Hyaluronic acid - polyethylene glycol medical hydrogel

Abstract

The present invention discloses a hyaluronic acid - polyethylene glycol hydrogel. By preparing a hyaluronic acid - polyethylene glycol double - network hydrogel from hyaluronic acid, polyethylene glycol derivatives and their cross - linker, the stability and mechanical properties of hyaluronic acid are significantly improved, and it can be used clinically as a tissue filler, tissue adhesion preventer, tissue engineering scaffold, sealant or embolization agent.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to a hyaluronic acid-polyethylene glycol medical hydrogel, which can be widely used in clinical practice as a tissue filler, a tissue adhesion preventer, a tissue engineering scaffold, a sealant or an embolization agent. Background Art

[0002] Hyaluronic acid is a main component of connective tissues such as human extracellular matrix, vitreous humor of the eye, and synovial fluid of joints, and plays important physiological functions in the body such as water retention, maintaining the extracellular space, regulating osmotic pressure, lubrication, and promoting cell repair. At higher concentrations, due to the complex three-dimensional network structure formed by intermolecular interactions, its aqueous solution has significant viscoelasticity. As a main component of the extracellular matrix, hyaluronic acid directly participates in the regulation of electrolyte exchange inside and outside cells, and plays the role of a filter for physical and molecular information. Hyaluronic acid has unique physical and chemical properties and physiological functions, and has been widely used in medicine. Existing hyaluronic acid can be directly injected into the body as a soft tissue filler or a joint lubricant, etc., or can be cross-linked by a small molecule cross-linking agent (Langmuir, 2004, 20, 5153-5156) and then injected into the body. However, hyaluronic acid and cross-linked hyaluronic acid are prone to enzymatic hydrolysis in the body, and their stability is relatively low, usually requiring multiple injections. In addition, the mechanical properties of hyaluronic acid gels are relatively low and cannot be used for filling harder tissues including cartilage.

[0003] Polyethylene glycol is a class of water-soluble synthetic polymers and has great application value in the biomedical field. This benefits from its several unique advantages, including good biocompatibility, easy chemical modification, and good controllability of properties, etc. Chinese patent document CN109646723A discloses a medical hydrogel, which forms a gel by using a multi-arm polyethylene glycol capped with aldehyde groups and a mixture of polyethyleneimine and polylysine. Polyethylene glycol hydrogels have good mechanical strength. At the same time, due to the in-situ gelation method, the hydrogel can be conveniently injected into the body through a dual syringe. The in-situ gelation method of PEG hydrogels has the following advantages compared with the pre-gelled method: 1. The hydrogel formed by in-situ gelation has better biocompatibility; 2. The precursor of the in-situ gelled hydrogel is liquid and can be evenly distributed around the target; 3. The in-situ gelation method will not introduce a foreign body sensation in the body. Summary of the Invention

[0004] Aiming at the disadvantages of existing hyaluronic acid gels in the prior art, the present invention provides a hyaluronic acid-polyethylene glycol medical hydrogel, which has good stability and mechanical properties and can be used in clinical practice as a tissue filler, a tissue adhesion preventer, a tissue engineering scaffold, a sealant or an embolization agent.

[0005] The specific technical solution of the present invention is as follows:

[0006] A hyaluronic acid - polyethylene glycol hydrogel is formed by mixing hyaluronic acid with aldehyde - terminated star - shaped multi - arm polyethylene glycol and then in - situ cross - linking with polyethyleneimine and polylysine. The hyaluronic acid is embedded in the three - dimensional network formed by the in - situ cross - linking of polyethylene glycol, polyethyleneimine and polylysine.

[0007] Preferably, in the mixed solution of hyaluronic acid and aldehyde - terminated star - shaped multi - arm polyethylene glycol, the concentration of hyaluronic acid is 0.5% - 5% (w / v), the molecular weight of hyaluronic acid is 50000 - 2500000 Da, and the concentration of aldehyde - terminated star - shaped multi - arm polyethylene glycol is 2 - 30%.

[0008] More preferably, in the mixed solution of hyaluronic acid and aldehyde - terminated star - shaped multi - arm polyethylene glycol, the concentration of hyaluronic acid is 1% - 5% (w / v), the molecular weight of hyaluronic acid is 50000 - 2500000 Da, and the concentration of aldehyde - terminated star - shaped multi - arm polyethylene glycol is 15 - 30%.

[0009] The aldehyde group and the star - shaped multi - arm polyethylene glycol are connected by chemical bonds such as ester bonds, ether bonds, amide bonds, urethane bonds, imine bonds or urea bonds, preferably connected by amide bonds, ester bonds or ether bonds.

[0010] Preferably, the number of arms of the star - shaped multi - arm polyethylene glycol is 2 - 8, more preferably 4 - 8, and the molecular weight of a single arm is 1000 - 5000 Da.

[0011] Preferably, the molar ratio of the aldehyde group of the aldehyde - terminated star - shaped multi - arm polyethylene glycol to the amino groups in polylysine and polyethyleneimine is 1:0.2 - 5:0.2 - 5.

[0012] The aldehyde group is preferably one or more of aromatic aldehyde groups and alkyl aldehyde groups.

[0013] Another object of the present invention is to provide the application of the hydrogel of the present invention in the preparation of tissue fillers, tissue adhesion preventives, tissue engineering scaffolds, sealants or embolization agents. It can be used as a temporary tissue filler or a permanent implant in clinical applications.

[0014] Another object of the present invention is to provide a preparation method of the hydrogel of the present invention. Dissolve aldehyde - terminated star - shaped multi - arm polyethylene glycol and hyaluronic acid in a pH 4 - 10 buffer solution to prepare an aldehyde - terminated star - shaped multi - arm polyethylene glycol - hyaluronic acid solution, where the concentration of hyaluronic acid is 0.5% - 5% and the concentration of aldehyde - terminated star - shaped multi - arm polyethylene glycol is 2 - 30%; dissolve polyethyleneimine and polylysine in a pH 4 - 10 buffer solution, with the concentration of polyethyleneimine and polylysine being 0.5 - 20%, to prepare a solution; mix the two to obtain the hyaluronic acid - polyethylene glycol hydrogel.

[0015] When the present invention is specifically applied, a two-component hydrogel is first prepared. The first component is hyaluronic acid and aldehyde-capped star-shaped multi-arm polyethylene glycol, preferably 8-arm, 6-arm, and 4-arm polyethylene glycol (the molecular weight of a single arm is 1000-5000 Da), and the aldehyde group is one or more of aromatic aldehydes and alkyl aldehydes, preferably benzaldehyde group. The aldehyde group and the polymer can be connected by stable or hydrolyzable chemical bonds. The second component is a mixed component including polylysine (including ε-polylysine and polylysine) and polyethyleneimine.

[0016] The two components of the hydrogel can be stored separately in a double-barrel syringe. When in use, the two components are ejected through a mixing head or injected into a designated site to form a gel.

[0017] The present invention utilizes the reaction between the aldehyde group at the end of multi-arm polyethylene glycol and the amino group of a multi-amino compound to generate a Schiff base, thereby producing cross-linking and forming a medical injectable gel.

[0018] Advantages of the present invention:

[0019] By preparing a hyaluronic acid-polyethylene glycol double-network hydrogel from hyaluronic acid, polyethylene glycol derivatives and their cross-linking agents, the stability and mechanical properties of hyaluronic acid are significantly improved. Further, the gelation time, degradation time, mechanical properties and swelling ratio of the system can be adjusted by adjusting the contents of polyethylene glycol and hyaluronic acid, or by selecting polyethylene glycol and hyaluronic acid with different molecular weights, and by adjusting the molar ratio of the aldehyde group of aldehyde-capped star-shaped multi-arm polyethylene glycol to the amino group in polylysine and polyethyleneimine to meet different clinical needs. The viscosity of the hydrogel precursor solution can also be adjusted by adjusting the concentration of hyaluronic acid to ensure that the precursor solution does not leak from the target tissue. Specific embodiments

[0020] The following illustrates the specific steps of the present invention through examples, but is not limited by the examples.

[0021] The terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art unless otherwise specified.

[0022] The present invention will be further described in detail below with reference to specific examples and data. It should be understood that these examples are only for illustrating the present invention and do not limit the scope of the present invention in any way.

[0023] In the following examples, various processes and methods not described in detail are conventional methods well known in the art.

[0024] Example 1 Preparation of hyaluronic acid-polyethylene glycol hydrogel

[0025] Dissolve 3 g of 8-armed phenylaldehyde-terminated polyethylene glycol in 20 mL of 0.02 M phosphate buffer solution (pH 5.6) to obtain Solution A; prepare a phosphate buffer solution (pH 8.9) containing polylysine (the molar ratio of the aldehyde group of polyethylene glycol to the amino group of polylysine is 1:1) and polyethyleneimine (M.W. 1.8 kDa) (the molar ratio of the aldehyde group of polyethylene glycol to the amino group of polyethyleneimine is 1:1) as Solution B. Separately dissolve 0.2 g of high molecular weight hyaluronic acid (molecular weight 1.8 million) into Solution A or Solution B, and mix Solutions A and B through a double syringe to observe the gel formation. The results are shown in Table 1.

[0026] Table 1

[0027]

[0028] The experimental results show that when hyaluronic acid is dissolved in polyethylene glycol solution and mixed with the cross-linking agent solution, a gel can be formed in a short time; while when hyaluronic acid is dissolved in cross-linking agent Solution B, a white precipitate will form and no gel can be formed. Compared with the polyethylene glycol hydrogel without hyaluronic acid, the addition of hyaluronic acid will slow down the gelation rate.

[0029] Example 2

[0030] Dissolve 3 g of 8-armed phenylaldehyde-terminated polyethylene glycol and 0.2 g of high molecular weight hyaluronic acid (molecular weight 1.8 million Da) in 20 mL of 0.02 M phosphate buffer solution (pH 5.6) to obtain Solution A; prepare a phosphate buffer solution (pH 8.9) containing polylysine (the molar ratio of the aldehyde group of polyethylene glycol to the amino group of polylysine is 1:1) and polyethyleneimine (M.W. 1.8 kDa) (the molar ratio of the aldehyde group of polyethylene glycol to the amino group of polyethyleneimine is 1:1) as Solution B. Mix Solutions A and B through a double syringe to form a gel.

[0031] Prepare a hyaluronic acid gel without polyethylene glycol: dissolve 0.2 g of high molecular weight hyaluronic acid (molecular weight 1.8 million Da) in 20 mL of 0.02 M phosphate buffer solution.

[0032] Place the hydrogel in 10 times its volume of PBS and test the stability of the hydrogel at 37 degrees, that is, the time required for the hydrogel to completely degrade in vitro. The results are shown in Table 2.

[0033] Table 2

[0034]

[0035] The above experimental results confirm that the stability of a single hyaluronic acid solution is poor, and it is completely degraded within 24 hours, and no residual hyaluronic acid gel is found. The composite gel formed by hyaluronic acid and polyethylene glycol has significantly higher stability. The degradation time of the gel can be regulated by adjusting the molecular weight of polyethylene glycol or the connecting bond between the aldehyde group and polyethylene glycol to meet different clinical needs. It is suitable for temporary tissue filling or permanent implants.

[0036] Example 3 Regulation of the mechanical properties of hyaluronic acid-polyethylene glycol hydrogel

[0037] Dissolve 8-arm phenyl aldehyde-terminated polyethylene glycol and high molecular weight hyaluronic acid (molecular weight 1.8 million Da) in a 0.02 M phosphate buffer solution (pH 5.6) with a concentration of 20 mL as solution A; prepare a phosphate buffer solution (pH 8.9) containing polylysine and polyethyleneimine as solution B. Mix solutions A and B through a dual syringe to obtain the corresponding hydrogel. Measure the compressive modulus of the hydrogel, and the results are shown in Table 3.

[0038] Table 3

[0039]

[0040]

[0041] The experimental results show that the hyaluronic acid-polyethylene glycol hydrogel has a high compressive modulus. The mechanical strength of the hydrogel can be easily adjusted by regulating the molecular weight, concentration of polyethylene glycol, and the ratio of polyethylene glycol to the cross-linking agent; compared with the polyethylene glycol hydrogel, the addition of hyaluronic acid will reduce the compressive modulus of the hydrogel. The possible reason is that hyaluronic acid will interfere with the formation of the polyethylene glycol hydrogel network. The elastic modulus of most tissues in the human body is between a few kilopascals and a few hundred kilopascals, which has a good modulus matching with the hyaluronic acid-polyethylene glycol hydrogel.

[0042] Example 4 Regulation of the swelling ratio of hyaluronic acid-polyethylene glycol hydrogel

[0043] Dissolve a certain mass of 8-arm phenyl aldehyde-terminated polyethylene glycol connected by an ester bond and 0.2 g of high molecular weight hyaluronic acid (molecular weight 1.8 million) in a 0.02 M phosphate buffer solution (pH 5.6) with a concentration of 20 mL as solution A; prepare a phosphate buffer solution (pH 8.9) containing polylysine (the molar ratio of the aldehyde group of polyethylene glycol to the amino group of polylysine is 1:1) and polyethyleneimine (M.W. 1.8 KDa) (the molar ratio of the aldehyde group of polyethylene glycol to the amino group of polyethyleneimine is 1:1) as solution B. Mix solutions A and B through a dual syringe to obtain the corresponding hydrogel. Measure the swelling ratio of the hydrogel after 24 hours at 37 degrees. The results are shown in Table 4.

[0044] Table 4

[0045]

[0046] The experimental results show that the swelling ratio of the hyaluronic acid-polyethylene glycol hydrogel can be adjusted by the molecular weight and concentration of polyethylene glycol. The higher the molecular weight and concentration of polyethylene glycol, the higher the corresponding swelling ratio. Compared with the polyethylene glycol hydrogel without hyaluronic acid, the addition of hyaluronic acid will increase the swelling ratio of the hydrogel.

[0047] Example 5 Viscosity Regulation of Hyaluronic Acid-Polyethylene Glycol Hydrogel Prepolymer Solution

[0048] In-situ injection hydrogels require that the hydrogel prepolymer solution can remain at the target position before gelation, so as to control the physical shape of the hydrogel and avoid gel leakage to other parts. In this example, a certain mass of 8-arm benzaldehyde-capped polyethylene glycol linked by an ester bond and hyaluronic acid were dissolved in a 0.02 M phosphate buffer solution (pH 5.6) with a concentration of 20 mL, and the viscosity of the solution was measured using a viscometer. The results are shown in Table 5.

[0049] Table 5

[0050]

[0051] The results show that compared with the polyethylene glycol solution, the addition of hyaluronic acid significantly increases the viscosity of the solution. The higher the molecular weight and concentration of hyaluronic acid, the higher the corresponding viscosity.

Claims

1. A hyaluronic acid - polyethylene glycol hydrogel, characterized in that it is formed by in - situ cross - linking of hyaluronic acid and aldehyde - terminated star - shaped multi - arm polyethylene glycol with polyethyleneimine and polylysine. The concentration of hyaluronic acid in the mixed solution of hyaluronic acid and aldehyde - terminated star - shaped multi - arm polyethylene glycol is 0.5% - 5% w / v, the molecular weight of hyaluronic acid is 50000 - 2500000 Da, the concentration of aldehyde - terminated star - shaped multi - arm polyethylene glycol is 2 - 30% w / v, the number of arms of the star - shaped multi - arm polyethylene glycol is 2 - 8, the molecular weight of a single arm is 1000 - 5000 Da, and the molar ratio of the aldehyde group of the aldehyde - terminated star - shaped multi - arm polyethylene glycol to the amino groups in polylysine and polyethyleneimine is 1:0.2 - 5:0.2 - 5.

2. The hydrogel according to claim 1, characterized in that the aldehyde group and the star - shaped multi - arm polyethylene glycol are chemically connected by an ester bond, an ether bond, an amide bond, a urethane bond, an imine bond or a urea bond.

3. The hydrogel according to claim 2, characterized in that the aldehyde group and the star - shaped multi - arm polyethylene glycol are connected by an amide bond, an ester bond or an ether bond.

4. The hydrogel according to claim 1, characterized in that the number of arms of the star - shaped multi - arm polyethylene glycol is 4 - 8.

5. The hydrogel according to claim 1, characterized in that the aldehyde group is selected from one or more of aromatic aldehyde groups and alkyl aldehyde groups.

6. Use of the hydrogel tissue according to any one of claims 1 - 5 in the preparation of a tissue filler, a tissue adhesion preventer, a tissue engineering scaffold, a sealant or an embolization agent.

7. The preparation method of the hydrogel according to any one of claims 1 - 5, characterized in that dissolve aldehyde - terminated star - shaped multi - arm polyethylene glycol and hyaluronic acid in a pH 4 - 10 buffer solution to prepare an aldehyde - terminated star - shaped multi - arm polyethylene glycol - hyaluronic acid solution, where the concentration of hyaluronic acid is 0.5% - 5% w / v and the concentration of aldehyde - terminated star - shaped multi - arm polyethylene glycol is 2 - 30% w / v; dissolve polyethyleneimine and polylysine in a pH 4 - 10 buffer solution, with the concentration of polyethyleneimine and polylysine being 0.5 - 20%, to prepare a solution; mix the two to obtain the hyaluronic acid - polyethylene glycol hydrogel.

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