluronic acid structure is: HA-SS-HA or HA-Se-Se-HA, wherein the ROS-responsive bond is a disulfide bond or a diselenide bond.

[0089] The second aspect of the present invention provides a method for preparing the hyaluronic acid nanogel described in the first aspect, wherein the hyaluronic acid nanogel is prepared from the three modified hyaluronic acids described in the first aspect by an O / W emulsification method.

[0090] The hyaluronic acid modification method comprises the following steps:

[0091] (1) Take 500 mg of 100KD sodium hyaluronate HA, dissolve it in 20 ml of water, place it in a dialysis bag, dialyze it in a 0.01M HCl dilute hydrochloric acid environment, dialyze it overnight for 24 hours, freeze-dry it, and take it out;

[0092] (2) 50 mg of the acidified HA (379,1 eq) obtained in step (1) was dissolved in 10 ml of DMSO, 125 mg of carbodiimide hydrochloride (191,5 eq) and 75 mg of N-hydroxysuccinimide (115,5 eq) were weighed, and the carboxyl group was activated for 4 h; 30 mg of tert-butyl 2-((2-aminoethyl)amino)-2-oxoethoxycarbamate (or 26.5 mg of tert-butyl N-(5-aminopentyl)carbamate), 21 mg of selenocystamine hydrochloride, 28.5 mg of galactosamine, and 12.5 mg of diamine were added, and the reaction was allowed to proceed overnight; the above DMSO was placed in a 10KD dialysis bag, dialyzed with water three times for 24 h, and lyophilized to obtain hyaluronic acid modified with different functional groups: HA-ONH2, HA-Se-Se-HA, and HA-Gal;

[0093] Preferably, the molar ratio of the modified molecule to the hyaluronic acid monomer is 1:1;

[0094] Preferably, the activator is selected from carbodiimide hydrochloride and N-hydroxysuccinimide;

[0095] Preferably, the reaction temperature is 18-25°C, more preferably 20-25°C, most preferably 25°C;

[0096] Preferably, the reaction time is 12 to 24 hours, more preferably 18 to 24 hours, and most preferably 24 hours. The step (2) further comprises: removing the unreacted cross-linking agent and the modified molecule from the prepared hyaluronic acid modified product with a dialysis bag;

[0097] Preferably, the dialysis bag is a dialysis bag with a molecular weight cut-off of 10KDa;

[0098] The preparation of the hyaluronic acid nanogel comprises the following steps:

[0099] (1) Weigh 20 mg of HA raw material (HA-ONH2:HA-Se-Se-HA:HA-Gal=16:3:1), dissolve in 1 ml of PBS, and sonicate if necessary; take 180 mg of Tween 80, 180 mg of Span 80, and 4 ml of n-hexane, place in a 10 ml EP tube, and sonicate; mix the above two samples thoroughly, and sonicate with a probe at 30%, 5 min, on for 1 s, off for 2 s;

[0100] (2) After ultrasonication, centrifuge at 12000 rpm for 10 min, discard the supernatant, and resuspend and wash with n-hexane; resuspend with 15 ml of water three times, and evaporate for 20 min to remove the organic solvent; centrifuge at 15000 rpm for 45 min, discard the supernatant, wash with water three times, and centrifuge at 15000 rpm for 45 min, discard the supernatant, and resuspend with 1 ml of PBS to obtain HA-Gal-Se-ON nanogel.

[0101] The third aspect of the present invention provides the use of the hyaluronic acid nanogel described in the first aspect or the hyaluronic acid nanogel prepared according to the method described in the second aspect in the treatment of alcoholic liver disease;

[0102] Application of hyaluronic acid nanogel in acute alcohol poisoning

[0103] The hyaluronic acid nanogel prepared according to the method described in the second aspect is used in a mouse model of acute alcohol poisoning, which can effectively reduce the content of acetaldehyde in the blood and is beneficial to liver protection;

[0104] Application of hyaluronic acid nanogel in chronic alcoholic liver disease

[0105] The hyaluronic acid nanogel prepared according to the method described in the second aspect is used in a mouse model of chronic alcoholic liver disease to repair the functional damage of chronic alcoholic liver.

[0106] The present invention relates to the field of nano drug preparations, and provides a new idea for the intervention and treatment of alcoholic liver disease. The hyaluronic acid nanogel is combined with an oxyamine group, which can remove acetaldehyde produced by alcohol metabolism, reduce oxidative stress, reduce ROS levels, reduce lipid production, and achieve the purpose of treating alcoholic liver. The hyaluronic acid nanogel for removing aldehyde groups provided by the present invention has a stable structure, a particle size of about 130nm, a surface potential of -15mV, and a uniform particle size distribution; in vivo experiments show that the liver has a high uptake of HA-Gal-Se-ON nanogel; HA-Gal-Se-ON nanogel can remove acetaldehyde produced by alcohol metabolism in liver parenchymal cells, and can be used for the treatment of acute alcohol poisoning and chronic alcoholic hepatitis. The preparation method of the present invention is simple, the biocompatibility is good, and it has good industrial application prospects.

[0107] The hyaluronic acid nanogel for removing aldehyde groups of the present invention may have but is not limited to the following beneficial effects:

[0108] The hyaluronic acid nanogel of the present invention has high dispersibility, good stability and good biocompatibility. The different types of functional groups carried give the gel excellent acetaldehyde scavenging ability, ROS responsiveness, and hepatocyte-specific targeting. The hyaluronic acid nanogel provided by the present invention has potential application value in the field of alcoholic liver disease treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0109] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0110] Figure 1 The synthesis process of hyaluronic acid nanogel in Example 1 of the present invention is shown.

[0111] Figure 2 The synthesis process of hyaluronic acid modified with aldehyde scavenging groups, hyaluronic acid modified with targeting groups and hyaluronic acid cross-linked with ROS response bonds in Example 1 of the present invention is shown; wherein, Figure 2 A shows the synthesis process of hyaluronic acid modified with aldehyde scavenging groups; Figure 2 B shows the synthesis process of ROS-responsive cross-linked hyaluronic acid; Figure 2 C shows the synthesis process of hyaluronic acid modified with targeting groups.

[0112] Figure 3 The morphology of the hyaluronic acid nanogel prepared in Example 1 of the present invention observed by transmission electron microscopy is shown; wherein the white color is a ruler, and the length of the ruler represents 100 nm.

[0113] Figure 4 The protective effect of the hyaluronic acid nanogel prepared in Example 1 of the present invention on alcohol-damaged AML12 cells is shown.

[0114] Figure 5 The figure shows the evaluation of the acetaldehyde scavenging ability of the hyaluronic acid nanogel prepared in Example 1 of the present invention in the blood of mice with acute alcohol poisoning.

[0115] Figure 6 HE staining of a mouse liver sample injected with the hyaluronic acid nanogel prepared in Example 1 is shown.

[0116] Figure 7 The liver function results in the blood biochemistry of mice injected with the hyaluronic acid nanogel prepared in Example 1 are shown. DETAILED DESCRIPTION

[0117] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed and specific description and should not be understood as limiting the present invention in any form.

[0118] This section generally describes the materials and test methods used in the experiments of the present invention. Although many materials and operating methods used to achieve the purpose of the present invention are well known in the art, the present invention is still described as detailed as possible. It is clear to those skilled in the art that in the context, if not specifically stated, the materials and operating methods used in the present invention are well known in the art.

[0119] The reagents and instruments used in the following examples are as follows:

[0120] Reagents:

[0121] Sodium hyaluronate was purchased from Shanghai Myrel Biochemical Technology Co., Ltd.

[0122] Dimethyl sulfoxide, n-hexane, carbodiimide hydrochloride, N-hydroxysuccinimide, tert-butyl 2-((2-aminoethyl)amino)-2-oxoethoxycarbamate, selenocystamine hydrochloride, and galactosamine were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0123] PBS, Tween 80, and Span 80 were purchased from Beijing Solebow Co., Ltd.

[0124] CCK-8 kit was purchased from Dojin Chemical Co., Ltd., Japan.

[0125] instrument:

[0126] Transmission electron microscope, model HT7700, purchased from Hitachi.

[0127] Example 1

[0128] This example is an exemplary description of the synthesis and preparation method of the hyaluronic acid nanogel for removing aldehyde groups of the present invention.

[0129] (1) Take 500 mg of 100KD sodium hyaluronate, dissolve it in 20 ml of water, place it in a dialysis bag, dialyze it in a 0.01M HCl dilute hydrochloric acid environment, dialyze it overnight for 24 hours, freeze-dry it, and take it out;

[0130] (2) Take 50 mg of the acidified hyaluronic acid (379,1 eq) obtained in step (1) and dissolve it in 10 ml of dimethyl sulfoxide, weigh 125 mg of the activator carbodiimide hydrochloride (191,5 eq) and 75 mg of N-hydroxysuccinimide (115,5 eq), and activate the carboxyl group for 4 hours; add 30 mg of tert-butyl 2-((2-aminoethyl)amino)-2-oxoethoxycarbamate (233,1 eq, 0.6 times the mass) and react overnight; place the above dimethyl sulfoxide in a 10KD dialysis bag, dialyze with water three times for 24 hours, and lyophilize to obtain oxyamine-modified hyaluronic acid: HA-ONH2.

[0131] (3) Take 50 mg of the acidified hyaluronic acid (379,1 eq) obtained in step (1) and dissolve it in 10 ml of dimethyl sulfoxide, weigh 125 mg of the activator carbodiimide hydrochloride (191,5 eq) and 75 mg of N-hydroxysuccinimide (115,5 eq), and activate the carboxyl group for 4 hours; add 21 mg of selenocystamine hydrochloride (318, 0.5 eq, 0.42 times the mass); place the above dimethyl sulfoxide in a 10KD dialysis bag, dialyze with water three times for 24 hours, and lyophilize to obtain diselenide cross-linked hyaluronic acid: HA-Se-Se-HA.

[0132] (4) Take 50 mg of the acidified hyaluronic acid (379,1 eq) obtained in step (1) and dissolve it in 10 ml of dimethyl sulfoxide, weigh 125 mg of the activator carbodiimide hydrochloride (191,5 eq) and 75 mg of N-hydroxysuccinimide (115,5 eq), and activate the carboxyl group for 4 hours; add 28.5 mg of galactosamine (215,1 eq, 0.57 times the mass); place the above dimethyl sulfoxide in a 10KD dialysis bag, dialyze with water three times for 24 hours, and lyophilize to obtain galactosamine-modified hyaluronic acid: HA-Gal.

[0133] (5) Weigh 20 mg of raw material (HA-ONH2:HA-Gal:HA-Se-Se-HA=16:1:3), dissolve in 1 ml PBS, and sonicate if necessary; take 180 mg Tween 80, 180 mg Span 80, 4 ml n-hexane, place in a 10 ml EP tube, and sonicate; mix the above two samples thoroughly, and sonicate with a probe at 30%, 5 min, on for 1 s, off for 2 s;

[0134] (6) After ultrasonication, centrifuge at 12000 rpm for 10 min, discard the supernatant, and resuspend and wash once with n-hexane; resuspend with 15 ml of water three times, and evaporate for 20 min to remove the organic solvent; centrifuge at 15000 rpm for 45 min, discard the supernatant, wash three times with water, and centrifuge at 15000 rpm for 45 min, discard the supernatant, and resuspend with 1 ml of PBS to obtain HA-Gal-Se-ON nanogel.

[0135] Example 2-13

[0136] This example is another exemplary description of the synthesis and preparation method of the hyaluronic acid nanogel for removing aldehyde groups of the present invention.

[0137] The preparation methods of Examples 2 to 13 are the same as those of Example 1, except for Table 1.

[0138] Table 1 Hyaluronic acid nanogels for removing aldehyde groups prepared in Examples 2 to 13

[0139]

[0140]

[0141]

[0142] Embodiment 14

[0143] This example is used to illustrate the morphology of the hyaluronic acid nanogel for removing aldehyde groups of the present invention.

[0144] The hyaluronic acid nanogels prepared in Examples 1 to 13 were dissolved in water three times, stained with uranyl acetate, and the prepared spherical structures were observed under a transmission electron microscope.

[0145] Figure 3 The morphology of the hyaluronic acid nanogel observed by transmission electron microscopy in Example 1 of the present invention is shown. The particle size is 130 nm; wherein the scale is 100 nm.

[0146] Table 2 Morphological characteristics of hyaluronic acid nanogels prepared in Examples 2 to 13

[0147]

[0148]

[0149] Embodiment 15

[0150] This example is used to illustrate the protective effect of the hyaluronic acid nanogel for removing aldehyde groups of the present invention on AML12 cells damaged by alcohol.

[0151] This example uses the hyaluronic acid nanogels prepared in Examples 1 to 13.

[0152] In this example, a CCK-8 kit was used to perform cell viability analysis. AML12 cells were inoculated into a 96-well plate and cultured to 50% confluence, treated with 200 mM ethanol solution, and fresh culture medium containing hyaluronic acid nanogels with different functions (HA-Gal-Se-NH2, HA-Gal-C-ON, HA-NT-Se-ON, HA-Gal-Se-ON) was added to treat the cells for 24 hours. Finally, the cells were cultured with fresh culture medium containing 10% CCK-8 reagent for 1 hour, and the absorbance was measured at a wavelength of 450 nm to calculate the cell viability.

[0153] Figure 4 The results of the evaluation of cytotoxicity in Example 1 are shown. The results show that compared with other control nanogels, the cell viability value of the HA-Gal-Se-ON nanogel group was 85%, which significantly alleviated the cytotoxicity caused by alcohol and had a good protective effect.

[0154] As shown in Table 3, the nanogels described in Examples 2-13 all have good protective effects on hepatic parenchymal cells.

[0155] Table 3 Evaluation results of cytotoxicity of hyaluronic acid nanogels prepared in Examples 2 to 13

[0156]

[0157]

[0158] Example 16

[0159] This example is used to illustrate the acetaldehyde-scavenging effect of the hyaluronic acid nanogel for scavenging aldehyde groups of the present invention.

[0160] This example uses the hyaluronic acid nanogels prepared in Examples 1 to 13.

[0161] In this example, male C57BL / 6J mice weighing 20-23 were randomly divided into six groups, one group was the control group, and the remaining five groups were given a single alcohol gavage and intravenous injection of different nanogels. Blood was collected at 4 and 8 hours to monitor the acetaldehyde content in the blood.

[0162] Figure 5 The results of the evaluation of the acetaldehyde scavenging ability of the hyaluronic acid nanogel prepared in Example 1 are shown. The results show that compared with other control nanogels, the HA-Gal-Se-ON nanogel in Example 1 significantly reduced the acetaldehyde content in the blood of mice after alcohol gavage (reduced by 75%), and can well alleviate the symptoms of acute alcohol poisoning.

[0163] As can be seen from Table 4, the nanogels described in Examples 2-13 all showed a certain acetaldehyde scavenging ability.

[0164] Table 4 Acetaldehyde removal effect of hyaluronic acid nanogels prepared in Examples 2 to 13

[0165]

[0166]

[0167] Embodiment 17

[0168] This example is used to illustrate the therapeutic effect of the hyaluronic acid nanogel for removing aldehyde groups of the present invention on chronic alcoholic hepatitis.

[0169] This example uses the hyaluronic acid nanogels prepared in Examples 1 to 13.

[0170] In this example, female C57BL / 6J mice weighing 20-23 were randomly divided into six groups, one group was a control group, and the remaining five groups were fed with chronic alcohol for eleven days, and different nanogels were intravenously injected every day. At the end of the modeling on the eleventh day, liver samples and blood samples were collected for analysis.

[0171] Figure 6 HE staining of a mouse liver sample injected with the hyaluronic acid nanogel prepared in Example 1 is shown. Figure 7 The results of liver function in blood biochemistry of mice injected with the hyaluronic acid nanogel prepared in Example 1 are shown. The results show that HA-Gal-Se-ON nanogel can alleviate liver inflammation and restore liver function damage in chronic alcoholic liver disease, and can be used to treat chronic alcoholic liver disease.

[0172] The nanogels described in Examples 2-13 all had good protective effects on the liver of chronic alcoholic liver disease. HE staining and liver function tests showed that liver inflammation and damage were reduced, and liver function was restored.

[0173] Although the above embodiments / test examples show the effects of some embodiments, those skilled in the art should understand that according to the concept of the present invention, the above-mentioned other embodiments without specific effects or other technical solutions of the present invention not shown in the embodiments or test examples can also achieve the following technical effects stated in the invention content section that are equivalent to the above embodiments / test examples:

[0174] The hyaluronic acid nanogel of the present invention has high dispersibility, good stability and good biocompatibility. The different types of functional groups carried give the gel excellent acetaldehyde scavenging ability, ROS responsiveness, and hepatocyte-specific targeting. The hyaluronic acid nanogel provided by the present invention has potential application value in the field of alcoholic liver disease treatment.

[0175] Although the present invention has been described to a certain extent, it is obvious that appropriate changes in various conditions can be made without departing from the spirit and scope of the present invention. It is understood that the present invention is not limited to the embodiments described, but belongs to the scope of the claims, which includes equivalent replacements of each factor described.

Claims

1. A hyaluronic acid nanogel for removing aldehyde groups, characterized in that: The hyaluronic acid nanogel is prepared from the following raw materials via an O / W emulsification method: Contains hyaluronic acid modified with aldehyde scavenging groups, Targeting group-modified hyaluronic acid, and Hyaluronic acid cross-linked with reactive oxygen species responsive bonds; Preferably, the particle size of the hyaluronic acid nanogel is 100 to 200 nm, more preferably 110 to 180 nm, and further preferably 120 to 150 nm; and / or Preferably, the potential of the hyaluronic acid nanogel is -10 mV to -20 mV, more preferably -12 mV to -18 mV, and further preferably -14 mV to -16 mV.

2. The hyaluronic acid nanogel according to claim 1, characterized in that: The ratio of the hyaluronic acid raw material components is: the mass ratio of hyaluronic acid modified with aldehyde scavenging groups: hyaluronic acid modified with targeting groups: hyaluronic acid cross-linked with active oxygen response bonds is 16:1-3:1-3, preferably 16:1-2:2-3, and most preferably 16:1:3; The hyaluronic acid modified with the targeting group is hyaluronic acid containing galactosamine; and / or The active oxygen responsive bond cross-linked hyaluronic acid is hyaluronic acid containing a diselenide bond or a disulfide bond.

3. The hyaluronic acid nanogel according to claim 1 or 2, characterized in that: The general structural formula of the hyaluronic acid modified with the aldehyde scavenging group is HA-X, wherein X is selected from one or more of the following: NH2, ONH2, NHNH2, preferably NH2 or ONH2, and most preferably ONH2; The general structural formula of the hyaluronic acid modified with the targeting group is: HA-Y, wherein Y is a targeting group, preferably a liver targeting group, more preferably one or more selected from the following: galactose, N-acetylgalactose, mannose, 6-phosphomannose, N-acetylmannose, lactose, further preferably one or more selected from the following: galactose, N-acetylgalactose, mannose, further preferably galactose or N-acetylgalactose; and / or The general structural formula of the active oxygen responsive bond cross-linked hyaluronic acid is HA-Z-HA, wherein Z is selected from one or more of the following: sulfur, selenium, tellurium, disulfide bond, diselenide bond, thioacetal, preferably selected from one or more of the following: disulfide bond, diselenide bond, thioacetal, more preferably disulfide bond and / or diselenide bond.

4. A method for preparing the hyaluronic acid nanogel according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: 1) preparing hyaluronic acid modified with an aldehyde scavenging group, hyaluronic acid modified with a targeting group, and hyaluronic acid cross-linked with an active oxygen responsive bond; 2) mixing the three hyaluronic acids prepared in step 1) in proportion and dissolving them in a buffer solution to obtain a hyaluronic acid mixed solution; 3) preparing an organic mixed solution by taking a nonionic surfactant and a diluent; 4) The hyaluronic acid mixed solution prepared in step 2) and the organic mixed solution prepared in step 3) are mixed and ultrasonically homogeneously, and then subjected to an O / W emulsification method to obtain the hyaluronic acid nanogel.

5. The method according to claim 4, characterized in that: The hyaluronic acid modified with the aldehyde scavenging group in step 1) is prepared by coupling an oxyamine group to hyaluronic acid; and / or The step 1) comprises: a) dissolving sodium hyaluronate in water and placing the mixture in a dialysis bag for overnight dialysis, and freeze-drying the mixture to obtain acidified sodium hyaluronate, dissolving the acidified sodium hyaluronate in an organic solvent, and activating the hydroxyl groups to obtain a hyaluronic acid monomer; b) adding modified molecules with different modification functions to the hyaluronic acid monomer prepared in step a), reacting overnight, dialyzing and freeze-drying to obtain the hyaluronic acid modified with the aldehyde scavenging group, the hyaluronic acid modified with the targeting group and / or the hyaluronic acid cross-linked with the active oxygen response bond, respectively; Preferably, in step a): The external environment of the dialysis is an acid environment, and the acid in the acid environment is preferably hydrochloric acid; and / or the concentration of the acid is preferably 0.01M to 0.1M, and most preferably 0.01M; and / or the organic solvent is selected from one or more of the following: dimethyl sulfoxide, N,N-dimethylformamide, dichloromethane, preferably dimethyl sulfoxide or N,N-dimethylformamide, and most preferably dimethyl sulfoxide; and / or the hydroxyl-activated activator is selected from one or more of the following: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, N,N,N ',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)urea hexafluorophosphate, 1,3-dicyclohexylcarbodiimide, 1,3-diisopropylcarbodiimide, 1-hydroxybenzotriazole, preferably one or more selected from the following: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)urea hexafluorophosphate, more preferably 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and / or N-hydroxysuccinimide; and / or Preferably, in step b): The modified molecules are selected from one or more of the following: aldehyde scavenging group modified molecules, targeting group modified molecules, and active oxygen response bond cross-linked modified molecules; wherein the molar ratio of the aldehyde scavenging group modified molecules to the hyaluronic acid monomer is 1-4:1, preferably 1-2:1, and most preferably 1:1; the molar ratio of the targeting group modified molecules to the hyaluronic acid monomer is 1-4:1, preferably 1-2:1, and most preferably 1:1; the molar ratio of the active oxygen response bond cross-linked modified molecules to the hyaluronic acid monomer is 1-2:1-2, preferably 1:1-2, and most preferably 1:2; the reaction temperature is 18-25°C, preferably 20-25°C, and most preferably 25°C; the reaction time is 12-24h, preferably 18-24h, and most preferably 24h; and / or the dialysis time is 12-24h, preferably 18-24h, and most preferably 24h; wherein: Preferably, the aldehyde scavenging group modification molecule is selected from one or more of the following: tert-butyl 2-((2-aminoethyl)amino)-2-oxoethoxycarbamate, tert-butyl N-(5-aminopentyl)carbamate, 3-aminopropylhydrazine, more preferably tert-butyl 2-((2-aminoethyl)amino)-2-oxoethoxycarbamate or tert-butyl N-(5-aminopentyl)carbamate, most preferably tert-butyl 2-((2-aminoethyl)amino)-2-oxoethoxycarbamate; Preferably, the targeting group modification molecule is selected from one or more of the following: galactosamine, N-acetylgalactosamine, mannosamine, 6-phosphomannosamine, N-acetylmannosamine, lactosamine, more preferably selected from one or more of the following: galactosamine, N-acetylgalactosamine, mannosamine, further preferably galactosamine or N-acetylgalactosamine; and / or Preferably, the active oxygen responsive bond cross-linking modification molecule is selected from one or more of the following: selenocystamine dihydrochloride, cystamine dihydrochloride, cysteamine hydrochloride, selenocystamine hydrochloride, tellurylcystamine dihydrochloride, tellurylcysteamine hydrochloride, and thioketal diamine, more preferably selected from one or more of the following: selenocystamine hydrochloride, cystamine dihydrochloride, cysteamine hydrochloride, more preferably selenocystamine hydrochloride or cystamine dihydrochloride.

6. The method according to claim 5, characterized in that The step 1) also includes: c) using a dialysis bag to remove the unreacted activator and the modified molecules from the hyaluronic acid modified with the aldehyde scavenging group, the hyaluronic acid modified with the targeting group and the hyaluronic acid cross-linked with the active oxygen responsive bond prepared in step b).

7. The method according to any one of claims 4 to 6, characterized in that In the step 2): The mass ratio of the hyaluronic acid modified with the aldehyde scavenging group, the hyaluronic acid modified with the targeting group and the hyaluronic acid cross-linked with the active oxygen response bond is 16:1-3:1-3, preferably 16:1-2:2-3, more preferably 16:1:3; and / or The buffer is selected from one or more of the following: PBS, Hank's balanced salt solution, Earle's balanced salt solution, preferably PBS or Hank's balanced salt solution, most preferably PBS.

8. The method according to any one of claims 4 to 6, characterized in that In the step 3): The nonionic surfactant is selected from one or more of the following: Tween, Span, sucrose fatty acid ester, polyoxyethylene fatty acid ester, polyoxyethylene fatty alcohol ether, poloxamer, preferably selected from one or more of the following: Tween 80, Span 80, polyoxyethylene fatty alcohol ether, poloxamer, more preferably Tween 80 or Span 80; The diluent is selected from one or more of the following: n-hexane, glyceride, vegetable oil, beeswax, fatty alcohol, preferably selected from one or more of the following: n-hexane, glyceride, vegetable oil, more preferably n-hexane or glyceride.

9. The method according to any one of claims 4 to 6, characterized in that In the step 4): The O / W emulsification method further comprises: mixing the hyaluronic acid mixed solution and the organic mixed solution uniformly and ultrasonicating them, and then sequentially performing: a first centrifugation to discard the supernatant, a first resuspension, rotary evaporation, discarding the supernatant, a second centrifugation, discarding the supernatant, and a second resuspension to obtain the hyaluronic acid nanogel; Preferably, the mixing ultrasound is probe ultrasound.

10. Use of the hyaluronic acid nanogel according to any one of claims 1 to 3 in the preparation of a medicament for treating liver disease; Preferably, the liver disease is alcoholic liver disease; More preferably, the alcoholic liver disease is selected from one or more of the following: chronic alcoholic liver disease, acute alcoholic liver disease, acute alcohol poisoning, preferably chronic alcoholic liver disease or acute alcoholic liver disease.

Images