Compound temperature-sensitive recombinant humanized collagen gel stable at low temperature as well as preparation method and application of compound temperature-sensitive recombinant humanized collagen gel

Abstract

The invention discloses low-temperature stable compound temperature-sensitive recombinant humanized collagen gel as well as a preparation method and application thereof. The compound temperature-sensitive recombinant humanized collagen gel comprises the following components in percentage by weight: 17%-20% of poloxamer 407, 0.1%-0.4% of recombinant humanized collagen, 10%-20% of glycerol, 1%-5% of a humectant and the balance of water. The stability of the recombinant humanized collagen gel is improved by adding moisturizers such as glycerol and PEG-8 into a poloxamer 407 gel system, the controllable adjustment of the gel forming temperature of the gel is realized by adjusting the composition proportion, and the gel forming temperature of the gel is regulated and controlled to be 10-30 DEG C. The compound temperature-sensitive recombinant humanized collagen gel disclosed by the invention can stably form gel under different acidic and neutral pH conditions, has good cell compatibility, is free of vaginal irritation and mild implantation reaction, and has wide application prospects in the fields of vaginal in-situ gel medical instruments, biological materials and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a low-temperature stable composite temperature-sensitive recombinant humanized collagen gel, and a preparation method and application thereof. Background Art

[0002] Vaginal in situ gel is a new drug delivery system. After being administered in solution form, it triggers a phase change in the vagina due to environmental stimuli (temperature, pH value, etc.), transforming from a liquid to a semi-solid gel. This characteristic enables it to combine the ease of administration of liquid preparations with the sustained-release and long-lasting effect of gel preparations. Based on environmental response factors, vaginal in situ gels are divided into three categories: temperature-sensitive, pH-sensitive, and composite. Among them, the main matrix materials used in temperature-sensitive vaginal in situ gels are poloxamer (Poloxamer 407 / 188), chitosan, etc. Poloxamer is a liquid solution at low temperatures. When the temperature rises to body temperature, the hydrophobic PPO chain segments dehydrate and aggregate to form a hydrophobic core, and the hydrophilic PEO chain segments extend outward to form a three-dimensional network gel structure. Poloxamer 407 is the most commonly used model, and its gelation temperature can be regulated by its concentration (concentration 15-30%), but high concentration may result in excessive viscosity. Therefore, it is often compounded with poloxamer 188 to optimize the gelation temperature, gelation time and mechanical properties (Feng Tianqi, Wang Chunli, Wang Siyao, et al. Preparation of chitosan / poloxamer composite thermosensitive hydrogel [J]. Journal of Jilin Agricultural University, 2021, 43(06): 679-684. DOI: 10.13327 / j.jjlau.2021.1074.).

[0003] Due to its excellent biocompatibility and bioactivity, recombinant humanized collagen has broad application prospects in the fields of medical devices and biomaterials, and has been studied for its application in in situ vaginal gelation. Chinese patent CN202211621563.3 discloses a thermosensitive composite recombinant humanized collagen hydrogel comprising: poloxamer 407 (20-25%), poloxamer 188 (2.5-7.5%), and recombinant human type III collagen (0.05-5%). Studies have found that the dosage of each component is crucial. If the dosage of poloxamer 407 is too low or poloxamer 188 is too high, the final hydrogel product will not gel easily even at temperatures above normal human body temperature, resulting in direct excretion after injection, completely ineffective. If the dosage of poloxamer 407, poloxamer 188, or recombinant human type III collagen is too high, the hydrogel product will gel at lower temperatures, making it difficult to inject. For example, when the mass fraction of poloxamer 407 is 17.5%, poloxamer 188 is 10%, and recombinant human type III collagen is 0.5%, the gelation temperature is as high as 44±0.2°C, which cannot meet the requirements of in situ gelation in vivo. Summary of the Invention

[0004] The inventors have discovered that recombinant humanized collagen in a poloxamer gel system is prone to problems such as turbidity and protein precipitation at low temperatures (10°C), affecting the stability and performance of the product. To address the above problems, the present invention provides a low-temperature stable composite thermosensitive recombinant humanized collagen gel, its preparation method, and application. The present invention improves the low-temperature stability of the thermosensitive recombinant humanized collagen gel by adding ingredients such as glycerol and polyethylene glycol-8 (PEG-8) to the poloxamer 407 gel system, while ensuring the biocompatibility of the thermosensitive recombinant humanized collagen gel.

[0005] The technical solutions of the present invention are as follows:

[0006] The invention discloses a low-temperature stable composite thermosensitive recombinant humanized collagen gel, which comprises the following components by weight: 17% to 20% of poloxamer 407, 0.1% to 0.4% of recombinant humanized collagen, 10% to 20% of glycerol, 1% to 5% of a humectant, and the balance is water. The recombinant humanized collagen is produced by fermentation of Pichia pastoris with a deposit number of CGMCC No. 5021, and the humectant is PEG-8, propylene glycol, butylene glycol, or hexylene glycol.

[0007] Furthermore, the above-mentioned compound temperature-sensitive recombinant humanized collagen gel also includes antibacterial agents 0.05% of octanoylhydroxamic acid, 0-1% of butanediol, and 0-0.5% of p-hydroxyacetophenone.

[0008] Furthermore, the pH of the composite temperature-sensitive recombinant humanized collagen gel is 3.5-7.5.

[0009] Furthermore, the composite thermosensitive recombinant humanized collagen gel comprises the following components by weight: 17% poloxamer 407, 0.4% recombinant humanized collagen, 20% glycerol, 1% to 5% moisturizer, and the balance water; the gel can gel at 30°C.

[0010] Furthermore, the composite thermosensitive recombinant humanized collagen gel comprises the following components by weight: 19% poloxamer 407, 0.4% recombinant humanized collagen, 20% glycerol, 1% to 5% moisturizer, and the balance water; the gel can gel at 20°C.

[0011] Furthermore, the composite thermosensitive recombinant humanized collagen gel comprises the following components by weight: 20% poloxamer 407, 0.4% recombinant humanized collagen, 20% glycerol, 1% to 5% moisturizer, and the balance water; the gel can gel at 10°C.

[0012] The method for preparing the above-mentioned low-temperature stable composite temperature-sensitive recombinant humanized collagen gel comprises the following steps:

[0013] According to the ratio, poloxamer 407 is first added to part of the water and dissolved at 0-4°C until it is clear. The recombinant humanized collagen, glycerin, and moisturizer are mixed and dissolved in the remaining water. The two solutions are mixed evenly. After degassing at a low temperature of -15°C to 4°C, the solution is filled to obtain a low-temperature stable compound temperature-sensitive recombinant humanized collagen gel.

[0014] Furthermore, the above preparation method further comprises irradiating the prepared low-temperature stable composite thermosensitive recombinant humanized collagen gel with Co60 or electron beam at 8.5 to 15 kgy.

[0015] The present invention also provides use of the composite temperature-sensitive recombinant humanized collagen gel in the preparation of vaginal in situ gel.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] (1) The present invention improves the low-temperature stability of recombinant humanized collagen in a poloxamer gel system by adding moisturizers such as glycerol and PEG-8 when the addition amount of poloxamer 407 does not exceed 20%, thereby preventing protein precipitation.

[0018] (2) The present invention regulates the gelling temperature of the final compound thermosensitive recombinant humanized collagen gel within the range of 10°C to 30°C by adjusting the addition amount of each component. When the gelling temperature is 10°C, the gel can be evenly distributed on the surface of local application. When the gelling temperature is 30°C, the gel can satisfy the requirements of uniform self-flow distribution of liquid during use and long retention time after gelling.

[0019] (3) The normal pH value of the human vagina is between 3.8 and 4.5, which is an acidic environment. This helps inhibit the invasion of pathogens and protect the health of the female reproductive system. The pH value of the uterine cavity is around 7.0, which is a neutral environment. The composite thermosensitive recombinant humanized collagen gel of the present invention can stably gel under different pH conditions, such as acidic and neutral, and has broad application prospects in the field of vaginal in situ gelation. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The gelation conditions of samples 1 to 9 at 10°C;

[0021] Figure 2 The gelation conditions of samples 1 to 9 at 20°C;

[0022] Figure 3 The gelation conditions of samples 1 to 9 at 30°C;

[0023] Figure 4 The gelation conditions of samples 10 to 17 at 10°C;

[0024] Figure 5 The gelation conditions of samples 10 to 17 at 20°C;

[0025] Figure 6 The gelation conditions of samples 10 to 17 at 30°C;

[0026] Figure 7 The gelation conditions of samples 18 to 19 at 10°C are shown;

[0027] Figure 8 The gelation conditions of samples 18 to 19 at 20°C are shown;

[0028] Figure 9 The gelation conditions of samples 18 to 19 at 30°C are shown;

[0029] Figure 10 This is the pathological image of the vaginal mucosal irritation test of sample 9;

[0030] Figure 11 This is the pathological picture of the subcutaneous implant reaction test of sample 9. DETAILED DESCRIPTION

[0031] The present invention will be further described below in conjunction with specific embodiments and accompanying drawings.

[0032] The recombinant humanized collagen used in the present invention is produced by fermentation of Pichia pastoris with a deposit number of CGMCC No. 5021, has been fully disclosed in Chinese Patent ZL201110327865.5, and can also be purchased commercially.

[0033] Example 1

[0034] By mass percentage, first add poloxamer 407 to a portion of the water and dissolve it at 0-4°C overnight until clear. Then, dissolve recombinant humanized collagen, glycerol, and the remaining water, or recombinant humanized collagen, glycerol, PEG-8, and the remaining water. Mix the two solutions, stir evenly, and adjust the pH to 3.5-4.5 with 10% citric acid to prepare a composite temperature-sensitive recombinant humanized collagen gel. The formulas for each sample are as follows:

[0035] Sample 1: Poloxamer 407 17%, recombinant humanized collagen 0.1%, glycerol 0%, citric acid to adjust pH to 3.5-4.5, water to 100%.

[0036] Sample 2: Poloxamer 407 17%, recombinant humanized collagen 0.1%, glycerol 10%, citric acid to adjust pH to 3.5-4.5, water to 100%.

[0037] Sample 3: Poloxamer 407 17%, recombinant humanized collagen 0.1%, glycerol 15%, citric acid to adjust pH to 3.5-4.5, water to 100%.

[0038] Sample 4: Poloxamer 407 17%, recombinant humanized collagen 0.1%, glycerol 20%, citric acid to adjust pH to 3.5-4.5, water to 100%.

[0039] Sample 5: Poloxamer 407 17%, recombinant humanized collagen 0.4%, glycerol 0%, citric acid to adjust pH to 3.5-4.5, water to 100%.

[0040] Sample 6: Poloxamer 407 17%, recombinant humanized collagen 0.4%, glycerol 10%, citric acid to adjust pH to 3.5-4.5, water to 100%.

[0041] Sample 7: Poloxamer 407 17%, recombinant humanized collagen 0.4%, glycerol 15%, citric acid to adjust pH to 3.5-4.5, water to 100%.

[0042] Sample 8: Poloxamer 407 17%, recombinant humanized collagen 0.4%, glycerol 20%, citric acid to adjust pH to 3.5-4.5, and water to 100%.

[0043] Sample 9: Poloxamer 407 17%, recombinant humanized collagen 0.4%, glycerol 20%, PEG-85%, citric acid to adjust pH to 3.5-4.5, water to 100%.

[0044] Take 5g of each sample and put it into a vial. Place it in a constant temperature water bath at 10℃, 20℃ and 30℃ for 10min respectively. Then take out the sample and observe its clarity and gelation. The results are shown in Table 1 and Figures 1 to 3 .

[0045] Table 1 Gelling and Clarity Results of Samples 1-9

[0046]

[0047]

[0048] According to Table 1 and Figures 1 to 3As can be seen from Samples 1 and 5, in the gel system containing only poloxamer 407 and recombinant humanized collagen, when the poloxamer 407 addition amount is ≤20% (17%), no gel can be formed at 10°C, regardless of whether the recombinant humanized collagen addition amount is 0.1% or 0.4%, and protein precipitation will occur, resulting in turbidity of the solution. Comparing Sample 1 with Samples 2 to 4, it can be seen that at a low recombinant humanized collagen addition amount (0.1%), the addition of glycerol to the gel system can improve the stability of the gel at 10°C, and as the amount of glycerol added increases, the gelation temperature of the formed gel decreases (from 30°C to 20°C). However, for a high recombinant humanized collagen addition amount (0.4%), the addition of glycerol does not improve the stability of the gel at 10°C, and the formed gel still experiences protein precipitation at 10°C, resulting in turbidity of the solution. Comparison of Sample 8 and Sample 9 shows that for the gel system with a high amount of recombinant humanized collagen added (0.4%), further adding PEG-8 on the basis of adding glycerol can improve the low-temperature stability of the formed gel at 10°C.

[0049] Example 2

[0050] First, add poloxamer 407 to a portion of water, calculated by mass percentage, and dissolve overnight at 0-4°C until clear. Then, dissolve the recombinant humanized collagen, the remaining ingredients, and water. Combine the two solutions and stir evenly until the pH is between 5.0 and 7.5 to prepare a composite temperature-sensitive recombinant humanized collagen gel. The formulas for each sample are as follows:

[0051] Sample 10: Poloxamer 407 17%, recombinant humanized collagen 0.4%, glycerol 20%, PEG-85%, water to 100%, pH 5.0-7.5.

[0052] Sample 11: Poloxamer 407 19%, recombinant humanized collagen 0.4%, glycerol 20%, PEG-85%, water to 100%, pH 5.0-7.5.

[0053] Sample 12: Poloxamer 407 19%, recombinant humanized collagen 0.4%, glycerol 25%, water to 100%, pH 5.0-7.5.

[0054] Sample 13: Poloxamer 407 19%, recombinant humanized collagen 0.4%, PEG-8 25%, water to 100%, pH 5.0-7.5.

[0055] Sample 14: Poloxamer 407 19%, recombinant humanized collagen 0.4%, glycerol 20%, PEG-8 1%, water to 100%, pH 5.0-7.5.

[0056] Sample 15: Poloxamer 407 19%, recombinant humanized collagen 0.4%, glycerol 20%, propylene glycol 1%, water to 100%, pH 5.0-7.5.

[0057] Sample 16: Poloxamer 407 19%, recombinant humanized collagen 0.4%, butylene glycol 25%, water to 100%, pH 5.0-7.5.

[0058] Sample 17: Poloxamer 407 19%, recombinant humanized collagen 0.4%, glycerol 20%, hexylene glycol 1%, water to 100%, pH 5.0-7.5.

[0059] Sample 18: Poloxamer 407 20%, recombinant humanized collagen 0.4%, glycerol 20%, PEG-85%, water to 100%, pH 5.0-7.5.

[0060] Take 5g of each sample and put it into a vial. Place it in a constant temperature water bath at 10℃, 20℃ and 30℃ for 10min respectively. Then take out the sample and observe its clarity and gelation. The results are shown in Table 2 and Figures 4 to 9 .

[0061] Table 2 Gelling and Clarity Results of Samples 10-19

[0062]

[0063] According to Tables 1, 2 and Figures 4 to 9 Samples 9 and 10 are gels made with the same formula and are stable at low temperatures under acidic conditions of pH 3.5-4.5 and neutral conditions of pH 5.0-7.5.

[0064] Samples 13 and 16 were turbid at 10°C and did not gel at 30°C. Long-term storage may lead to protein precipitation and poor gelation at body temperature, indicating that simply increasing PEG-8 25% and butanediol 25% cannot improve the stability of recombinant humanized collagen in the gel, and butanediol and PEG-8 do not significantly reduce the gelation temperature.

[0065] Sample 10 does not gel at 20°C, but gels at 30°C. It is free-flowing when stored at room temperature, but gels after entering the lesion site, prolonging the retention time of collagen and facilitating in situ injection.

[0066] Although Sample 12 gelled at 10°C, the glycerol content was too high. The remaining samples, 11, 14, 15, and 17, were based on 19% Poloxamer 407 and 20% glycerol, with adjustments made to 1-5% PEG-8, 1% propylene glycol, or 1% hexylene glycol. All of these solutions were clear and fluid at temperatures as low as 10°C and gelled above 20°C, suitable for topical application at room temperatures above 20°C.

[0067] Sample 18 can gel and clarify at 10°C, and can be used for local application at indoor temperatures above 10°C.

[0068] Comparative Example 1

[0069] By weight percentage, poloxamer 407 was first dissolved in water, followed by the addition of poloxamer 188. After stirring evenly, the mixture was placed at 0-4°C overnight to dissolve until clear. Finally, recombinant humanized collagen was added, stirred and dissolved, and the pH was adjusted to 5.0-7.5 with 10% citric acid to prepare a composite thermosensitive recombinant humanized collagen gel. The formula of this sample is as follows:

[0070] Sample 19: Poloxamer 407 19%, recombinant humanized collagen 0.4%, Poloxamer 18 85%, water to 100%, citric acid to adjust pH to 5.0-7.5.

[0071] Take 5g of sample and put it into a vial. Place it in a constant temperature water bath at 10℃, 20℃ and 30℃ for 10min respectively. Then take out the sample and observe its clarity and gelation. The results are shown in Table 2 and Figures 7-9 .

[0072] As shown in Table 2, in the gel system of poloxamer 407 and 188, even if the ratio of poloxamer 407 and 188 is optimized, the problems of turbidity and protein precipitation in the feed solution cannot be solved.

[0073] Example 3

[0074] The compounded thermosensitive recombinant humanized collagen hydrogel sample 9 prepared in Example 1 was subjected to comprehensive biocompatibility testing in terms of safety, including cytotoxicity, vaginal irritation and implantation reaction. The specific methods are as follows.

[0075] 1. Cytotoxicity Test

[0076] Cytotoxicity testing was conducted using in vitro cultured mammalian L-929 cells in accordance with GB / T 16886.5-2117. After 24 hours of incubation with the 100% concentration extract of the test sample, the cells maintained essentially intact morphology and had a cell viability of 76.5%, indicating that the test sample had no potential cytotoxicity.

[0077] 2. Vaginal irritation test

[0078] According to GB / T 16886.10-2117, vaginal irritation test was conducted using 0.9% sodium chloride injection and sesame oil extract. The test sample or control solution was gently injected into the animal vagina, and the operation was repeated every day (24±2 hours) for 5 consecutive days. Figure 10As shown, the vaginal mucosal skin of the animals in the test group was intact and no vaginal irritation reaction occurred, indicating that the test product had no vaginal mucosal irritation.

[0079] 3. Implantation reaction test

[0080] According to GB / T 16886.6-2122, a rat subcutaneous implantation test of medical recombinant humanized collagen gel was conducted. Healthy SD rats were taken and their backs were shaved 24 hours before the test. After the animals were anesthetized, 0.2 mL of the stock solution was drawn with a syringe and injected subcutaneously into the left side of the rat spine. A silk braided non-absorbable suture was implanted on the other side as a negative control. Four weeks after implantation, the implants and surrounding tissues (within 5 mm) were cut out and fixed with 10% neutral formalin. Microscopic evaluation was performed using a semi-quantitative system. The results are shown in Figure 2. Figure 11 As shown in the figure, the score of microscopic observation of subcutaneous tissue of SD rats at week 4 was 8.7, and the subcutaneous implantation reaction type was mild irritation, indicating that the test article had good biocompatibility.

Claims

1. A low-temperature stable composite thermosensitive recombinant humanized collagen gel, characterized in that: The invention comprises the following components by weight percentage: 17% to 20% poloxamer 407, 0.1% to 0.4% recombinant humanized collagen, 10% to 20% glycerol, 1% to 5% humectant, and the balance is water. The recombinant humanized collagen is produced by fermentation of Pichia pastoris with a deposit number of CGMCC No. 5021. The humectant is PEG-8, propylene glycol, butylene glycol, or hexylene glycol.

2. The composite temperature-sensitive recombinant humanized collagen gel according to claim 1, characterized in that: It also includes antibacterial agents 0.05% octanoylhydroxamic acid, 0-1% butylene glycol, and 0-0.5% p-hydroxyacetophenone.

3. The composite temperature-sensitive recombinant humanized collagen gel according to claim 1, characterized in that: The pH of the complex thermosensitive recombinant humanized collagen gel is 3.5~7.

5.

4. The composite temperature-sensitive recombinant humanized collagen gel according to claim 1, characterized in that: The composition comprises the following components by weight percentage: 17% of poloxamer 407, 0.4% of recombinant humanized collagen, 20% of glycerol, 1% to 5% of moisturizing agent, and the balance of water.

5. The composite temperature-sensitive recombinant humanized collagen gel according to claim 1, characterized in that: The composition comprises the following components by weight percentage: poloxamer 407 19%, recombinant humanized collagen 0.4%, glycerin 20%, moisturizer 1%-5%, and the balance is water.

6. The composite temperature-sensitive recombinant humanized collagen gel according to claim 1, characterized in that: The composition comprises the following components by weight percentage: 20% of poloxamer 407, 0.4% of recombinant humanized collagen, 20% of glycerol, 1% to 5% of moisturizing agent, and the balance of water.

7. The method for preparing the composite temperature-sensitive recombinant humanized collagen gel according to any one of claims 1 to 6, characterized in that: The following steps are involved: According to the ratio, poloxamer 407 is first added to part of the water and dissolved at 0-4°C until it is clear. The recombinant humanized collagen, glycerol, and preservative are mixed and dissolved in the remaining water. The two solutions are mixed evenly. After degassing at a low temperature of -15°C to 4°C, the solution is filled to obtain a low-temperature stable compound temperature-sensitive recombinant humanized collagen gel.

8. The preparation method according to claim 7, wherein The method also includes irradiating the prepared low-temperature stable composite thermosensitive recombinant humanized collagen gel with Co60 or electron beam for 8.5-15 kgy.

9. Use of the composite thermosensitive recombinant humanized collagen gel according to any one of claims 1 to 6 in the preparation of vaginal in situ gel.

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