Transdermal photocurable hydrogel having biological activity, method for preparing same, and use thereof

Abstract

The present invention relates to a transdermal photocurable hydrogel having biological activity, its preparation method and use. The hydrogel can release a recombinant collagen having biological activity, and the recombinant collagen comprises a sequence shown in SEQ ID No.1, and the amino acid sequence of the recombinant collagen comprises N basic repeating units, and the basic repeating units comprise n1 of the following characteristic amino acid sequence: "G-Xaa1-Xaa2-GE-Xaa3", and the 3'-end and 5'-end of the basic repeating units are linked to form the characteristic amino acid sequence. The recombinant collagen provided by the present invention has significant integrin binding activity and has the effect of promoting cell adhesion, proliferation and differentiation. The hydrogel of the present invention has good biocompatibility, stable quality, can be solidified in situ by transdermal photocrosslinking after injection, is easy to operate and controllable, meets the clinical requirements for filling irregular defects, and has a better soft tissue filling effect.

Description

[Technical field]

[0001] This invention claims priority to (1) Application No. 2022112245930, filed with the China Patent Office on October 8, 2022, and (2) Application No. 2023103463184, filed with the China Patent Office on April 3, 2023. The entire contents of the prior applications are incorporated herein by reference.

[0002] The present invention relates to the technical field of biotechnology, specifically to a transdermal photocurable moldable hydrogel with biological activity, its preparation method and use. [Background technology]

[0003] The maxillofacial soft tissue has a special anatomical and physiological structure, plays an important role in the chewing, pronunciation, aesthetics, etc. of the human body, and is susceptible to trauma, infection, congenital diseases, etc. Abnormalities and deficiencies in the morphology and volume of the maxillofacial soft tissues are often seen in clinical practice, causing facial hollows, asymmetry, aging, etc., which not only bring functional problems to patients, but also seriously affect the image and psychological health of patients, causing great inconvenience to their lives. Current clinical repair methods are mainly autologous soft tissue transplantation repair, which causes secondary trauma and is unfavorable to orthopedics. In recent years, the demand for cosmetic repair has increased, and the key to maxillofacial soft tissue repair is to restore the external shape of the defective tissue as much as possible while restoring the normal function of the defective tissue, and to functionally reconstruct the defective tissue to achieve a good cosmetic repair effect.

[0004] Soft tissue filling is the main item of non-surgical cosmetic treatment, which achieves the purpose of tissue repair, deformity correction, and juvenile cosmetic treatment by implanting soft tissue filling materials into the body to occupy and replace the cavities and defects caused by tissue damage or lesions, and perform or enhance the original functions. It is the patient's demand for minimally invasive surgical techniques, and is also one of the current trends in the development of clinical techniques in plastic surgery. Non-invasive facial filling using injectable soft tissue filling materials has become the most popular operation method, and the injection filling technique is easy to operate, only takes a few minutes of injection to achieve immediate results, is painless, does not affect work and life, is very convenient, leaves no traces, is highly confidential, and can protect the privacy of customers. However, the ideal injectable soft tissue filler must simultaneously possess biocompatibility, safety, ease of handling, fixation, and durability, which has traditionally been a very challenging task for scholars who research and develop cosmetic cosmetic products.

[0005] The clinical repair effect of soft tissue defects is closely related to the source and performance of materials. In order to realize the functional reconstruction and aesthetic reconstruction of defective tissues, not only is it required that the injectable filling material has good biocompatibility and immediate filling effect, but also that the graft material has good tissue repair and regeneration function, wide source, and easy operation. At present, injectable soft tissue fillers such as hyaluronic acid and carboxymethylcellulose have been widely developed and applied, but they lack various biological signal molecules necessary for cell adhesion, proliferation and differentiation, and have limited ability to resist their own enzymatic degradation and free radical degradation, making it difficult to regulate cell behavior and promote tissue repair. For example, cross-linked hyaluronic acid and its derivatives, as physical volume fillers, are not favorable for cell adhesion, have limited ability to induce tissue regeneration, have a fast metabolic rate, and have limited effect maintenance. In addition, cross-linked hyaluronic acid-based products are formed intraoperatively by the surgeon after implantation, which places high demands on the surgeon's anatomical theoretical knowledge and operation skills, and at the same time, the filler may cause complications such as displacement, which has a great impact on the satisfaction of transplantation.

[0006] Therefore, conventional injectable materials have problems such as insufficient mechanical strength, short duration of injection effect, poor biocompatibility with human tissue, and unnatural post-injection effects, making it difficult to meet the requirements of actual application environments. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention has been made in consideration of the disadvantages of the prior art, and aims to provide a bioactive transdermal photocurable hydrogel which has excellent biocompatibility, can be cured in situ after injection by a transdermal photocrosslinking method, can achieve the purpose of tissue filling while releasing biologically active recombinant collagen, and has the effect of promoting tissue repair, as well as a method for preparing and using the same. [Means for solving the problem]

[0008] In order to achieve the above object, the present invention employs the following technical aspects. In a first aspect, the present invention provides a transdermal photocurable moldable hydrogel having biological activity, the synthesis raw materials of the hydrogel include an auxiliary acid anhydride, a natural polysaccharide material, an activator, an amino orthodiol, and an oxidizing agent.

[0009] The amino acid sequence of the recombinant collagen includes N basic repeating units, and the basic repeating units include n1 of the following characteristic amino acid sequence: "G-Xaa1-Xaa2-GE-Xaa3," and the 3' end and 5' end of the basic repeating units are linked to form the characteristic amino acid sequence.

[0010] Here, N is an integer of 4 or greater, and n1 is an integer of 3 or greater.

[0011] Furthermore, the value of N is an integer from 4 to 300.

[0012] Furthermore, the N value is an integer between 4 and 200, including but not limited to 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30......200.

[0013] Furthermore, the N value is such that the molecular weight of the recombinant collagen is 1 to 200 kDa, and further 3 to 150 kDa.

[0014] Furthermore, the characteristic amino acid sequences are arranged consecutively or at intervals in the basic repeat unit.

[0015] Furthermore, the amino acid sequence of the recombinant collagen exhibits the following characteristics: [-GE-Xaa3-Yaa1-(G-Xaa1-Xaa2-GE-Xaa3) n2 -Yaa2-(G-Xaa1-Xaa2-GE-Xaa3) n3 -Yaa3-(G-Xaa1-Xaa2-GE-Xaa3) n3 - Yaa4-(G-Xaa1-Xaa2-GE-Xaa3) n4 -…………-Yaa n -(G-Xaa1-Xaa2-GE-Xaa3) n -Yaa n+1 -G-Xaa1-Xaa2-] N

[0016] wherein Xaa1 is a non-polar hydrophobic amino acid, Xaa2 is one of serine (S), alanine (A), proline (P), or hydroxyproline (O), and Xaa3 is a basic amino acid.

[0017] Yaa1, Yaa2, Yaa3, Yaa4, ......, Yaa n , Yaa n+1 is independently selected from absent, one or more different or the same amino acid at each occurrence.

[0018] n2, n3, n4, ………, n are integers greater than or equal to 0, and both cannot be 0 at the same time.

[0019] Preferably, the recombinant collagen sequence does not include a protein tag.

[0020] Preferably, the amino acid sequence of said recombinant collagen is SEQ ID NO.1.

[0021] Preferably, the acid anhydride is one of cyclic unsaturated acid anhydrides or carboxyl group-containing unsaturated acid anhydrides or 4-pentenoic anhydride, crotonic anhydride, and methacrylic anhydride. Furthermore, the anhydride is one of maleic anhydride, citraconic anhydride, cis-3-carboxyglutaconic anhydride (cis-aconitic anhydride), 4-pentenoic anhydride, crotonic anhydride, and methacrylic anhydride. Furthermore, the anhydride is methacrylic anhydride.

[0022] Preferably, the natural polysaccharide material is selected from hyaluronic acid, carboxymethylcellulose, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, alginic acid, dextran, agarose, heparin, chondroitin sulfate, ethylene glycol chitosan, propylene glycol chitosan, chitosan lactate, carboxymethyl chitosan, or chitosan quaternary ammonium salt. Preferably, the natural polysaccharide material is carboxymethylcellulose.

[0023] Preferably, the activating agent is NHS and EDC. Furthermore, the amino ortho-diol is 3-amino-1,2 propanediol, and the side chain group containing the ortho-diol is specifically oxidized to generate an aldehyde group, thereby effectively avoiding the destruction of the main chain structure of natural polysaccharides.

[0024] In the present invention, EDC means 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and NHS means N-hydroxysuccinimide.

[0025] Furthermore, the oxidizing agent is selected from periodate and hypochlorite, and preferably, the oxidizing agent is sodium periodate.

[0026] In a second aspect, the present invention provides a method for preparing a transdermal photocurable moldable hydrogel having biological activity, said hydrogel being obtainable by transdermal photocuring.

[0027] Furthermore, the specific steps are as follows: S1. Natural polysaccharide substances are appropriately chemically modified with acid anhydrides to obtain natural polysaccharide derivatives with photocrosslinking properties. S2. The natural polysaccharide derivative having photocrosslinking ability is mixed with an activator to react with each other, and the pH value of the reaction solution is 4.65 to 5.2. S3. Add amino ortho-diol to the reaction system of S2 to carry out a side chain carboxyl activation reaction, purify by dialysis, and after completion of dialysis, freeze-dry to obtain a natural polysaccharide derivative containing ortho-diol side chains. S4. The ortho-diol side chain-containing natural polysaccharide derivative is dissolved, reacted with an oxidizing agent, and purified by dialysis. After the dialysis is completed, the natural polysaccharide derivative having aldehyde side chains is obtained by freeze-drying. S5. After dissolving the aldehyde side chain-containing natural polysaccharide derivative, mix it with the recombinant collagen and a photoinitiator to obtain a repair matrix precursor liquid, and then irradiate it with transdermal light to crosslink it to obtain a hydrogel.

[0028] The present invention adjusts the composition and structure of natural polysaccharide substances through moderate chemical modification and rational blending of materials, and allows the chemically modified natural polysaccharide substances to contain photoinducible groups, which has the effect of enabling transdermal photoirradiation molding through subcutaneous injection into the body, and its mechanical and degradable properties are also significantly improved. Such a special structure can also support cell survival and proliferation, and the final tissue filler (hydrogel) has good thermal stability, mechanical strength, and anti-enzymatic degradation properties, and has a longer retention time in the body.

[0029] In the field of soft tissue filling, carboxymethylcellulose, like other polysaccharide-based polymer compounds, lacks cell adhesion sites, making it difficult for cells to adhere to and spread on the hydrogel, and therefore it is difficult for cells to migrate and proliferate in the hydrogel, and they can only proliferate, aggregate and grow in situ. The present invention utilizes the biological safety, high adhesive activity and biological effect of recombinant collagen, which is favorable for cell adhesion and growth on the composite hydrogel, and the recombinant collagen is continuously released with the degradation of carboxymethylcellulose, which regulates the biological behavior of cells and induces the expression of extracellular matrix, thereby achieving the repair effect of maintaining the initial morphology and inducing the regeneration of soft tissue matrix in the later stage.

[0030] The hydrogels of the present invention can be used to improve defects in soft tissue contour and can be applied to fill the dermis layer (e.g., mid and deep layers), shallow to deep subcutaneous layers, and to restore depressed areas of the skin, such as wrinkles, scars, depressed defects or lesions.

[0031] Preferably, in step S1, the degree of photocrosslinking modification of the natural polysaccharide substance is 30 to 60%. The molar ratio of the natural polysaccharide derivative having photocrosslinking ability to NHS and EDC in the activator is 1:(1-3):(2-5).

[0032] Furthermore, in the step S2, the molar ratio of the natural polysaccharide derivative having photocrosslinking ability:NHS:EDC is 1:2:3.

[0033] Preferably, the pH of the solution to which the amino ortho-diol is added in step S3 to carry out the activation reaction is 7 to 7.7, the molar ratio of amino ortho-diol:natural polysaccharide derivative having photocrosslinking ability is (1 to 10):1, the dialysis time in steps S3 and S4 is 20 to 72 hours, and the molecular weight of the dialyzed fraction is 8,000 to 14,000 kDa.

[0034] The natural polysaccharide derivative of the present invention contains an aldehyde group, and can be bound to recombinant collagen through a Schiff base reaction and released gradually in the hydrogel. Preferably, in step S5, the mass / volume ratio of the natural polysaccharide derivative containing an aldehyde side chain is 1-5%, the mass / volume ratio of the recombinant collagen is 0.5-2%, and the mass / volume ratio of the photoinitiator is 0.01-0.05%.

[0035] In a third aspect, the present invention provides the use of a bioactive percutaneous light-cured moldable hydrogel in soft tissue filling and repair.

[0036] The hydrogel according to the present invention may be used in combination with other functional materials, such as components having antibacterial or bacteriostatic properties, antiaging components, anticoagulant components, antioxidant components, growth factors, etc., as necessary.

[0037] All of the features disclosed herein, or any method or process steps disclosed herein, may be combined in any manner, except for those features and / or steps that are mutually exclusive.

[0038] When a mass-to-volume ratio or other value or parameter is expressed as a range, a preferred range, or a range limited by an upper preferred value and a lower preferred value, it should be understood to specifically disclose all ranges formed by any combination of the upper range limit or preferred value with the lower range limit or preferred value, regardless of whether the range is disclosed individually. For example, if a range "1-5" is disclosed, the range described should be interpreted to include the ranges "1-4", "1-3", "1-2", "1-2 and 4-5", "1-3 and 5", etc. When a numerical range is described herein, unless otherwise specified, the range is intended to include its limits and all integers and fractions within the range.

[0039] The present invention has the following beneficial effects: (1) The present invention provides a method for chemically modifying natural polysaccharides with unsaturated acid anhydrides to introduce unsaturated carbon-carbon double bonds, activating the carboxyl groups of the modified natural polysaccharides with EDC / NHS, then adding amino orthodiol to introduce adjacent hydroxyl groups, obtaining aldehyde groups under oxidation with an oxidizing agent, and finally reacting the aldehyde groups with the amino groups of recombinant collagen with a Schiff base, followed by transdermal light irradiation crosslinking to obtain a recombinant collagen activated soft tissue filling hydrogel. The hydrogel obtained by the present invention has good biocompatibility, is easy to operate, and at the same time has the properties of being injectable and in situ curing moldable, and is also satisfactory for filling clinically irregular tissue defects. (2) The present invention uses natural polysaccharide materials such as carboxymethylcellulose as a base to complete the construction of a novel composite tissue filler through only three steps: pre-mixing of the materials, local injection, and light curing. The preparation method is simple, the degree of the denaturation reaction can be controlled, and the defect of the lack of cell adhesion sites of natural polysaccharide materials such as carboxymethylcellulose is resolved. (2) The photoinitiator of the present invention is easy to operate, has a mild reaction, undergoes transdermal photocrosslinking, does not require invasive surgery, and can be shaped intraoperatively and molded postoperatively. [Brief description of the drawings]

[0040] It should be noted that the drawings described herein are intended to provide a further understanding of the present invention and constitute a part of the present invention, and the exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and are not intended to limit the present invention. [Figure 1] This shows the results of measuring the cytotoxicity of a natural polysaccharide derivative (CMC-MA) with photocrosslinking ability. [Diagram 2] Morphology and mechanical strength of CMC-MA hydrogels. A: Gel formation state of 2% CMC-MA material at different times of in vitro transdermal irradiation. B: Storage modulus, loss modulus, and Tan Delta of 2% CMC-MA material gel formation at different times of in vitro transdermal irradiation. [Diagram 3]The gel formation status of recombinant collagen-activated transdermal light-curing soft tissue filler (CMC-MA-AP-CHO-rhCol III) before and after subcutaneous injection and transdermal light irradiation in SD rats. [Figure 4] Release curves of recombinant collagen; A: rhCol III sustained release curves of CMC-MA / rhCol III hydrogel at different time points; B: rhCol III sustained release curves of CMC-MA-AP-CHO-rhCol III hydrogel at different time points. [Diagram 5] CMC-MA-AP-CHO-rhCol III indentation force test, where Sample 1, Sample 2, and Sample 3 represent samples repeated three times. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] In order to make the object, technical means and advantages of the present invention clearer, the present invention will be described in more detail with reference to examples. It should be understood that the specific embodiments described herein are merely for the purpose of illustrating the present invention and are not intended to limit the present invention.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The terms used herein are for the purpose of describing specific examples only and are not intended to be limiting of the present invention.

[0043] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0044] As used herein, the terms "comprise," "include," "have," "contain," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, or product of elements recited by inclusion is not necessarily limited to only those elements, but may include other elements not expressly recited or inherent to such composition, process, method, or product.

[0045] <Example 1> (1) Preparation of Percutaneous Photocurable Soft Tissue Filling Matrix Carboxymethylcellulose (CMC, 250KD, DS=0.7) was modified with methacrylic anhydride (MA). 1g of CMC raw material was dissolved in 100mL of deionized water to a concentration of 1% (wt), stirred with a mechanical stirrer for 6 hours, then left in a 4°C refrigerator for 24 hours and stirred at room temperature for 2 hours to fully dissolve the CMC and fully extend the molecular chain, which is favorable for the progress of the modification reaction. The dissolved 1% CMC solution was placed in a low-temperature water bath controlled by a low-temperature circulation pump, and the reaction temperature was set and equilibrated for 2 hours. At the start of the reaction, 2mL of methacrylic anhydride was dropped into the CMC solution, and the pH was adjusted with 5M NaOH during the reaction to maintain the reaction pH at 8.0-8.5. When the pH of the reaction solution stabilized, the solution was reacted overnight. Next, the reaction solution was poured into 500 mL of ethanol to precipitate the reaction product, and unreacted methacrylic anhydride and a portion of the by-products were removed. The product was obtained by filtration, washed three times with ethanol, and the product was placed in an 8000-14000 kDa dialysis bag and dialyzed with deionized water for four days, changing the water three times per day. After dialysis, the product was freeze-dried in a vacuum freeze dryer to obtain CMC-MA, a natural polysaccharide derivative with photocrosslinking properties, which was then stored at 4 °C.

[0046] (2) Preparation of recombinant collagen 1. Production of recombinant collagen by E. coli fermentation Genetic modification and transcription: The collagen DNA fragment was codon optimized and spliced ​​by PCR, and pET-32a was selected to construct an expression vector, which was then transferred into E. coli expression strain BL21, cultured, and selected to obtain E. coli recombinant bacteria with high protein expression.

[0047] Fermentation culture: A single colony of the desired E. coli recombinant bacteria is picked from the LB plate, placed in a 100mL Erlenmeyer flask containing 10mL of LB medium, and cultured at 37℃ and 220 rpm for 12 to 16 hours. The bacterial liquid is inoculated into a fermenter containing LB medium at a ratio of 1:100 for amplification culture. When the culture is cultured at 37℃ and 220 rpm until the OD600 reaches approximately 0.6, 0.5mM IPTG is added, and induction culture is performed at 16℃ for 20 hours, followed by centrifugation to collect the bacterial cells.

[0048] Protein isolation and purification: After reselecting the bacterial cells in Tris buffer, the bacterial cells were completely dissolved by high-speed stirring, and the supernatant was collected by centrifugation and cooled to 4°C. The supernatant was successively filtered through 1 μm, 0.45 μm, and 0.22 μm filters, and then further purified by affinity chromatography to obtain recombinant collagen.

[0049] The amino acid sequence of the recombinant collagen is characterized in that it has a repeating unit in which multiple characteristic amino acid sequences are combined, i.e., GER GAP GFR GPA GPN GIP GEK GPA GER GAP, which is directly linked 16 times to obtain the recombinant collagen of the present invention, and the amino acid sequence is as shown in SEQ ID NO.1. GERGAPGFRG PAGPNGIPGE KGPAGERGAP GERGAPGFRG PAGPNGIPGE KGPAGERGAP GERGAPGFRG PAGPNGIPGE KGPAGERGAP GERGAPGFRG PAGPNGIPGE KGPAGERGAP GERGAPGFRG PAGPNGIPGE KGPAGERGAP GERGAPGFRG PAGPNGIPGE KGPAGERGAP GERGAPGFRG PAGPNGIPGE KGPAGERGAP GERGAPGFRG PAGPNGIPGE KGPAGERGAP GERGAPGFRG PAGPNGIPGE KGPAGERGAP GERGAPGFRG PAGPNGIPGE KGPAGERGAP GERGAPGFRG PAGPNGIPGE KGPAGERGAP GERGAPGFRG PAGPNGIPGE KGPAGERGAP GERGAPGFRG PAGPNGIPGE KGPAGERGAP GERGAPGFRG PAGPNGIPGE KGPAGERGAP GERGAPGFRG PAGPNGIPGE KGPAGERGAP GERGAPGFRG PAGPNGIPGE KGPAGERGAP.

[0050] 2.Cell culture method 1) Sample preparation: Recombinant collagen is prepared in a solution at a certain concentration (e.g., 0.5 mg / mL) in PBS, and animal-derived collagen solution is diluted to the same concentration (e.g., 0.5 mg / mL) in PBS, with PBS as the blank control group. 2) Plating: Experimental solutions were added separately to 96-well plates, 100 μL per well, 5 wells per set, and incubated overnight at 4°C. 3) Blocking: After plating, the liquid was removed from the well plate, which was then washed twice with 200 μL of PBS solution. 100 μL of heat-inactivated 1% BSA-PBS solution was added, and the plate was incubated for 1 hour in an incubator at 37°C and 5% CO2. 4) Cell inoculation: After incubation, remove the liquid from the well plate, wash twice with 200 μL of PBS solution, and inoculate cells at a density of 5 × 10 at 100 μL per well. 4 ~ 1×10 6 A cell suspension at 100 / mL was added and incubated at 37° C. in a 5% CO 2 incubator for 1 h. 5) Detection: After incubation, the liquid was removed from the well plate, which was then washed twice with 200 μL of PBS solution. 150 μL of CCK-8 was added, and the plate was incubated for 1 hour in an incubator at 37°C and 5% CO2. 100 μL of the detection solution was then taken and added to a new 96-well plate, and the optical density (OD) was detected at 450 nm.

[0051] (3) Preparation of recombinant collagen-activated percutaneous light-cured soft tissue filler (3.1) Carboxymethylcellulose (CMC-MA), a natural polysaccharide derivative with photocrosslinking ability and a degree of modification of 30-40%, was dissolved in deionized water to obtain a 0.5% methacrylated carboxymethylcellulose solution. (3.2) Solid NHS and solid EDC were added sequentially to the mixed solution (the molar ratio of CMC-MA, EDC, and NHS was 1:2:3), and the pH of the mixed solution was maintained at 4.75–5 using 5.0 M NaOH solution or 5.0 M HCl solution. (3.3) The reaction was stirred at room temperature for 2 hours. (3.4) The pH of the reaction system was adjusted to 7.4 with 1.0 M NaOH solution. (3.5) 3-Amino-1,2-propanediol (AP) was added to the reaction system so that the molar ratio of 3-amino-1,2-propanediol to methacrylated carboxymethylcellulose in the mixed solution was 5:1, and the reaction was carried out with stirring for 24 hours. (3.5) The solution after the reaction was placed in a dialysis bag with a molecular weight cutoff of 8,000 to 14,000, dialyzed against deionized water for 3 days, and then freeze-dried in a freeze dryer to obtain the ortho-diol side chain-containing natural polysaccharide derivative CMC-MA-AP. (3.6) The freeze-dried sponge from step (3.5) was dissolved in deionized water, and 0.5 M sodium periodate solution was added dropwise to the solution such that the molar ratio of 0.5 M sodium periodate to CMC-MA-AP was 0.2:1, and the mixture was reacted with stirring for 24 h. (3.7) The solution after the reaction was placed in a dialysis bag with a molecular weight cutoff of 8,000 to 14,000, dialyzed against deionized water for 3 days, and then freeze-dried in a freeze dryer to obtain the natural polysaccharide derivative containing aldehyde side chains, CMC-MA-AP-CHO. (3.8) The CMC-MA-AP-CHO material obtained by lyophilization in step (3.7) was dissolved in deionized water to a concentration of 2%, and recombinant humanized type III collagen (rhCol III) was added to a final concentration of 1%, followed by the addition of LAP photoinitiator solution (final concentration 0.02% by mass / volume). (3.9) The precursor liquid obtained in step (3.8) was poured into a silica gel mold with a standard size of Φ6 mm × 2.5 mm, and then irradiated with ultraviolet light for 1 minute to obtain a recombinant collagen-activated transdermal photocurable soft tissue forming filler: CMC-MA-AP-CHO-rhCol III hydrogel.

[0052] Test Example 1: Detection of cytotoxicity of CMC-MA and CMC-MA-AP-CHO-rhColIII hydrogels CMC-MA cytotoxicity detection 1. Preparation of material leaching solution: After sterilizing CMC-MA, the concentration was adjusted to 2%. Then, it was sealed in a syringe under sterile conditions, and the hardened material was prepared under sterile conditions, with the size Φ=8±0.1mm,h=2.0±0.2mm. The material was placed in a 48-well plate, and α-MEM medium was added to completely immerse the composite material to a leaching rate of 0.2g / mL as recommended in GB / T 16886.12-2017 "Medical Device Biological Evaluation Part 12: Preparation of Samples and Reference Materials". After leaching for 24h in an incubator at 37℃ and 5% CO2, the supernatant was extracted, and then an appropriate amount of the supernatant was taken and 10% newborn fetal calf serum (FBS) was added sequentially to prepare leaching solutions with concentrations of 100%, 50%, 25% and 12.5%, respectively. 2. Resuscitate L929 cells, subculture to generation P3, digest the cells, and reduce the cell concentration to 5 × 10 4The cell suspension was adjusted to 100 cells / mL, and then aspirated into a 96-well plate, dropped to 100 μL per well, and cultured in an incubator for 24 hours using α-MEM containing 10% newborn calf serum as the medium. The medium was then aspirated and discarded, and 150 μL of sample elution solutions of concentrations of 100%, 50%, 25% and 12.5% ​​were added, as well as 20% DMSO (positive control) and 10% FBS-containing medium (negative control). At this time, 10% FBS-containing medium was dropped into the surrounding wells as a blank control. Five parallel samples were placed for each set of samples and incubated for 24 h. 3. After 24 h incubation, the supernatant was aspirated and discarded, and 150 μL of CCK-8 solution was added to each well, placed in an incubator, and incubated for 2 h in the dark. 100 μL of the supernatant was aspirated from each well and placed into a new 96-well plate, and the optical density value (OD value) at 450 nm was measured using a microplate reader. The cell viability was calculated according to the data analysis in ISO 10993-5:2017 Appendix C. The results are shown in Figure 1.

[0053] The safety of the CMC-MA composite material is acceptable in accordance with the cytotoxicity provisions of GB / T 16886.5-2017 "Part 5 of the Biological Evaluation of Medical Devices: In Vitro Cytotoxicity Testing".

[0054] CMC-MA-AP-CHO-rhColIII hydrogel toxicity detection According to the method of "CMC-MA cytotoxicity detection", the toxicity of CMC-MA-AP-CHO-rhColIII hydrogel is detected, and according to the regulation on cytotoxicity in GB / T16886.5-2017 "Medical Device Biological Evaluation Part 5: In Vitro Cytotoxicity Test", the safety of the hydrogel composite is accepted.

[0055] Test Example 2: Gel formation by in vitro subcutaneous light irradiation of CMC-MA SD rats A 1.2% CMC-MA solution was filled into a 1 mL syringe, and the composite material was injected into a mold (d: 8 mm, h: 2 mm). The back skin of a 12-week-old rat was shaved, and the material in the mold was placed under the ex vivo skin and irradiated with a 5 W blue flashlight (wavelength = 405 nm) for different times (1, 2, 3, 4, 5, 7, 10, 15, 20 min). 2. The mold was removed, photographed immediately, and the mechanical strength (storage modulus, loss modulus) of the hydrogel was measured by DMA. As shown in Figure 2, the material can form a gel during 1 min of transdermal light irradiation. The mechanical strength of the hydrogel gradually increased with the extension of transdermal light irradiation time, and no significant statistical difference was observed in the hydrogel strength when the light irradiation time exceeded 5 min.

[0056] Test Example 3: Gel formation by subcutaneous light irradiation of CMC-MA-AP-CHO-rhCol III in SD rats in vivo A 1.2% CMC-MA-AP-CHO-rhCol III solution was filled into a 1 mL syringe, and the back of an SD rat was shaved. 0.2 mL of the CMC-MA-AP-CHO-rhCol III solution was subcutaneously injected, appropriately trimmed, and the changes in the condition of the dorsal skin before light irradiation and the condition of the subcutaneous material before light irradiation were recorded (Figure 3). The skin was irradiated with a 2.5 W blue flashlight (wavelength = 405 nm) for different times (1 min), and the condition of the skin after irradiation and the state of gel formation of the subcutaneous material were recorded (Figure 3).

[0057] From Figure 3, it can be seen that after transdermal injection, without light irradiation, the material did not form a gel but was mucus-like, while after 1 min of transdermal irradiation, the material obviously formed a gel and had an obvious fixed form.

[0058] Test Example 4: CMC-MA-AP-CHO-rhCol III hydrogel protein release test The composite solutions of 1.2% CMC-MA-AP-CHO-rhCol III (rhCol III final concentration 10 mg / mL) and CMC-MA / rhCol III (rhCol III final concentration 10 mg / mL) were injected into 1 mL syringes, respectively, and the composites were poured into molds (d: 8 mm, h: 2 mm). They were irradiated with a 5 W blue flashlight (wavelength = 405 nm) for 1 min. The masses of CMC-MA-AP-CHO-rhCol III and CMC-MA / rhCol III hydrogels were accurately weighed, and the volume of the leaching solution (0.01 M PBS) was determined with a leaching ratio of 0.2 g / mL (see GB / T16886.12). The hydrogels were immersed in the exudate and placed on a thermostatic air shaker (37°C, 70 rpm / min). Three parallel samples were collected for each group, and 100 μL of exudate was collected from each parallel sample at the designated time points and replenished with 100 μL of fresh exudate.

[0059] 2. The concentration of rhCol III (1, 2, 4, 6, 8, 12, 24, 36, 48, 72h) in the infusion solution was measured by the Coomassie Brilliant Blue method. The absorbance value (Abs) at a wavelength of 595nm was measured using a UV-Visible spectrophotometer. A calibration curve was created from the standard protein solution concentration and the corresponding absorbance value, and the protein content in the test solution was calculated. For specific procedures, please refer to the instruction manual (Solarbio, PC0015).

[0060] The results are shown in Figure 4. In Figure 4A, the release rate of rhCol III for CMC-MA / rhCol III hydrogel gradually increased with time, and at 24 hours, the release rate reached a maximum of 73.59%, showing a tendency to continue increasing, reaching 80% at 72 hours. In Figure 4B, the release rate of CMC-MA-AP-CHO-rhCol III hydrogel was only 2.73% at 24 hours, and the release rate of rhCol III slowly increased with time. It can be seen that the hydrogel prepared by the method of the present invention can achieve a sustained release effect of recombinant collagen, effectively solving the problem of rapid release of recombinant collagen alone, with a short treatment period and long-lasting effect.

[0061] Test Example 5 CMC-MA-AP-CHO-rhColIII compression test The injectability of an injectable material is an important physicochemical indicator regarding the ease of use for practitioners, the presence or absence of blockages during the extrusion process, discontinuities, etc. To evaluate the injectability, a universal material tester (AGS-X, SHIMADZU, Japan) was used to test the push-in force of the composite material in a syringe as follows: 1. Sample preparation: According to the expected technical requirements of the injectable composite material, 2% CMC-MA-AP-CHO-rhColIII precursor solution was placed in a 1 mL syringe with a 30-gauge needle and kept cryogenically stored at 4°C. 2. Preliminary preparation: Before the extrusion test, the air at the tip of the syringe needle was pushed out and the material was extruded. 3. Test: The syringe containing the material was fixed in a special mold and a compressive load was applied vertically using a universal material tester at a constant speed of 30 mm / min. The force-displacement curve was recorded, and the test was repeated three times for each sample.

[0062] The results are shown in Figure 5. The extrusion force of 2% CMC-MA-AP-CHO-rhColIII precursor solution reached a relatively stable level after a rapid linear increase until the material was exhausted. At a certain extrusion stage, the extrusion force of the sample was stable at 2.76 ± 0.96 N, and the entire curve was smooth with a small drop, suggesting that the material system components were uniform and there were no significant bubbles. In addition, the material had a small extrusion force, which made it easy to inject and handle by the surgeon.

[0063] In conclusion, the natural polysaccharide derivative prepared in the present invention has significant gel formation after 1 min of transdermal injection light irradiation in vitro and in vivo, the gel formation time is fast, and there is no damage to skin tissue. CMC-MA-AP-CHO, a natural polysaccharide derivative with aldehyde side chains obtained by the reaction of CMC-MA, a natural polysaccharide derivative with photocrosslinking ability, with 3-amino-1,2-propanediol and subsequent oxidation, can reduce the destruction of the sugar ring main chain during the oxidation process and reduce the decrease in mechanical strength to a certain extent. The test results show that the hydrogel of such modified natural polysaccharide derivative has good safety, injectability and handling properties. With the decomposition of carboxymethylcellulose, the recombinant collagen-activated CMC-MA-AP-CHO-rhCol III hydrogel can sustainably release rhCol III, induce the biological expression behavior of the extracellular matrix, thereby achieving the maintenance of the initial morphology, and induce the repair effect of soft tissue matrix regeneration in the later stage, with a longer action time.

[0064] The above examples are merely illustrative to describe some features of the method of the present invention. The appended claims are intended to claim the broadest possible scope, and the examples presented herein are merely descriptions of embodiments selected according to all possible combinations of examples. Therefore, it is the applicant's intention that the appended claims are not selectively limited by the examples that describe the features of the present invention. The numerical ranges used in the claims should be interpreted to include subranges within the ranges, and to the extent possible, modifications within the ranges are also encompassed by the appended claims.

Claims

1. The raw materials include recombinant collagen, acid anhydride, natural polysaccharide substances, activators, amino orthodiols, and oxidants. The amino acid sequence of the recombinant collagen includes N basic repeating units, and the basic repeating units have the following characteristic amino acid sequence: "G-Xaa １ -Xaa ２ -G-E-Xaa ３ " n1 times, the 3' end and the 5' end of the basic repeating unit are linked to form the characteristic amino acid sequence, A biologically active transdermal photocurable moldable hydrogel, wherein N is an integer of 4 or more, and n1 is an integer of 3 or more.

2. The hydrogel according to claim 1, wherein N is an integer from 4 to 300.

3. The hydrogel according to claim 1, wherein N is an integer from 4 to 200.

4. The hydrogel according to claim 1, characterized in that the characteristic amino acid sequence is arranged consecutively or at intervals in the basic repeating unit.

5. The amino acid sequence of the recombinant collagen is a hydrogel that exhibits the following characteristics: [-G-E-Xaa ３ -Yaa １ -(G-Xaa １ -Xaa ２ -G-E-Xaa ３ ) ｎ２ -Yaa ２ -(G-Xaa １ -Xaa ２ -G-E-Xaa ３ ) ｎ３ -Yaa ３ -(G-Xaa １ -Xaa ２ -G-E-Xaa ３ ) ｎ３ - Yaa ４ -(G-Xaa １ -Xaa ２-G-E-Xaa ３ ) ｎ４ --Yaa ｎ -(G-Xaa １ -Xaa ２ -G-E-Xaa ３ ) ｎ -Yaa ｎ＋１ -G-Xaa １ -Xaa ２ -] Ｎ , Here, the Xaa １ is a non-polar hydrophobic amino acid, ２ is one of serine (S), alanine (A), proline (P), and hydroxyproline (O); ３ is a basic amino acid, Yaa １ , Yaa ２ , Yaa ３ , Yaa ４ , ………, Yaa ｎ , Yaa ｎ＋１ is independently selected from absent, one or more different or the same amino acid, The hydrogel according to claim 1, characterized in that n2, n3, n4, ...., n are integers of 0 or more, and both are not 0 at the same time.

6. The hydrogel of claim 1, wherein the recombinant collagen sequence does not contain a protein tag.

7. 2. The hydrogel according to claim 1, wherein the amino acid sequence of the recombinant collagen is SEQ ID NO.

8. 2. The hydrogel according to claim 1, wherein the acid anhydride is one of a cyclic unsaturated acid anhydride, a carboxyl group-containing unsaturated acid anhydride, 4-pentene anhydride, crotonic anhydride, and methacrylic anhydride.

9. 9. The hydrogel of claim 8, wherein the acid anhydride is one of maleic anhydride, citraconic anhydride, cis-3-carboxyglutaconic anhydride, 4-pentene anhydride, crotonic anhydride, and methacrylic anhydride.

10. 2. The hydrogel of claim 1, wherein the natural polysaccharide material is selected from hyaluronic acid, carboxymethylcellulose, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, alginic acid, dextran, agarose, heparin, chondroitin sulfate, ethylene glycol chitosan, propylene glycol chitosan, chitosan lactate, carboxymethyl chitosan, or chitosan quaternary ammonium salt.

11. 2. The hydrogel of claim 1, wherein the activating agents are NHS and EDC, the amino orthodiol is 3-amino-1,2-propanediol, and the oxidizing agent is selected from periodate and hypochlorite, preferably the oxidizing agent is sodium periodate.

12. The hydrogel is prepared by transdermal photocuring, and the specific steps are as follows: S1. A process for appropriately chemically modifying a natural polysaccharide substance with an acid anhydride to obtain a natural polysaccharide derivative having photocrosslinking ability; S2. A step of mixing and reacting the photocrosslinkable natural polysaccharide derivative with an activator, and the pH value of the reaction solution is 4.65 to 5.2; S3. A step of adding amino ortho-diol to the reaction system of S2 to perform a side chain carboxyl activation reaction, purifying the product by dialysis, and freeze-drying the product after the dialysis to obtain a natural polysaccharide derivative having ortho-diol side chains; S4. A step of dissolving the ortho-diol side chain-containing natural polysaccharide derivative, reacting it with an oxidizing agent, purifying it by dialysis, and freeze-drying it after the dialysis to obtain an aldehyde side chain-containing natural polysaccharide derivative; S5. A step of dissolving the aldehyde side chain-containing natural polysaccharide derivative, mixing it with recombinant collagen and a photoinitiator to obtain a repair matrix precursor liquid, and then irradiating the liquid with transdermal light to crosslink the liquid to obtain a hydrogel. A method for preparing the transdermal photocurable moldable hydrogel according to any one of claims 1 to 11, comprising:

13. The preparation method according to claim 12, characterized in that the degree of modification of the natural polysaccharide derivative having photocrosslinking ability in S1 is 30-60%, and the molar ratio of the natural polysaccharide derivative having photocrosslinking ability to NHS and EDC in the activator is 1:(1-3):(2-5).

14. The preparation method according to claim 12, characterized in that the molar ratio of the natural polysaccharide derivative having photocrosslinking ability:NHS:EDC in S2 is 1:2:

3.

15. The preparation method according to claim 12, characterized in that in step S3, the pH value of the solution to which amino ortho-diol is added to carry out the activation reaction is 7 to 7.7, the molar ratio of amino ortho-diol to the natural polysaccharide derivative having photocrosslinking ability is (1 to 10):1, the dialysis time in steps S3 and S4 is 20 to 72 hours, and the molecular weight of the dialyzed fraction is 8,000 to 14,000 kDa.

16. The preparation method according to claim 12, characterized in that in S5, the mass / volume ratio of the aldehyde side chain-containing natural polysaccharide derivative is 1-5%, the mass / volume ratio of the recombinant collagen is 0.5-2%, and the final mass / volume ratio of the photoinitiator is 0.01-0.05%.

17. Use of the bioactive transdermal photocurable moldable hydrogel according to any one of claims 1 to 11 in filling and repairing soft tissues.

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