CN109627463B

CN109627463B - Hydrogel and protein drug

Info

Publication number: CN109627463B
Application number: CN201811565784.7A
Authority: CN
Inventors: 唐键
Original assignee: Shenzhen Puluo Meikang Materials Co ltd
Current assignee: Shenzhen Puluo Meikang Materials Co ltd
Priority date: 2016-06-22
Filing date: 2016-06-29
Publication date: 2022-04-01
Anticipated expiration: 2036-06-29
Also published as: CN106046398A; CN109627463A

Abstract

The invention provides a hydrogel and a protein drug. The hydrogel comprises the following raw materials in parts by weight: 1-50 parts of chitosan, 0.1-20 parts of cross-linking agent and 10-100 parts of water; the cross-linking agent is prepared by combining a non-degradable raw material A and a degradable raw material B to form a block copolymer and then connecting a raw material C to the tail end of the block copolymer. The hydrogel is injectable, coatable and degradable in vivo, can well maintain the biological activity of medicinal protein, and has no toxic or side effect on the hydrogel and degradation products thereof. The protein drug adopts the hydrogel to carry and disperse the drug protein, and can well maintain the biological activity and the functional stability of the drug protein.

Description

Hydrogel and protein drug

Technical Field

The invention relates to the technical field of biomedical materials, in particular to a hydrogel and a protein drug.

Background

The therapeutic role of proteins has long been recognized, for example in the 19 th century for the preparation of antisera for diphtheria; insulin was isolated from the pancreas of cattle and swine in the early 20 th century to treat diabetes and the like. However, these human-or animal-derived proteins have limitations in terms of applications, such as limited yields, cross-viral infections, immune responses in patients, and the like. Therefore, before the birth of gene recombination technology, protein medicines cannot enter clinical application on a large scale. In 1982, insulin prepared by using gene recombination technology is used as a recombinant protein drug which is approved by the FDA in the United states and is on the market for the first time, and the door for large-scale entry of therapeutic proteins into the clinic is opened. To date, over a hundred protein drugs have entered clinical use, the vast majority of which are recombinant proteins.

However, the protein has a remarkable weakness that its three-dimensional structure is fragile and susceptible to physical and chemical factors in the environment, resulting in being destructured or degraded to reduce or even completely lose biological activity. The protein drugs can be classified into the following categories according to their functions: a therapeutic protein that modulates activity using the enzymatic activity or signaling pathway of the protein; therapeutic proteins with specific targeting activity; a protein vaccine; proteins for diagnostic use, and the like. The first two basic therapeutic proteins are administered clinically by intravenous injection, and strict and complicated measures such as low temperature are required to avoid deterioration and inactivation of the proteins during storage and transportation.

With the continuous expansion and deepening of the application field of therapeutic protein, a way other than intravenous injection needs to be developed to utilize protein drugs; moreover, for some protein drugs that are cleared quickly in the blood circulation, frequent injections not only increase the economic burden on the patient, but also severely reduce the quality of life of the patient. Therefore, the development of new formulations of protein drugs, such as in vivo slow release, slow release at ulcer and wound sites, or application to skin care and the like, can expand the application scope of the protein and improve the use efficiency of the protein; meanwhile, the compliance of patients can be improved, and the medication cost can be reduced, so that the development of various sustained-release formulations of protein drugs is an effective demand in the field of medical treatment. Research and development of sustained-release formulations of some proteins have been reported, and some have even entered clinical use.

In order to prolong the blood circulation clearing period of protein, the currently adopted main technical means is a micro-nano drug release carrier, typically liposome or phospholipid micro-micelle wraps and carries protein, and the administration route is mainly intravenous injection. The defects are that the liposome micro-nano drug release carrier has limited drug loading capacity to protein drugs, and long-term intravenous injection can cause blood fat accumulation.

In addition, it has been reported that collagen, chitosan, inorganic or synthetic polymer materials such as PLA, PLGA, etc. are used to prepare protein carriers for micro-nano particles, but the steps of organic solvents, high-speed stirring, heating, etc. used in the preparation process are easy to destroy the structure of protein, resulting in protein inactivation. And the cost of the sustained-release medicine is increased sharply due to the fact that a large amount of medicine is lost due to an overlong preparation process, the medicine-loading rate is limited and the like.

Secondly, the fusion protein modified by polyethylene glycol (PEG) utilizes long chains or multi-branched chains of PEG with large molecular weight to cover the enzyme cutting sites of the protein, thereby avoiding the rapid degradation of the protein in blood. The challenge lies in the proper choice of protein binding sites, as well as the homogeneity, stability and effectiveness of the fusion protein.

In addition, tissue repair in the field of regenerative medicine may consider using a scaffold carrier containing a protein factor that promotes cell growth to guide tissue regeneration repair by sustained release of the protein. Common carrier materials include collagen of animal origin, natural polymeric materials such as chitosan, cellulose, alginic acid, polyphosphates; synthetic polymer materials such as PLGA, PLA, etc. The prepared bracket is a mixture of one or more of the materials. The preparation process of the scaffold carrier also uses a plurality of steps such as organic solution, acid-base environment, high salt, violent stirring and the like, for example, collagen needs acid dissolution; inorganic materials need stirring, high pressure, sintering and the like; the synthetic polymer needs to be dissolved in an organic solvent, and high salt is added. These steps are all prone to disrupt the higher order structure of the protein, leading to inactivation of the protein. If the scaffold is mainly prepared from degradable synthetic polymers, the degraded polymers have more acid accumulation, and the side effects of damaging the structure of protein, causing tissue inflammation and the like can be caused.

The hydrogel carrier has the advantages of direct injection, convenient use, no need of implantation operation, free filling of pathological changes or defects of tissues with hydrogel, and no need of cutting according to actual shape. Injectable hydrogels currently reported, one type is a high viscosity sol that does not undergo a crosslinking process, such as a solution or mixture of collagen, chitosan, cellulose or PLGA, etc.; the other is temperature-sensitive physical hydrogel, the basic material of which contains chemically synthesized multi-block PEG, PLGA, PLA and other high molecules or needs thermal polymerization conditions (Novartis, WO2015/092690A1), and the temperature-sensitive physical hydrogel is characterized by being in a sol state at low temperature and normal temperature; injecting into body, and converting into gel state after body temperature is raised; still another type is crosslinking of natural polymer materials such as chitosan using highly toxic crosslinking agents such as glyoxal, glutaraldehyde, etc. (JP 2012-092137, a). The disadvantages include that the non-crosslinked sol is rapidly eliminated in vivo; the block of the temperature-sensitive physical hydrogel contains a large amount of synthetic macromolecules, and the potential risk of acid accumulation also exists in degradation products; the highly toxic cross-linking agent is not easy to be completely removed, and has the potential safety hazards of carcinogenesis and pathogenicity.

In summary, there is still a contradiction between improving the efficiency or convenience of protein drug use and maintaining the biological activity of protein. The technology of the invention aims to solve the problem, and the developed protein sustained-release gel can fully maintain the biological activity of the drug protein and can also be used for the sustained release of the polypeptide and a delivery carrier of therapeutic cells.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: providing an injectable, coatable, in vivo degradable hydrogel; the hydrogel can well maintain the biological activity of the drug protein, and the hydrogel and the degradation product thereof have no toxic or side effect.

In order to meet the technical requirements, the invention provides a hydrogel which can be crosslinked under mild conditions and has good biocompatibility, the viscosity and the degradation rate of the hydrogel can be adjusted, and the hydrogel can be used as a drug carrier for in vivo injection and body surface coating.

The hydrogel provided by the invention can be crosslinked into gel at 0-60 ℃ under mild conditions, namely in an aqueous solution with the pH value within the range of 4.5-9.0.

The hydrogel provided by the invention can be crosslinked into hydrogel without adding an additional chemical catalyst or extreme conditions such as photocatalysis, thermocatalysis, acid-base catalysis and the like.

The hydrogel provided by the invention is suitable for being used as carriers of various water-soluble biological macromolecules and micromolecular medicines, particularly protein therapeutic and cosmetic active substances.

The hydrogel provided by the invention is composed of two basic components, namely water-soluble chitosan and a cross-linking agent. The molecular weight of the chitosan is between 50,000 and 500,000 Da. The chitosan or its derivative and the crosslinking agent can be prepared from a weak acid, a weak base or a neutral aqueous solution such as pure water, physiological saline, a cell culture solution, a phosphate buffer solution and the like.

The cross-linking agent in the hydrogel provided by the invention is a block copolymer, and comprises a non-degradable segment (A) and a degradable segment (B), wherein the composition sequence is AB, ABA and BAB, 1-5 parts of A and 0.1-5 parts of B. The non-degradation section selects polyethylene glycol (PEG) with the molecular weight of 300-20000Da and the content of 50-90 wt%; the degradable segment is degradable polymer selected from one or more of polyglycolide, polylactide, polyglycolide lactide and polycaprolactone, and the content is 50-10 wt%. After A, B segments are combined, an active aldehyde group is connected at the terminal end, and the aldehyde group is selected from one or more of glyoxylic acid, uronic acid and aldehyde benzoic acid.

The hydrogel generally comprises 1-50 parts of chitosan, 0.1-20 parts of cross-linking agent and 10-100 parts of water; the crosslinking time is adjustable within the range of 1-100 minutes; the viscosity can be adjusted within the range of 0.1-10.0 Pa.s.

In summary, the invention provides a hydrogel, which comprises the following raw materials in parts by weight: 1-50 parts of chitosan, 0.1-20 parts of cross-linking agent and 10-100 parts of water, wherein the cross-linking agent is prepared from the following raw materials in parts by weight: 1-5 parts of non-degradable raw material A and 0.1-5 parts of degradable raw material B, wherein the raw material A is selected from polyethylene glycol, and the raw material B is degradable polymer and can be selected from one or more of polyglycolide, polylactide, polyglycolide-lactide and polycaprolactone; the combination sequence of the raw materials A and B can be AB, ABA and BAB; the raw material C which is jointed at the terminal ends of the A and B blocks is one or more selected from glyoxylic acid, uronic acid and aldehyde benzoic acid; the molecular weight ratio of A and B is 1: 0.1-1.

Further, the chitosan is one or more of chitosan with deacetylation degree of 50% -80%, chitosan oligosaccharide, chitosan sulfate, carboxymethyl chitosan and hydroxyethyl chitosan.

Further, the molecular weight of the polyethylene glycol is 300-; the average molecular weight of the polylactide is 300-; the average molecular weight of the polyglycolide lactide is 3000-20000 Da; the average molecular weight of the polycaprolactone is 3000-20000Da, and the ratio of the polylactide to the polyglycolide in the polyglycolide lactide is 50: 50, 65: 35, 75: 25 or 85: 15.

The invention provides a preparation method of hydrogel, which comprises the following steps:

step 1, placing polyethylene glycol with two ends both provided with hydroxyl groups at 160 ℃ at 120 ℃ under the protection of nitrogen, adding a degradable polymer and a catalyst I, reacting for 6-8h, collecting insoluble substances, and drying at 20-35 ℃ to constant weight to obtain a solid A; or dissolving polyethylene glycol in DCM or THF at room temperature, adding a catalyst II and a degradable polymer, reacting for 5-60min, precipitating with diethyl ether, collecting the product, dialyzing, purifying, and freeze-drying to obtain a solid B, wherein the degradable polymer is one or more selected from polyglycolide, polylactide, polyglycolide-lactide and polycaprolactone, the molecular weight ratio of the polyethylene glycol to the degradable polymer is 1: 0.1-1, the catalyst I is stannous octoate, and the catalyst II is DBU;

step 2, dissolving the solid A or the solid B and a raw material C in an organic solvent according to a molar ratio of 1: 2-10, adding a catalyst III, reacting for 24-48h, and concentrating to obtain a solid D, namely the cross-linking agent, wherein the raw material C is selected from one or more of glyoxylic acid, uronic acid and aldehyde benzoic acid, the organic solvent is ethanol, DCM or THF, and the catalyst III is DMAP, EDC or DCC;

and 3, respectively dissolving the chitosan and the cross-linking agent obtained in the step 2 in water and mixing, wherein the weight ratio of the chitosan to the cross-linking agent to the water is 1-50: 0.1-20: 10-100 during mixing, so that the hydrogel is obtained.

Further, the uronic acid is D-glucuronic acid, D-galacturonic acid or D-mannuronic acid, and the aldehyde benzoic acid is o-aldehyde benzoic acid, m-aldehyde benzoic acid or p-aldehyde benzoic acid.

Further, the hydrogel has a viscosity of 0.1 to 10.0Pa · s.

The present invention also provides another method for preparing the above hydrogel, comprising the steps of:

step 1, placing polyethylene glycol with one end sealed by methoxy group and one end sealed by hydroxyl group into a catalyst I and a degradable polymer at 160 ℃ under the protection of nitrogen, reacting for 6-8h, collecting insoluble substances, and drying at 20-35 ℃ to constant weight to obtain a solid A; or dissolving polyethylene glycol in an organic solvent at room temperature, adding a catalyst II and a degradable polymer, reacting for 5-60min, precipitating with diethyl ether, collecting a product, dialyzing, purifying, and freeze-drying to obtain a solid product B, wherein the degradable polymer is one or more selected from polyglycolide, polylactide, polyglycolide-lactide and polycaprolactone, the molecular weight ratio of the polyethylene glycol to the degradable polymer is 1: 0.1-1, the catalyst I is stannous octoate, the catalyst II is DBU, and the organic solvent is DCM or THF;

step 2, dissolving the solid A or the solid B in sulfur ethanol, adding a catalyst III, reacting at room temperature for 6-24h, concentrating and collecting a product C; or dissolving the solid A or the solid B in DCM, adding a catalyst IV, refluxing for 2-12h at 100-;

step 3, dissolving the product C or D in an organic solvent, adding the raw material E and the catalyst V in a molar ratio of 1: 2-10, reacting for 24-48h, and concentrating to obtain a solid F, namely the cross-linking agent, wherein the organic solvent is ethanol, DCM or THF; the raw material E is glyoxylic acid, uronic acid or aldehyde benzoic acid; the catalyst V is DMAP, EDC or DCC.

And 4, respectively dissolving the chitosan and the cross-linking agent obtained in the step 3 in water and mixing, wherein the weight ratio of the chitosan to the cross-linking agent to the water is 1-50: 0.1-20: 10-100 during mixing, so that the hydrogel is obtained.

Further, the hydrogel has a viscosity of 0.1 to 10.0Pa · s.

The hydrogel prepared by the preparation method can be used for injecting or implanting carriers of drugs or bioactive substances into functional organs and tissue parts such as joints, muscles, subcutaneous tissues, brains and the like, and the degradation time in vivo can be adjusted within the range of 1-180 days; can also be applied to body surface ulcer, wound, eye, nose, face, etc.

The hydrogel of the invention has drug loading:

(1) for drugs that are aqueous, the maximum drug loading is a saturated aqueous solution.

(2) The water-insoluble or low water-soluble drug and the solid drug can be directly stirred to mix the hydrogel.

The hydrogel of the invention is prepared by the following specific preparation process:

(1) preparing chitosan or its water-soluble derivative solution with weak acid, water or water solution with neutral or physiological pH value, and the concentration is 0.1-10.0%.

(2) The cross-linking agent is prepared by water or aqueous solution with the same pH value, and the concentration is 0.1-25.0%.

(3) According to actual requirements, taking a proper amount of chitosan solution, adding the medicine according to the requirements, uniformly mixing, adding the cross-linking agent solution according to the requirements, lightly stirring, uniformly mixing, standing or rotating, and crosslinking to form the gel within 1-100 minutes in an environment with the temperature of 0-60 ℃.

The invention has the advantages that:

(1) the hydrogel provided by the invention has wider application range than temperature-sensitive hydrogel, and can be used in vivo and on body surface;

(2) the hydrogel provided by the invention can be injected by using needles with different numbers before sol crosslinking, so that micro-injection is facilitated;

(3) the hydrogel provided by the invention has adjustable viscosity and degradation time, and further can adjust the degradation rate and the drug release rate of the hydrogel in a wider range;

(4) the hydrogel provided by the invention has mild gelling conditions, and is beneficial to preserving the activity of bioactive substances; the storage time and storage conditions after freeze-drying are loose, and the freeze-dried powder can be transported at normal temperature, so that the cost in the processes of storage, transportation and use is reduced;

(5) the main component of the hydrogel provided by the invention is chitosan or derivatives thereof, and the chitosan has a certain antibacterial and bacteriostatic effect, so the hydrogel naturally has a certain antibacterial effect.

Further analysis shows that the main material of the hydrogel is chitosan which is a natural source, and the hydrogel is widely applied to the fields of medical dressings, cosmetics and the like at present. The other important component is a cross-linking agent, wherein the polyethylene glycol is also widely applied to the fields of medicines, biochemical reagents, cosmetics and the like; the selected degradable polymer is a medical biomaterial approved by governments of all major countries; the selected aldehyde group substance has low toxicity or no toxicity, and the toxicity is completely eliminated after being combined on a polymer chain, and the cytotoxicity test verifies the good biocompatibility of the cross-linking agent. Finally, the content of degradable materials and aldehyde groups in the gel system is extremely low, so that the biological safety of the hydrogel is ensured again.

The preparation method of the hydrogel is simple and convenient, namely aqueous solutions of chitosan and the cross-linking agent are respectively prepared, and the chitosan and the cross-linking agent are spontaneously cross-linked at low temperature, normal temperature or body temperature to obtain the gel after being mixed according to a required proportion before use. The gel can be automatically crosslinked in a physiological environment, so that the structure and the biological activity of the drug protein can be protected to the maximum extent.

In conclusion, the beneficial effects of the invention are as follows: the chitosan has no biological toxicity, and the cross-linking agent also has no biological toxicity; the chitosan and the cross-linking agent are spontaneously cross-linked in a mild aqueous solution, which is particularly beneficial to protect the biological activity of the protein; the cross-linking agent can be degraded, so that the hydrogel network structure is disintegrated; the degradation products can be metabolized, so that the degradation products do not need to be taken out after a secondary operation; the viscosity and degradation rate of the hydrogel can be adjusted according to the ratio of chitosan to the cross-linking agent in the gel; before the monomer is crosslinked, needles with different numbers can be selected to complete in vivo injection; the hydrogel is in an indefinite form, and can be applied to the body surface after carrying a drug.

Detailed Description

In order to explain the technical content, the objects and the effects of the present invention in detail, the following description will be given with reference to the embodiments.

The most key concept of the invention is as follows: the reaction product of polyethylene glycol and degradable polymer is reacted with glyoxylic acid, uronic acid or aldehyde benzoic acid, and the obtained cross-linking agent and chitosan spontaneously form hydrogel in water, wherein the hydrogel is automatically cross-linked under physiological conditions, is degradable, safe and nontoxic.

Based on the above concept, the invention provides a hydrogel which can be crosslinked under mild conditions and has good biocompatibility, the viscosity and degradation rate of the hydrogel can be adjusted, and the hydrogel can be used as a drug carrier for in vivo injection and body surface coating.

Further, the hydrogel has a viscosity of 0.1 to 10.0Pa · s.

The hydrogel of the invention has drug loading:

The invention has the advantages that:

Example 1: synthesis of Cross-linker 1

0.5g mPEG (5000) was dissolved in 1mL DCM, 10mg DBU was added; 0.8g Lactide (LA) was dissolved in 5mL DCM; transferring the LA solution into the mPEG solution, and stirring for 10 min; precipitation with diethyl ether gave product I. Dissolving the product I in 60mL of sulfur ethanol, and adding equimolar AlCl3 for reaction for 24 hours; extraction with DCM and drying gave product II. Dissolving the product II in 100mL of DCM, then adding 5 times of aldehyde benzoic acid and EDC in molar weight and 0.5 times of DMAP in molar weight, and reacting for 24 hours; the product III is precipitated by ether, namely the cross-linking agent 1. The number average and weight average molecular weights (Mn, Mw) of crosslinker 1 were 10619 and 10848, respectively, and the polydispersity index (Mw/Mn) was 1.02, as determined by GPC.

Example 2: synthesis of Cross-linker 2

0.5g mPEG (5000) was dissolved in 1mL DCM, 10mg DBU was added; 0.75g Lactide (LA) and 0.3g Glycolide (GA) were dissolved in 5mL DCM; transferring LA/GA solution into mPEG solution, and stirring for 10 min; precipitation with diethyl ether gave product I. Dissolving the product I in 60mL of sulfur ethanol, and adding equimolar AlCl3 for reaction for 24 hours; extraction with DCM and drying gave product II. Dissolving the product II in 100mL of DCM, then adding 5 times of aldehyde benzoic acid and EDC in molar weight and 0.5 times of DMAP in molar weight, and reacting for 24 hours; the product III, namely the cross-linking agent 2, is precipitated by ether. The number average and weight average molecular weights (Mn, Mw) of crosslinker 2 were 9820 and 10017, respectively, and the polydispersity index (Mw/Mn) was 1.03, as determined by GPC.

Example 3: synthesis of Cross-linker 3

0.5g PEG (5000) was dissolved in 1mL DCM, 10mg DBU was added; 0.05g LA in 5mL DCM; transferring the LA solution into the mPEG solution, and stirring for 10 min; precipitation with diethyl ether gave product I. Dissolving the product I in 60mL of sulfur ethanol, and adding equimolar AlCl3 for reaction for 24 hours; extraction with DCM and drying gave product II. Dissolving the product II in 100mL of DCM, then adding 5 times of aldehyde benzoic acid and EDC in molar weight and 0.5 times of DMAP in molar weight, and reacting for 24 hours; the product III, namely the cross-linking agent 3, is precipitated by ether. The crosslinking agent 3 was found by GPC to have a number average and weight average molecular weight (Mn, Mw) of 5403 and 5587, respectively, and a polydispersity index (Mw/Mn) of 1.03.

Example 4: synthesis of Cross-linker 4

Vacuumizing polyethylene glycol at 120 ℃ to remove water, placing the dehydrated polyethylene glycol at 140 ℃ under the protection of nitrogen, adding polyglycolide and stannous octoate with the mass fraction of 0.2%, reacting for 6 hours, collecting insoluble substances, and drying at 20 ℃ to constant weight to obtain a product I, wherein the molecular weight ratio of the polyethylene glycol to the polyglycolide is 1: 0.1, and the mass ratio of the polyethylene glycol to the polyglycolide is 8: 5;

and respectively dissolving the product I and glyoxylic acid in an organic solvent according to the weight ratio of 4: 3, mixing, reacting for 14 hours, and concentrating to obtain a product II, namely the cross-linking agent 4.

Example 5: synthesis of Cross-linker 5

A method for preparing a hydrogel, comprising the steps of:

step 1, carrying out vacuum pumping treatment on polyethylene glycol at 120 ℃ to remove moisture, placing the dehydrated polyethylene glycol at 160 ℃ under the protection of nitrogen, adding polylactide and stannous octoate with the mass fraction of 0.2%, reacting for 8 hours, collecting insoluble substances, and drying at 35 ℃ to constant weight to obtain a product I, wherein the molecular weight ratio of the polyethylene glycol to the polylactide is 1: 1.5, and the mass ratio of the polyethylene glycol to the polylactide is 5: 2;

respectively dissolving the product I and uronic acid in an organic solvent according to the weight ratio of 5: 2, mixing, reacting for 16h, and concentrating to obtain a product II, namely the cross-linking agent 5.

Example 6: synthesis of Cross-linker 6

Vacuumizing polyethylene glycol at 120 ℃ to remove water, placing the dehydrated polyethylene glycol at 150 ℃ under the protection of nitrogen, adding polyglycolide-lactide and stannous octoate with the mass fraction of 0.2%, reacting for 7 hours, collecting insoluble substances, and drying at 28 ℃ to constant weight to obtain a product I, wherein the molecular weight ratio of the polyethylene glycol to the polyglycolide-lactide is 1: 0.8, and the mass ratio of the polyethylene glycol to the polyglycolide-lactide is 2: 1; and respectively dissolving the product I and p-aldehyde benzoic acid in an organic solvent according to the weight ratio of 9:5, mixing, reacting for 15 hours, and concentrating to obtain a product II, namely the cross-linking agent 6.

Example 7: synthesis of Cross-linker 7

Dissolving 1g PEG (2000) in DCM, adding 10mg DBU, adding 0.5g polylactide glycolide, and stirring for 10 min; the product I is precipitated by ethyl ether, and the mass ratio of the polyethylene glycol to the polylactide is 2: 1. Dissolving the product I and p-aldehyde benzoic acid in an organic solvent according to the weight ratio of 9:5, reacting for 24h, and concentrating to obtain a product II, namely a cross-linking agent 7.

Example 8: hydrogel preparation

Taking 2g carboxymethyl chitosan solution in 100mL water; 0.025g of crosslinker was dissolved in 1mL of water. Adding 480 microliters of water and 20 microliters of cross-linking agent solution into 500 microliters of chitosan solution, gently stirring, standing at room temperature, and gelling after 5 min.

Example 9: preparation of protein-containing hydrogels

1g of bovine serum albumin was dissolved in 10mL of phosphate buffer. Adding 100 microliters of albumin solution, 380 microliters of water and 20 microliters of cross-linking agent solution into 500 microliters of chitosan solution, gently stirring, standing at room temperature, and gelling after 5 min.

In summary, the hydrogel prepared by the preparation method of the hydrogel provided by the invention has the following beneficial effects: the hydrogel can bring good effects in the aspects of slow release of protein and polypeptide drugs, delivery carriers of stem cells and the like: in the preparation of protein drug sustained-release preparations, the maintenance of the activity of the protein to the maximum extent and the saving of the use of the protein are the primary concerns, because this is related to the feasibility and economy of protein drug sustained-release preparations. The hydrogel prepared by the technology can better solve the key problems, and can basically ensure that the activity of the protein is not damaged under the mild gel condition and the protein is not lost in the preparation process. Secondly, the hydrogel can be injected in vivo by using needles with different calibers, and is convenient to use. Finally, the hydrogel can be coated on the body surface after carrying drugs, so that the application range is expanded; the hydrogel is mild and has good biocompatibility, and is an excellent carrier for delivering therapeutic stem cells to a defective tissue part in regenerative medicine. The cells and the hydrogel can be simply mixed and then injected to the lesion position, and the use is simple and efficient; the hydrogel can also be expanded to the application fields of ointment, skin care and the like. The hydrogel is a good carrier for nursing gel, sticking film and the like which use high-efficiency and expensive protein to promote ulcer and wound healing, remove wrinkles and clean skin, and can effectively protect the activity of the protein. Due to the slow release and degradability of the hydrogel, the use amount of protein is saved, and the hydrogel is particularly beneficial to protein drugs with low yield and expensive unit price.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent modifications made by the present invention in the specification or directly or indirectly applied to the related technical field are included in the scope of the present invention.

Claims

1. A method of making a hydrogel, comprising:

placing the non-degradable raw material A at 160 ℃ at 120 ℃ under the protection of nitrogen, adding the degradable raw material B and a first catalyst, reacting for 6-8h, collecting insoluble substances, and drying at 20-35 ℃ to constant weight to obtain a block copolymer; or dissolving the non-degradable raw material A in a first organic solvent at room temperature, adding the degradable raw material B and a second catalyst, reacting for 5-60min, precipitating with diethyl ether, collecting the product, dialyzing, purifying, and freeze-drying to obtain a block copolymer;

when the non-degradable raw material A is polyethylene glycol, dissolving the block copolymer and the raw material C in a molar ratio of 1 (2-10) in a second organic solvent, adding a third catalyst, reacting for 24-48h, and concentrating to obtain a cross-linking agent;

when the non-degradable raw material A is polyethylene glycol monomethyl ether, dissolving the block copolymer in sulfur ethanol, adding a fourth catalyst, reacting at room temperature for 6-24h, concentrating and collecting a first intermediate; or dissolving the block copolymer in DCM, adding a fifth catalyst, refluxing at 100-130 ℃ for 2-12h, and concentrating and collecting a second intermediate; dissolving the first intermediate or the second intermediate in a second organic solvent, adding a raw material C and a third catalyst, reacting for 24-48h, and concentrating to obtain a cross-linking agent;

respectively dissolving chitosan and the cross-linking agent in water and mixing, wherein the weight ratio of the chitosan to the cross-linking agent to the water is (1-50) to (0.1-20) to (10-100) to obtain hydrogel;

the non-degradable raw material A is polyethylene glycol or polyethylene glycol monomethyl ether;

the degradable raw material B is a degradable polymer, and the degradable polymer is selected from one or more of polyglycolide, polylactide, polyglycolide-lactide and polycaprolactone;

the raw material C is selected from one or more of glyoxylic acid, uronic acid and aldehyde benzoic acid;

the combination sequence of the non-degradable raw material A and the degradable raw material B comprises one or more of AB, ABA and BAB;

the weight ratio of the non-degradable raw material A to the degradable raw material B is (1-5): (0.1-5);

the molecular weight ratio of the non-degradable raw material A to the degradable raw material B is 1: 0.1-1;

the first catalyst is stannous octoate;

the first organic solvent is DCM or THF;

the second catalyst is DBU;

the second organic solvent is ethanol, DCM or THF;

the third catalyst is DMAP, EDC or DCC;

the fourth catalyst is AlCl₃；

The fifth catalyst is HI.

2. The method for preparing the hydrogel according to claim 1, wherein the chitosan is one or more of chitosan with a deacetylation degree of 50-100%, chitosan oligosaccharide, chitosan sulfate, carboxymethyl chitosan, hydroxyethyl chitosan.

3. The method for preparing a hydrogel according to claim 1, wherein the molecular weight of said polyethylene glycol is 300-.

4. The method for preparing a hydrogel according to claim 1, wherein the uronic acid is D-glucuronic acid, D-galacturonic acid or D-mannuronic acid, and the aldehyde benzoic acid is o-, m-or p-aldehyde benzoic acid.

5. The method for producing a hydrogel according to claim 1, wherein the hydrogel has a viscosity of 0.1 to 10.0 Pa-s.

6. The method for preparing a hydrogel according to claim 1, wherein the molecular weight of the polyethylene glycol is 300-; the molecular weight of the chitosan is between 50,000-500,000 Da.

7. A protein drug comprising the hydrogel produced by the method for producing a hydrogel according to any one of claims 1 to 6 and a drug protein dispersed in the hydrogel.