Disclosure of Invention
In a first aspect, the invention provides a hydrogel, which consists of A, B polymers, wherein the main chains of A, B polymers are formed by forming an amide bond through polycondensation of lysine and glutamic acid, the value of n is 8-100, A, B polymers are polymers with arbitrary branching algebra respectively, and the structural formulas of A, B polymers are shown as a and b:
ra on the main chains of the A, B polymers is selected from (c) or (d), and the structural formulas are shown as (c) and (d):
Or alternatively
Wherein Ra1 is A, B, rb is A, B, rc is monosaccharide or oligosaccharide connected with Rb end of polymer A, rd is any phenylboronic acid structure connected with Rb end of polymer B.
Further, the functional molecule is selected from one or more of peptide fragments, fluorophores, antibacterial molecules, growth factors, functional oligosaccharides or polysaccharides.
Further, the peptide fragment is selected from cell-adhesive peptides, antibacterial peptides, hormone peptides, immunomodulatory peptides, and antioxidant peptides, and the functional oligosaccharide or polysaccharide may be one or more of glycosaminoglycan, fucan, alginate, hyaluronic acid, and chitosan.
Further, the Rb branched structure is a branching reaction that can be performed by A, B two polymers at the amino terminal of their side chains.
Further, the branching algebra is preferably 0 to 5 generations.
Further, the branching of the amino terminal is performed by branching with a compound containing an amino group to achieve a function of increasing the number of amino terminals.
Further, the branching method is selected from branching of compounds containing at least two amino groups and one carboxyl group or branching methods with polyamide-amine forming materials;
Further, the compound may be selected from one or more of lysine, diaminopropionic acid, diaminobutyric acid, diaminocaproic acid, diaminoheptanoic acid.
Further, the amino terminal of the branched structure of the polymer a may be linked to different kinds of mono-or oligosaccharides, respectively.
Further, the monosaccharide may be selected from one or more of mannose, glucose, galactose, fructose, xylose, fucose, gulose, idose, lyxose, mannuronic acid, glucuronic acid, galacturonic acid, guluronic acid, and the like, and derivatives thereof.
Further, the oligosaccharide may be one or more of any oligosaccharide.
Further, the amino terminal of the branched structure of the polymer B is connected with any phenylboronic acid structure, including one or more of phenylboronic acid half ester and carbonyl phenylboronic acid half ester.
Further, A, B polymers in the hydrogels are mixed in a 10% -20% solids solution.
Further, the proportion of A, B polymers in the hydrogel is 1-2:2-1.
Further, the strength of the hydrogel is 10Pa-100000Pa.
Further, the hydrogel has superior tissue adhesion, shear resistance, visible light transmittance, and/or biodegradability.
In a second aspect, the present invention provides the use of a hydrogel according to the first aspect for the preparation of a skin wound material.
Further, the skin wound refers to a wound of skin or mucous membrane caused by burn, mechanical injury or inflammation, and the mucous membrane comprises oral mucosa or nasal mucosa and the like.
Further, the trauma repair substance may be a drug or medical device.
In a third aspect, the present invention provides the use of a hydrogel according to the first aspect for the preparation of a suturing agent for conjunctival or corneal transplants.
Further, the use may be to improve the adhesion of conjunctival or corneal transplants in situ and closure of wounds.
Further, the application can also be used as a suture agent to replace a suture.
In a fourth aspect, the present invention provides a medical formulation comprising a hydrogel according to the first aspect of the invention.
Further, the preparation also contains preparations for diminishing inflammation, stopping bleeding and the like, pharmaceutical excipients and the like.
In a fifth aspect, the present invention provides a use of the hydrogel of the first aspect for preparing a bone repair material.
Further, the bone repair includes, but is not limited to, trauma, spinal, joint, orthopedic, and the like.
In a sixth aspect, the present invention provides a method for preparing the hydrogel according to the first aspect:
s1, preparing an intermediate product 1:
The starting materials Boc-Lys (Boc) -OH, H-Glc (OMe) -OMe.HCl undergo a condensation reaction under the action of reagent EDC, HOBT, DIPEA to give intermediate 1.
S2, preparing an intermediate product 2:
The intermediate product 1 undergoes deprotection reaction under the action of a reagent ethyl hydrogen chloride acetate solution to obtain an intermediate product 2.
S3, preparing an intermediate product 3:
The intermediate product 2 and raw material Boc-Lys (Boc) -OH undergo condensation reaction under the action of a reagent EDC, HOBT, DIPEA to obtain an intermediate product 3.
S4, preparation of intermediate products 4 and 5:
the intermediate product 1 or 3 is subjected to deprotection reaction under the action of lithium hydroxide reagent to obtain intermediate products 4 or 5 respectively.
S5, preparation of intermediate products 6, 7 and 8:
The raw material Boc-Glc-OH or the intermediate product 4 or 5 and the raw material pentafluorophenol are subjected to condensation reaction under the action of a reagent EDC to obtain intermediate products 6, 7 and 8 respectively.
S6, preparation of an intermediate product 9:
The raw material Boc-Lys (Boc) -OH and propargylamine are subjected to condensation reaction under the action of a reagent EDC, HOBT, DIPEA to obtain an intermediate product 9.
S7, preparation of intermediate 10 (Mb):
intermediate 9 undergoes deprotection under the action of ethyl hydrogen chloride solution to give intermediate 10 (Mb).
S8, preparation of intermediate 11 (Mc):
The raw materials Boc-aminoxyacetic acid and pentafluorophenol undergo condensation reaction under the action of a reagent EDC to obtain an intermediate product 11 (Mc).
S9, preparation of intermediate products 12, 13 and 14:
Intermediate 6 or 7 or 8 and intermediate 10 (Mb) are polymerized under the action of DIPEA reagent to give intermediates 12, 13, 14.
S10, preparation of intermediate products 15, 16 and 17:
intermediate 12 or 13 or 14 undergoes deprotection reaction under the action of trifluoroacetic acid reagent to give intermediates 15, 16, 17, respectively.
Preparation of the B component (products 18, 19, 20):
Intermediate 15, 16 or 17 and raw material Bob react under the action of sodium borohydride reagent to obtain product B components (products 18, 19 and 20) respectively.
S12, preparation of intermediate products 21, 22 and 23:
intermediate 15 or 16 or 17 and intermediate 11 (Mc) undergo a condensation reaction under the action of DIPEA reagent and further undergo a deprotection reaction under the action of trifluoroacetic acid reagent to give intermediates 21, 22, 23, respectively.
Preparation of component a (products 24, 25, 26):
the intermediate product 21 or 22 or 23 and the raw material monosaccharide or oligosaccharide undergo condensation reaction under the action of reagent aniline to respectively obtain a product A component (products 24, 25 and 26);
S14, synthesis of hydrogel
The solid content of the component A or the component B is 12-20wt%.
Detailed Description
EXAMPLE 1 Synthesis of hydrogel Pre-materials
In this example, the starting materials required for hydrogel synthesis include Boc-L-glutamic acid (Boc-Glc-OH), (S) -2, 6-di-tert-butoxycarbonylaminohexanoic acid (Boc-Lys (Boc) -OH), L-glutamic acid dimethyl ester hydrochloride (H-Glc (OMe) -OMe. HCl), pentafluorophenol, mannose (or different types of mono-or oligosaccharides), phenylboronic acid half ester (Bob), boc-aminooxyacetic acid, propargylamine, all commercially available.
The required reagents include 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), 1-Hydroxybenzotriazole (HOBT), N-Diisopropylethylamine (DIPEA), lithium hydroxide, ethyl hydrogen chloride acetate solution, sodium borohydride, trifluoroacetic acid, anhydrous diethyl ether, and aniline, all of which are commercially available.
1.1 Molecular Structure
1.1.1 Raw material molecular basic Structure
The structure diagram of the raw material A component and the raw material B component is shown in figure 1.
1.2 Molecular expansion Structure
The change in branching structure of 1.2.1A/B components is shown in FIG. 2.
1.2.2A component sugar changes
R=mannose (Man) Or r=glucose (Glc)
1.3 Preparation and characterization of Components A and B
1.3.1 Preparation of intermediates M0-a, M1-a, M2-a, mb, mc (see FIG. 3);
1. synthesis of Compound 1
(1) Boc-Lys (Boc) -OH, EDC, HOBT and H-Glc (OMe) -OMe.HCl were dissolved in dry dichloromethane.
(2) DIPEA or triethylamine is slowly added dropwise under ice water bath condition. The ratio of the amounts of starting materials was Boc-Lys (Boc) -OH: EDC: HOBT: H-Glc (OMe) -OMe.HCl: DIPEA or triethylamine=1:1:1:1:1.
(3) And removing the ice water bath, continuously stirring for 6-36 h, and washing with dilute hydrochloric acid, dilute sodium hydroxide and saturated saline water after the reaction is finished. After the organic phase was separated, the crude product was purified by silica gel column chromatography to give a white solid (see fig. 4).
2. Synthesis of Compound 2
(1) An excess of saturated ethyl hydrogen chloride acetate solution was slowly added dropwise to compound 1 under ice water bath conditions.
(2) The ice-water bath was removed and the reaction was stirred for 30min. During the reaction, a large amount of white precipitate was generated.
(3) After the reaction, a white solid was obtained by suction filtration, washed with ethyl acetate several times and dried under vacuum at room temperature (see fig. 5).
3. Synthesis of Compound 3
(1) Boc-Lys (Boc) -OH, EDC, HOBT and Compound 2 were dissolved in dry dichloromethane.
(2) Under the ice water bath condition, DIPEA or triethylamine is slowly added dropwise, and the ratio of the raw materials is that the compound 2:Boc-Lys (Boc) -OH is EDC:HOBT is DIPEA or triethylamine=1:2:2:2:2. The ice water bath was removed and the reaction was continued with stirring for 24h.
(3) After the completion of the reaction, the reaction mixture was washed with diluted hydrochloric acid, diluted sodium hydroxide and saturated brine, respectively. The organic phase was separated, dried over anhydrous magnesium sulfate, filtered and dried by spin. The crude product was purified by column chromatography on silica gel to finally obtain a white solid (see fig. 6).
4. Synthesis of Compound 5
(1) Compound 1 or 3 was dissolved in a mixed solvent of methanol/water=4:1.
(2) Under the ice water bath condition, slowly adding lithium hydroxide or sodium hydroxide or potassium hydroxide, wherein the mass ratio of the raw materials is that the compound 1 or3 is lithium hydroxide or sodium hydroxide or potassium hydroxide=1:2.
(3) The ice-water bath was removed and the reaction continued for 12h. Then, dilute hydrochloric acid was added to the reaction solution to adjust the pH to 5.
(4) After removing methanol from the system under reduced pressure, the product was extracted 3 times with ethyl acetate, and the organic phases were combined and dried by spin to finally obtain a white product (see fig. 7, 8).
5. Synthesis of Compounds 6 to 8 (Ma)
(1) Compound 4 or 5 or Boc-Glc-OH and EDC, respectively, were dissolved in dry dichloromethane and pentafluorophenol was slowly added under ice water bath.
(2) The ratio of the amounts of starting materials was compound 4 or 5 or Boc-Glc-OH: EDC: pentafluorophenol=1:2:2. After removing the ice water bath, stirring and reacting for 12h at normal temperature.
(3) After the reaction was completed, the excess solvent was distilled off under reduced pressure, and the crude product was separated by silica gel column chromatography to obtain a white solid (see FIGS. 9,10, 11).
6. Synthesis of Compound 9
(1) Boc-Lys (Boc) -OH, EDC, HOBT and propargylamine were dissolved in dry dichloromethane. DIPEA or triethylamine was slowly added dropwise under ice water bath.
(2) The ratio of the amounts of starting materials was Boc-Lys (Boc) -OH: EDC: HOBT: propargylamine: DIPEA or triethylamine=1:1:1:1:1. After removing the ice water bath, the reaction was continued for 24 hours.
(3) After the reaction, the system was washed with dilute hydrochloric acid and dilute sodium hydroxide solution, respectively, to separate out an organic phase. The crude product was purified by silica gel column chromatography to give the product ((see FIG. 12)).
7. Synthesis of Compound 10 (Mb)
(1) And cooling the compound 9 by using an ice water bath, slowly dropwise adding and dissolving excessive hydrogen chloride saturated ethyl acetate solution, removing the ice water bath, and continuing to react for 0.5-6 h.
(2) After the reaction, a white solid was obtained by suction filtration, washed with ethyl acetate several times and dried in vacuo to obtain the product (see fig. 13).
8. Synthesis of compound 11 (Mc):
(1) Boc-aminooxyacetic acid, EDC was dissolved in dry dichloromethane. Under ice water bath, pentafluorophenol was slowly added. The ratio of the amounts of the starting materials was Boc-aminoxyacetic acid to EDC to pentafluorophenol=1:1:1.
(2) After removing the ice water bath, stirring was continued for reaction for 24 hours. After the reaction was completed, the precipitate was filtered off, washed several times with methylene chloride, and dried in vacuo to give a white product (see fig. 14).
1.4P0, P1, P2, preparation of A component (A component comprising sugar molecule changed to glucose) (P0-Man (Glc), P1-Man (Glc), P2-Man (Glc)), B component (P0-Bob, P1-Bob, P2-Bob) (synthetic route see FIG. 15)
1.4.1 Synthesis of Compounds 12, 13, 14 (P0, P1, P2)
(1) Compound 6 or 7 or 8 and compound 10 were slowly dissolved in anhydrous DMSO or DMF. DIPEA or triethylamine was slowly added dropwise under ice water bath.
(2) The ratio of the amounts of the starting materials is compound 6 or 7 or 8, compound 10:dipea or triethylamine=1:1:2. After removing the ice water bath, stirring was continued for 48 hours.
(3) After the reaction, methanol or ethanol was added under ice water bath to dilute the solution, and then the product was precipitated in diethyl ether and centrifugally filtered. This procedure was repeated 3 times, and finally the precipitate was filtered off and dried in vacuo to give the product (see fig. 16).
1.4.2 Synthesis of Compounds 15, 16, 17
(1) Compound 12 or 13 or 14 was dissolved in excess trifluoroacetic acid under ice water bath conditions and stirring was continued for 4.5h after removal of the ice water bath.
(2) After the reaction, the solution was diluted with methanol, the product was precipitated in diethyl ether, and the product was filtered off, washed several times with diethyl ether and dried under vacuum to give a white product (see FIGS. 17,18, 19).
1.4.3 Synthesis of Compounds 19, 20 (P0-Bob, P1-Bob, P2-Bob)
(1) The flask was charged with compound 15 or 16 or 17, phenylboronic acid half ester and sodium borohydride and a vacuum was pulled.
(2) The ratio of the raw materials is that the compound 15 or 16 or 17 is phenylboronic acid half ester and sodium borohydride=1:1:4.
(3) Then slowly dropwise adding methanol under the ice water bath condition. The ice-water bath was then removed and the reaction stirred for an additional 24h.
(4) After the reaction, the mixture was dialyzed against deionized water using a dialysis bag having a molecular weight cut-off of less than 2000, and lyophilized to give a white solid (see FIGS. 20,21, 22).
1.4.4 Synthesis of Compounds 21, 22, 23
(1) Compound 15 or 16 or 17 and compound 11 (Mc) were dissolved in anhydrous DMSO or DMF.
(2) Under the ice water bath condition, DIPEA or triethylamine is slowly added dropwise. The ratio of the amounts of starting materials is compound 15 or 16 or 17: compound 11: dipea or triethylamine=1:1:2.
(3) After removing the ice water bath, stirring reaction is continued for 24 hours.
(4) After the reaction was completed, the solution was diluted with methanol under ice-water bath conditions, and the product was precipitated with diethyl ether. This procedure was repeated 3 times, and finally the precipitate was filtered off and the white solid was dried under vacuum at room temperature.
(5) The solid was then dissolved in excess trifluoroacetic acid under ice water bath conditions. The ice water bath was removed and stirring was continued for 4.5h.
(6) After the reaction, the reaction mixture was diluted with methanol in ice-water bath and the product was isolated by precipitation with diethyl ether. After filtration, the mixture was washed with diethyl ether several times and dried under vacuum at room temperature to give a white solid (see FIGS. 23,24, 25).
1.4.5 Synthesis of Compounds 24, 25, 26 (P0-Man (Glc), P1-Man (Glc), P2-Man (Glc) (synthetic route see FIG. 30)
(1) In an acetic acid/sodium acetate buffer solution with ph=4.2, compound 21 or 22 or 23, mannose (glucose) and aniline 10 to 100 μl are dissolved therein, and stirred for 6 to 24 hours at 37 ℃.
(2) The ratio of the amounts of the raw materials is that of the compound 21 or 22 or 23, mannose (glucose) =1:1.
(3) Dialyzing with dialysis bag with molecular weight cut-off less than 2000, and lyophilizing to obtain white solid (26, 27,28,29, 30). 1.4.6 Synthesis of Compound 27 (P0-Bob-1)
(1) Compound 15 (0.155 g,0.53 mmol) and Bob-1 (0.199g, 0.58 mmol) were dissolved in 1.5mL anhydrous DMF, cooled in an ice-water bath, and DIPEA (0.3 mL,1.63 mmol) was slowly added dropwise to the system, and the ice-water bath was removed and stirred for 6h. After the reaction was completed, the reaction system was diluted with 5mL of methanol and then precipitated with 250mL of diethyl ether. This step was repeated 3 times to substantially remove DIPEA. Finally, the product was dried in vacuo to give a white solid (0.197g, 82%).
1.4.7 Synthesis of Compound 28 (P1-Bob-1)
(1) Compound 16 (0.165 g,0.39 mmol) and Bob-1 (0, 295g,0.86 mmol) were dissolved in 1.5mL anhydrous DMSO, cooled in an ice-water bath, and DIPEA (0.3 mL,1.63 mmol) was slowly added dropwise to the system, and the ice-water bath was removed and stirred for 6h.
(2) After the reaction was completed, the reaction system was diluted with 5mL of methanol and then precipitated with 250mL of diethyl ether. This step is repeated 2 to 3 times to sufficiently remove the DIPEA.
(3) Finally, the product was dried in vacuo to give a white solid (0.219 g, 76%).
Synthesis of 1.4.8 Compound 29 (P2-Bob-1)
(1) Compound 17 (0.130 g,0.19 mmol) and Bob-1 (0.294 g,0.86 mmol) were dissolved in 1.5mL anhydrous DMF, cooled in an ice-water bath, and DIPEA (0.3 mL,1.63 mmol) was slowly added dropwise to the system, and the ice-water bath was removed and stirred for 6h.
(2) After the reaction was completed, the reaction system was diluted with 5mL of methanol and then precipitated with 250mL of diethyl ether. This step was repeated 3 times to substantially remove DIPEA. The product was finally dried in vacuo to give a white solid (0.182 g, 72%) (see fig. 31).
Synthesis of 1.4.9 Compound 30 (P2-Man-RGD)
(1) The flask was charged with compound 26, N3-GRGDSP peptide, DMF, cuprous bromide, PMDETA and deoxygenated.
(2) Heating to 50 ℃ and stirring to react for 24 hours.
(3) After the reaction is finished, dialyzing in deionized water by using a dialysis bag with the molecular weight cut-off of less than 2000, and freeze-drying to obtain white solid.
The white solid product P2-Man-RGD (FIG. 32).
1.5 Preparation and characterization of G0, G1, G2 with varying branching patterns (see FIG. 33)
1.5.1G1.0 Synthesis
(1) The polymer backbone G0 and monomer methyl acrylate MA are respectively dissolved in anhydrous methanol at room temperature, and then the methanol solution of G0 is slowly dripped into the methanol solution of MA, wherein the mass ratio of raw materials is G0 (n-NH 2): MA=1:30.
(2) Room temperature after the dripping is completed the reaction was stirred for 48h. After the reaction, the unreacted MA and the solvent methanol are dried by spin to obtain a product G0.5.
(3) Subsequently, G0.5 and ethylenediamine as reaction raw materials were dissolved in anhydrous methanol at room temperature, respectively, and then a methanol solution of G0.5 was slowly dropped into a methanol solution of ethylenediamine, the ratio of the amounts of the raw materials being G0.5 (n-COOR): ethylenediamine=1:50.
(4) Room temperature after the dripping is completed the reaction was stirred for 48h. After the reaction, unreacted ethylenediamine and methanol solvent were spin-dried to obtain a product G1.0 (see fig. 34, 35).
1.5.2G2.0 Synthesis
(1) The polymer G1.0 and the monomer methyl acrylate MA were dissolved in absolute methanol at room temperature, respectively, and then the methanol solution of G0 was slowly dropped into the methanol solution of MA, the ratio of the amounts of the raw materials being G1.0 (n-NH 2): ma=1:50. Room temperature after the dripping is completed the reaction was stirred for 48h.
(2) After the reaction was completed, unreacted MA and methanol solvent were spin-dried to obtain a product G1.5 (see fig. 36). Subsequently, G1.5 and ethylenediamine as reaction raw materials were dissolved in anhydrous methanol at room temperature, respectively, and then a methanol solution of G1.5 was slowly dropped into a methanol solution of ethylenediamine, the ratio of the amounts of the raw materials being G1.5 (n-COOR): ethylenediamine=1:50.
(3) Room temperature after the dripping is completed the reaction was stirred for 48h. After the reaction was completed, unreacted ethylenediamine and methanol solvent were spin-dried to obtain a product G2.0 (see fig. 37).
Example 2 hydrogel preparation and Properties
2.1 Gel preparation method
Firstly, preparing polymer solution of component A (Pn-Man, pn-Glc (n=0, 1, 2)), and component B (polymer solution of Pn-Bob (n=0, 1, 2): A, B, controlling the solid content to be 12-20wt%, respectively dissolving hyperbranched glycopeptide polymers (P0-Man, P1-Man and P2-Man) in pure water to obtain component A solution, respectively dissolving hyperbranched phenylboronic acid half-ester polypeptides (P0-Bob, P1-Bob and P2-Bob) in pure water, and regulating the pH to 8 to obtain component B solution, regulating the ratio of a donor/acceptor (sugar unit/phenylboronic acid unit) in component A, B solution to be 2:1, uniformly mixing the two solutions after the components are completely dissolved in a solvent, and standing to obtain a series of hyperbranched polypeptide hydrogel materials.
2.2 Performance test of gels
2.1.1 Hydrogel adhesion stability test
Pig skin was trimmed to 1cm x 1cm size, fixed with quick-drying adhesive to 1cm x 4cm size acrylic plates, and a total volume of 20 μl of hydrogel was applied between the two pig skins. After waiting 10 minutes for the gel to fully cure, the two ends of the spline were secured with clamps, the sample was pulled at a speed of 2mm/min using a ZP50 pulling machine until fully separated, and the maximum tissue adhesion strength reached before fully separating the sample was recorded.
The experimental results of the binding capacities of the two components show that the increase of the branching degree can effectively increase the number of the terminal functionalized sites, further increase the number of the binding sites, and finally further increase the molar binding enthalpy of the two components, namely better gel stability (see fig. 38a and 38 a), and the binding exotherms of P1-Glc+P1-Bob and P1-Man+P1-Bob are almost the same, so that the binding capacities are very close (see fig. 38 c).
2.1.2 Shear test
The gel provides effective adhesion in the 1:2 and 2:1 ratio ranges, has good tissue adhesion and shearing resistance, and is suitable for application in skin wound repair and conjunctival transplantation (see figure 39).
As can be seen from the rotameter test, the gel strength of A, B can be precisely controlled within the range of 10Pa to 100000Pa (see figures 40 and 41), and as can be seen from the rotameter test, the gel mechanical strength formed by P1-Glc+P1-Bob and P1-Man+P1-Bob is almost identical in the neutral state (see figure 42).
2.1.3 Torsion test-test of adhesion stability of hydrogels to pigskin in vitro under external force
The gel was first stained with markowski blue for convenient subsequent observation. And (3) dripping 20 mu L of A, B-component polymer solution on pigskin, waiting for 10 minutes until the solution is completely cured in situ to form hydrogel, applying different stresses such as torsion, folding, stretching and the like on the pigskin, observing whether the gel is stable in adhesion under the condition of being stressed, falling off and cracking, and taking a picture.
The gel coated on the surface of the pigskin can be stably attached to the surface of the pigskin under various deformation states without falling off and cracking. The gel has better adhesion stability (see fig. 43).
2.1.4 Adhesion stability-under water test gel adhesion stability test
The hydrogel was stained with markowski blue to facilitate subsequent observation. After in situ polymerization of the polymer solution droplets on pigskin to form hydrogel, the hydrogel was immersed in PBS buffer solution (ph=7.2), and the solution was changed daily until day 7. And taking out the sample after the soaking is finished, and observing whether the gel can be stably adhered to the surface of the pigskin after the soaking in water for a long time and the adhesion stability under water flow flushing.
The gel can still stably adhere to the tissue surface after being soaked in water for a long time. The gel has better adhesion stability (see fig. 44).
2.1.5 Light transmittance test
The light transmittance test results preliminarily showed that the visible light transmittance of the 0+0 formulation was close to that of carbomer, better than that of the 1+1 formulation, and that the 0+0 flatness was inferior to that of carbomer in practical imaging, and that small wrinkles were easily generated, so that the imaging permeability was slightly inferior Yu Kabo mu gel (see fig. 45), wherein the 0+0 formulation was a formulation of the a-component (Pn-Man, pn-Glc (n=0)) and B-component (Pn-Bob (n=0)), the 1+1 formulation was a formulation of the a-component (Pn-Man, pn-Glc (n=1)) and B-component (Pn-Bob (n=1)), and the 0+1 formulation was a formulation of the a-component (Pn-Man, pn-Glc (n=1)) and B-component (Pn-Bob (n=0)).
2.1.6 In vitro degradation experiments
The gel was shown to have a certain stability in a wet environment and medium-long-term degradability (see fig. 46).
2.1.7 Gel for autologous conjunctival transplantation test
The method comprises the steps of preparing a defect area with the size of 1cm multiplied by 1cm of a bilateral symmetry conjunctiva on a pig eyeball, injecting 12 mu L of hydrogel into the defect area of the conjunctiva in an experimental group, adhering the conjunctiva after waiting for 1min, and naturally covering the conjunctiva in a control group. The displacement areas of the conjunctival flaps were photographed at 0,10, 20, 30, 40min, respectively, and analyzed with Image J. Further, the same conjunctival defect as above was produced, the experimental group was adhered with the conjunctival flap hydrogel, the control group was covered with the conjunctival flap naturally, the conjunctival graft site was rinsed with a continuous water flow after the completion of the graft, and the condition of whether the graft conjunctival flap had fallen off was observed to judge the stability of the adhesion.
The 0+0 and 1+1 gel formulations have good adhesion and can effectively fix conjunctiva and sclera, and the rinsing experiments show that the 0+0 and 1+1 gel formulations have good adhesion and can enable the transplanted conjunctiva tissue to be adhered in situ and not washed away by water flow (see figure 47).
Biomechanical test of 2.1.8 hydrogel-enclosed in vitro porcine eyeball cornea incision
According to the cornea incision closure method, the pig eyes are randomly divided into 4 groups, namely, a suture group, gel0+0 (A component (Pn-Man, pn-Glc (n=0)) and B component (Pn-Bob (n=0))), gel1+1 (A component (Pn-Man, pn-Glc (n=1)) and B component (Pn-Bob (n=1)), a fibrin group, a cornea flap is removed by using a trephine under an operation microscope and placed in an artificial anterior chamber, a microinjection pump is connected to the water inlet end of the artificial anterior chamber, and a differential pressure gauge is connected to the water outlet end, after the artificial anterior chamber is filled to 21mmHg (normal eye pressure value), a linear incision of 2.8mm is made at the near-angle scleral margin by using a cornea main incision knife, a hydrogel of experimental group, a commercial fibrin glue or a suture suturing method is used, the microinjection pump is opened to fill the artificial anterior chamber with liquid, the pumping rate is 10mm/h, the peak reading of the video is recorded, and when the differential pressure reading is changed, whether the liquid leakage occurs or not is observed.
The results show that the 0+0 and 1+1 formulas have less leakage than the suture group and the commercial Fibrin, have better sealing effect and can tolerate the intraocular pressure of more than 200 mmHg. Suitable for closure of corneal wounds (see figure 48).
Example 3 biological experiments on gels
3.1 Biosafety test
SD rats were injected intradermally with 100. Mu.l of P1 Bob+P1Man100. Mu.l prepared in example 1 and sampled for 2 weeks, 1 month, 2 months and 3 months of injection for pathological sections.
The results showed no significant differences between the experimental group and normal skin, and no significant inflammatory reaction was induced, demonstrating good biocompatibility of the gel (see fig. 49).
Cell safety tests showed that the material did not affect cell activity and normal proliferation of cells (see figure 50).
3.2 Autologous conjunctival transplantation of rabbits
The suture, 0+0 (A-component (Pn-Man, pn-Glc (n=0)) and B-component (Pn-Bob (n=0)), 1+1 (A-component (Pn-Man, pn-Glc (n=1)) and B-component (Pn-Bob (n=1)), and commercial materials were compared, respectively.
The results primarily show that the gel group plays a role in transplanting, and tissues at the transplanted positions of the gel group have a restoration trend, so that conjunctival morphology multi-layered squamous epithelium and a small number of goblet cells are seen, and the epithelium regeneration effect is good, equivalent to that of a suture group, and better than that of commercial Fibrin (see figure 51).
Example 4 animal model for cornea injury repair
A single-plane linear main incision of 2.8mm is made on the near-angle scleral edge of the rabbit eye. Nylon suture stitching or gel closure was then performed separately per group.
The monitoring result of the intraocular pressure shows that the intraocular pressure is basically restored to normal intraocular pressure after all groups are adhered to the incision for 3 days, and the treatment means of all groups can effectively seal the incision to avoid anterior chamber leakage.
Anterior ocular segment optical coherence tomography showed that Gel 1+1 (a-component (Pn-Man, pn-Glc (n=1)) and B-component (Pn-Bob (n=1)) groups had more pronounced decrease in internal and external incision thickness at 3-7 days compared to the other groups, which could promote early healing of corneal tissue and edema regression earlier.
The slicing result shows that the Gel1+1 group has the best repairing effect, the tissue structure at the incision is continuous, and the arrangement rule structure of the collagen fibers of the cornea stroma layer is normal.
Overall, gel 1+1 effectively closes the corneal incision, favors early corneal tissue repair and edema regression, and favors correct morphological distribution of the post-operative cornea with best repair effect (see fig. 52).
EXAMPLE 5 bone defect repair animal model
SD rats were placed on the left femoral condyle and a dental drill was used to create 3mm diameter and 3mm deep bone defects. Four groups of rats received the following implants to repair bone defects:
(1) A pathological group, namely performing debridement treatment only on the defect;
(2) Gel1+1 (A-component (Pn-Man, pn-Glc (n=1)) and B-component (Pn-Bob (n=1)) groups, implantation of 40ul of the 1+1 Gel complex of the population at the defect;
(3) Gel2+2 (A-component (Pn-Man, pn-Glc (n=2)) and B-component (Pn-Bob (n=2)) groups, implantation of a total system of 40ul of 2+2 Gel complexes at the defect;
(4) The 2+2RGD group ((A-component (Pn-Man-RGD (n=2)) and B-component (Pn-Bob (n=2))) was implanted with 40ul of the 2+2RGD gel complex of the total system at the defect.
At week 4, rat left femoral condyles were taken and examined using Micro-CT.
CT reconstruction results show (see FIG. 53) that GEL2+2 and GEL2+2RGD treated groups significantly promote bone tissue defect repair relative to the blank and GEL1+1 groups.
The above description is a general description of the application. Variations in form and value may be substituted for the purpose of illustration and not limitation, as the terms are used herein, depending on the circumstances or actual requirements. Various changes and modifications may be made by one skilled in the art, and such equivalents are intended to fall within the scope of the application as defined in the following claims.