A natural undenatured collagen solution for injection and its preparation method

Abstract

The invention discloses a natural undenatured collagen solution for injection and a preparation method thereof. The preparation method uses NaOH to adjust the pH value of the collagen solution in stages, accurately controls the pH value, salt concentration and system temperature of the solution, so that the solution is quickly and evenly dissolved under low temperature, low pH value and salt dissolution conditions to obtain a high-concentration collagen solution; a small amount of collagen molecules are formed into aggregates and stabilized under physiological salt concentration, weak acidity and temperature rise; the pH value of the system is slowly increased to neutrality to obtain a natural undenatured collagen solution with a certain proportion of collagen molecule aggregates, so that the collagen solution can maintain a maximum pushing force of less than 20N and an average pushing force of less than 15N at a pushing speed of 20mm / min using a 30G needle, and can be used for injection; the collagen solution has an endotoxin content of less than 20EU / piece and is sterile, can be directly used for human tissue injection, and can improve the maintenance time of the injection effect.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials, and in particular relates to a natural undenatured collagen solution for injection and a preparation method thereof. Background Art

[0002] There are many ways to beautify and care for the skin, including food supplements, exercise, smearing, injections, etc. At present, the raw materials used in the field of beauty and skin care are mainly hyaluronic acid, collagen and various biological extract nutrients. Hyaluronic acid only has the effects of hydrating and moisturizing, and cannot achieve the ideal skin care effect; and various biological extracts have complex sources and unstable purification processes, which pose huge safety risks. Collagen, as the most abundant protein in the body, has received more and more attention due to its excellent biological activity. Injection beauty has been favored by the majority of beauty lovers for its advantages such as convenient operation and quick results. However, there are only collagen daily skin care products such as smears and patches on the market, and there are no natural collagen solution products for injection.

[0003] The special rod-shaped triple helix structure of collagen leads to its reduced solubility under neutral conditions. In order to solve the problem of collagen solubility, Chinese patent application 20111313770.8 discloses a method for preparing acylated type I collagen water light needle: by adopting the method of acylation modification of collagen molecules, compounding with hyaluronic acid to solve the problem of poor collagen solubility. However, the spatial structure of collagen molecules after side chain acylation modification has changed, resulting in a significant decrease in its biological activity; and the residual cross-linking agent in the production process also brings huge safety hazards. In addition, most of the current injection products choose phosphate as the final solvent system in order to obtain a certain pH buffering performance. However, according to the Hofmeister effect, phosphate belongs to a low-chaotropic sequence salt, which has weak charge delocalization and low polarizability, and has a high coagulation ability for collagen molecules, which is not conducive to the dissolution of collagen molecules under neutral conditions. Therefore, it is necessary to select a suitable chaotropic salt system to facilitate the stable dissolution of pure collagen molecules. Summary of the invention

[0004] In order to overcome the shortcomings of the prior art, the first purpose of the present invention is to provide a method for preparing a natural undenatured collagen solution for injection. This method maintains the triple helix structure of natural collagen and is free of any additives. By selecting a suitable chaotropic salt solvent system, a pure collagen solution that is sterile, endotoxin-controllable, and uniformly injectable under neutral conditions is obtained by controlling the preparation process.

[0005] The second object of the present invention is to provide a natural undenatured collagen solution for injection.

[0006] One of the purposes of the present invention can be achieved by adopting the following technical solution:

[0007] A method for preparing a natural undenatured collagen solution for injection comprises the following steps:

[0008] (1) Dissolving collagen solution or freeze-dried collagen in a HCl aqueous solution with a pH value of 2 to 3 at low temperature;

[0009] (2) After the collagen solution is evenly dissolved, dilute the collagen concentration with water for injection, use NaOH to adjust the pH value of the collagen solution to 4.5-5.0 in stages, and slowly stir to obtain a stable collagen solution;

[0010] (3) The pH value of the stable collagen solution is adjusted to 6-8, and the solution is stirred evenly to obtain the natural undenatured collagen solution for injection.

[0011] Furthermore, in step (1), the concentration of collagen after dissolution is 8 to 10 mg / mL; NaCl is added to the solution to a concentration of 0.2 to 0.4 M during dissolution; and the low-temperature dissolution condition is stirring and dissolving at 4 to 10° C. for 2 to 6 hours.

[0012] Furthermore, in step (2), the concentration of the diluted collagen is 2 to 6 mg / mL; and the concentration of NaCl is adjusted to 0.1 to 0.2 M after dilution.

[0013] Furthermore, the process of using NaOH to adjust the pH value of the collagen solution to 4.5-5.0 in stages in step (2) is specifically as follows:

[0014] The first stage: using NaOH to adjust the pH value of the collagen solution to 3-3.8, stirring, and then proceeding to the second stage: using NaOH to adjust the pH value of the collagen solution to 4.5-5.0.

[0015] Furthermore, the concentration of NaOH in the first stage is 0.4-0.8M; the stirring time is 1-3h; and the concentration of NaOH in the second stage is 0.05-0.2M.

[0016] Furthermore, in step (2), after adjusting the pH value of the collagen solution to 4.5-5.0 in stages using NaOH, the solution is stirred at a temperature of 8-15° C. and a stirring speed of 20-50 rpm for 12-20 hours.

[0017] Furthermore, the collagen solution or freeze-dried collagen is prepared by the following steps:

[0018] (11) Soaking the animal tissue in an acid or alkaline solution and then washing it with water;

[0019] (12) The cleaned animal tissue is placed in a weak organic acid and an enzyme is added for extraction;

[0020] (13) After the extraction is completed, the material is centrifuged to obtain the supernatant, and salt is added for salting out;

[0021] (14) Centrifuge to obtain the salt precipitation, fully dissolve the precipitation in acetic acid, and then dialyze until the conductivity of the dialyzate external fluid is 50 to 200 μs / cm. The dialyzed collagen solution is frozen or freeze-dried to obtain the collagen solution or freeze-dried collagen.

[0022] Furthermore, the animal tissue is animal skin or Achilles tendon that has been defatted, dehaired, and deproteinized; before soaking in step (11), the animal tissue is rinsed with ethanol; the concentration of the ethanol is 70-80%; after soaking in step (11), the pH value of the solution system is adjusted to 6-8 and then washed with water.

[0023] Furthermore, the concentration of the acid or alkali solution is 0.2-0.4M; the mass ratio of the material to the liquid during soaking is 1:40-80; and the soaking time is 4-6h.

[0024] The second purpose of the present invention can be achieved by adopting the following technical solutions:

[0025] A natural undenatured collagen solution for injection is prepared by any of the above-mentioned methods for preparing the natural undenatured collagen solution for injection.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. The method for preparing a natural undenatured collagen solution for injection of the present invention comprises the following steps: by precisely controlling the pH value, salt concentration and system temperature of the solution, the solution is rapidly and evenly dissolved under low temperature, low pH value and salt dissolution conditions to obtain a high-concentration collagen solution; a small amount of collagen molecules are aggregated and stabilized under physiological salt concentration, weak acidity and elevated temperature conditions; and the system pH is then slowly increased to neutral to obtain a natural undenatured collagen solution containing a certain proportion of collagen molecule aggregates, which can be used for injection and can increase the duration of the injection effect.

[0028] 2. The natural undenatured collagen solution for injection of the present invention contains a certain proportion of collagen molecular aggregates, which can increase the maintenance time of the injection effect; the collagen solution can maintain a maximum pushing force below 20N and an average pushing force below 15N at a pushing speed of 20mm / min using a 30G needle, and the endotoxin in the collagen solution is less than 20EU / piece and is sterile, and can be directly used for human tissue injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the electrophoresis diagram of the natural undenatured collagen solution of Example 5;

[0030] Figure 2 is the DSC thermal denaturation temperature diagram of the natural undenatured collagen solution of Example 5;

[0031] Figure 3 This is a curve diagram of the pushing force variation of the natural undenatured collagen solution of Example 5 using a 30G needle at a pushing speed of 20 mm / min;

[0032] Figure 4 This is a curve diagram of the pushing force variation of the collagen solution of Comparative Example 1 using a 30G needle at a pushing speed of 20 mm / min;

[0033] Figure 5 The pushing force variation curve of the collagen solution of Comparative Example 2 using a 30G needle at a pushing speed of 20 mm / min;

[0034] Figure 6 The pushing force variation curve of the collagen solution of Comparative Example 3 using a 30G needle at a pushing speed of 20 mm / min;

[0035] Figure 7 This is a curve diagram of the pushing force variation of the collagen solution of Comparative Example 4 using a 30G needle at a pushing speed of 20 mm / min;

[0036] Figure 8 is a distribution diagram of collagen particle size in the natural undenatured collagen solution of Example 5;

[0037] Fig. 9 This is the distribution diagram of collagen particle size in the natural undenatured collagen solution of Comparative Example 1;

[0038] Fig.10 This is the distribution diagram of collagen particle size in the natural undenatured collagen solution of Comparative Example 2;

[0039] Fig.11 This is the distribution diagram of collagen particle size in the natural undenatured collagen solution of Comparative Example 3;

[0040] Fig.12 This is the distribution diagram of collagen particle size in the natural undenatured collagen solution of Comparative Example 4;

[0041] Fig.13 This is a graph showing the results of culturing L929 cells in a high-glucose medium containing the natural undenatured collagen solution of Example 5;

[0042] Fig.14 The figure shows the results of culturing L929 cells in a high-glucose medium without the natural undenatured collagen solution of Example 5. DETAILED DESCRIPTION

[0043] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only partial embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0044] Among them, all reagents used in the preparation process of the present invention are of pharmaceutical grade or sterilized by drying in an oven at 250° C. for 2 hours to remove endotoxins; all containers are soaked in 0.2-0.4M HCl or NaOH solution to remove endotoxins and sterilized by wet heat.

[0045] A method for preparing a natural undenatured collagen solution for injection comprises the following steps:

[0046] (1) taking a collagen solution or freeze-dried collagen and dissolving it in an aqueous solution of HCl at a pH of 2 to 3 at low temperature;

[0047] (2) After the collagen solution is evenly dissolved, dilute the collagen concentration with water for injection, use NaOH to adjust the pH value of the collagen solution to 4.5-5.0 in stages, and slowly stir to obtain a stable collagen solution;

[0048] (3) The pH value of the stable collagen solution is adjusted to 6-8, and the solution is stirred evenly to obtain the natural undenatured collagen solution for injection.

[0049] The natural undenatured collagen solution for injection prepared by the present invention uses sodium chloride as the solvent system, which can stabilize the structural stability and solubility of collagen under neutral conditions. The osmotic pressure of the system is consistent with that of body fluids, meeting the injection requirements; the endotoxin in the collagen solution is less than 20EU / piece and is sterile, and can be directly used for human tissue injection.

[0050] In one embodiment, in step (1), the concentration of collagen after dissolution is 8 to 10 mg / mL; NaCl is added to the solution to a concentration of 0.2 to 0.4 M during dissolution; and the low-temperature dissolution condition is stirring and dissolving at 4 to 10°C for 2 to 6 hours.

[0051] NaCl is added to make the collagen solution or freeze-dried collagen dissolve quickly and evenly in the acidic solution, and the concentration of NaCl is 0.2-0.4M, which is the appropriate salt concentration to promote collagen salt dissolution under acidic conditions. Too high a concentration will cause collagen salting out. The temperature and stirring rate of the system affect the dissolution and dispersion of collagen, and under the conditions of the present invention, it is possible to achieve rapid and even dissolution under low temperature, low pH value, and salt dissolution conditions to obtain a high-concentration collagen solution.

[0052] In one embodiment, in step (2), the concentration of the diluted collagen is 2 to 6 mg / mL; and the concentration of NaCl is adjusted to 0.1 to 0.2 M after dilution.

[0053] The concentration of collagen is 2-6 mg / mL, which is the final concentration of pure collagen injection. The concentration of NaCl is 0.1-0.2 M, which is consistent with the concentration of injectable physiological saline in the human body and is also the final system salt concentration of pure collagen injection, and is at a salt concentration that promotes collagen dissolution.

[0054] In one embodiment, the process of adjusting the pH value of the collagen solution to 4.5-5.0 in stages using NaOH in step (2) is specifically as follows:

[0055] The first stage: using NaOH to adjust the pH value of the collagen solution to 3-3.8, stirring, and then proceeding to the second stage: using NaOH to adjust the pH value of the collagen solution to 4.5-5.0.

[0056] The pH value of the system is adjusted to between 4.5 and 5.0 in two stages because as the pH value of the solution increases, it gradually approaches the isoelectric point of the collagen molecule, which will cause the solubility of collagen to decrease. Therefore, adjusting the pH value in stages can prevent the pH from fluctuating too much in a short period of time and causing rapid precipitation of collagen.

[0057] In one embodiment, the concentration of NaOH in the first stage is 0.4-0.8M; the stirring time is 1-3h; and the concentration of NaOH in the second stage is 0.05-0.2M.

[0058] When adjusting the pH value in the second stage, choose NaOH with a lower concentration to slow down the pH rise and prevent the pH value from fluctuating too much and causing collagen to precipitate quickly.

[0059] In one embodiment, in step (2), when the pH value of the collagen solution is adjusted to 4.5-5.0 in stages using NaOH, the temperature is 8-15° C., the stirring speed is 20-50 rpm, and the stirring is performed for 12-20 hours.

[0060] When the pH value is increased in the second stage, the temperature of the solution system is simultaneously increased to increase the solubility of collagen and maintain the solubility stability of collagen in the system; long-term stirring for 12 to 20 hours in this state can allow some collagen molecules in the system to form a small amount of tiny aggregates, and under the condition of synchronously controlling the salt concentration and temperature, the aggregates are stabilized to prevent further aggregation.

[0061] In one embodiment, after the pH value of the stable collagen solution is adjusted to 6-8 in step (3), the temperature is controlled between 10-20°C and stirred for 2-4 hours to obtain the natural undenatured collagen solution for injection; preferably, 0.05-0.2M NaOH is used to adjust the pH value.

[0062] In one embodiment, the natural undenatured injectable collagen solution is bottled in a syringe.

[0063] In one embodiment, the collagen solution or freeze-dried collagen is purchased from active collagen having a molecular weight of 300 kDa, which is a collagen solution or freeze-dried collagen.

[0064] In one embodiment, the natural undenatured collagen solution for injection comprises, by mass percentage, 0.4 wt% of active collagen with a molecular weight of 300 kDa, 0.9 wt% of sodium chloride, and the remainder of water.

[0065] In one embodiment, the collagen solution or freeze-dried collagen is prepared by the following steps:

[0066] (11) Soaking the animal tissue in an acid or alkaline solution and then washing it with water;

[0067] (12) The cleaned animal tissue is placed in a weak organic acid and an enzyme is added for extraction;

[0068] (13) After the extraction is completed, the material is centrifuged to obtain the supernatant, and salt is added for salting out;

[0069] (14) Centrifuge to obtain the salt precipitation, fully dissolve the precipitation in acetic acid, and then dialyze until the conductivity of the dialyzate external fluid is 50 to 200 μs / cm. The dialyzed collagen solution is frozen or freeze-dried to obtain the collagen solution or freeze-dried collagen.

[0070] The collagen solution or freeze-dried collagen prepared by the preparation method of the present invention is natural undenatured collagen with a triple helix structure, no modified side chain, and no other additives; and the endotoxin content is small, meeting the injection requirements.

[0071] In one embodiment, the animal tissue is animal skin or Achilles tendon that has been defatted, dehaired, and deproteinized.

[0072] In one embodiment, before soaking in step (11), the animal tissue is rinsed with ethanol; the concentration of the ethanol is 70-80%; preferably, the ethanol is 75% ethanol.

[0073] In one embodiment, after soaking in step (11), the pH value of the solution system is adjusted to 6-8 and then washed with water. Preferably, the water used for washing is water for injection, and the number of washing times is 3-5 times. This helps to remove residual salts in animal tissues.

[0074] In one embodiment, the concentration of the acid or alkali solution is 0.2-0.4M; the material-liquid mass ratio during soaking is 1:40-80; and the soaking time is 4-6h.

[0075] If the concentration of acid or alkali is too high, the collagen molecules in the animal tissue will easily denature; and the liquid ratio should not be too low, because the tissue will easily swell during the soaking process, and a too low liquid ratio may lead to incomplete removal of endotoxins.

[0076] In one embodiment, in step (12), the organic acid is one or a combination of citric acid and acetic acid; the concentration of the organic acid is 0.2-0.8 M; and the mass ratio of the animal tissue to the liquid is 1:20-40.

[0077] In one embodiment, the enzyme is one or a combination of two or more of trypsin, chymotrypsin, pepsin, and papain; and the amount of the added enzyme is 1% to 3% of the wet weight of the animal tissue.

[0078] In one embodiment, the extraction process is: extracting for 3 days at a temperature of 4 to 10°C and a stirring speed of 80 to 200 rpm, then adding water to a solid-liquid ratio of 1:10 to 20 and continuing to extract for 2 days. The extraction efficiency can be improved by adding water in batches.

[0079] In one embodiment, in step (13), the salt is one or a combination of two or more of ammonium sulfate, sodium chloride, potassium chloride, and sodium sulfate; the amount of salt added is such that the salt concentration is 0.5 to 2.0 M.

[0080] In one embodiment, the salting-out conditions are: salting-out for 3 to 5 hours at a temperature of 4 to 10° C. and a stirring speed of 150 to 300 rpm, followed by standing for 5 to 10 hours.

[0081] In one embodiment, in step (14), the concentration of acetic acid is 0.2-0.4M; the mass content of the solution after the precipitate is dissolved is 0.05-0.1%; too low a concentration of the collagen solution will lead to gelation of collagen in the dialysis bag, while too high a concentration will lead to incomplete dialysis of the solution.

[0082] In one embodiment, the dialyzed molecular weight is 8000-14000Da; the dialysis time is 2-5 days, and preferably, the liquid is replaced 3 times a day.

[0083] Embodiment 1:

[0084] After degreasing, dehairing and deproteinizing, the cowhide used for collagen extraction was rinsed with 70% ethanol three times, and then soaked in 0.2M HCl for 6 hours, with a liquid ratio of 1:40 to remove endotoxins; after soaking, the pH value of the system was adjusted to 6. After pouring out the liquid, it was washed with injection water for 3 times, and then the treated animal tissue was added to 0.2M acetic acid, with a liquid ratio of 1:20, and then 1% of the wet weight of the animal tissue pepsin was added, the temperature was 4°C, the stirring speed was 80rpm, and after 3 days of extraction, the injection water was supplemented to a liquid ratio of 1:10 and the extraction was continued for 2 days. After the extraction was completed, the material was centrifuged, the supernatant was taken, and 0.5M sodium chloride was added for salting out for 4 hours, stirred at 150rpm, the temperature was 4°C, and then stood for 8 hours. The salt precipitation was centrifuged and dissolved in 0.2 M acetic acid. After fully dissolved, it was dialyzed against 0.05% acetic acid solution in a dialysis bag with a molecular weight of 8000-14000 Da for 4 days, with the solution changed 3 times a day. The conductivity of the dialyzed external fluid was finally measured to be 50-200 μs / cm. The dialyzed collagen solution was sterilized and frozen or freeze-dried for storage.

[0085] Embodiment 2:

[0086] After degreasing, dehairing and deproteinizing, the cowhide used for collagen extraction was rinsed with 75% ethanol three times, soaked in 0.3M HCl for 2.5h, and then soaked in 0.3M NaOH aqueous solution for 2.5h, with a liquid ratio of 1:60, to remove endotoxins; after soaking, the pH of the system was adjusted to 7. After pouring out the liquid, it was washed 4 times with injection water to remove the residual salt in the tissue. The treated animal tissue was in 0.5M acetic acid, with a liquid ratio of 1:30, and 2% of the wet weight of the tissue was added with pepsin, the temperature was 7°C, the stirring speed was 140rpm, and after 3 days of extraction, the injection water was supplemented to a liquid ratio of 1:15 and the extraction was continued for 2 days. After the extraction was completed, the material was centrifuged to take the supernatant, and 1.3M sodium chloride was added for salting out for 4h, stirred at 230rpm, the temperature was 7°C, and then left to stand for 8h. The salt precipitation was obtained by centrifugation and dissolved in 0.3 M acetic acid. After being fully dissolved, the product was dialyzed against 0.07% acetic acid solution in a dialysis bag with a molecular weight of 8000-14000 Da for 4 days, with the solution changed 3 times a day. The conductivity of the dialyzed external fluid was finally measured to be 50-200 μs / cm. The dialyzed collagen solution was sterilized and frozen or freeze-dried for storage.

[0087] Embodiment 3:

[0088] After degreasing, dehairing and deproteinizing, the cowhide used for collagen extraction was rinsed twice with 80% ethanol, and then soaked in 0.4M NaOH aqueous solution for 4 hours, with a liquid ratio of 1:80, to remove endotoxins; after soaking, the pH of the system was adjusted to 8. After pouring out the liquid, wash it with injection water 5 times to remove the residual salt in the tissue. The treated animal tissue was in 0.8M acetic acid, with a liquid ratio of 1:40, and 3% of the wet weight of the tissue was added with pepsin, the temperature was 10°C, and the stirring speed was 200rpm. After extraction for 3 days, the injection water was supplemented to a liquid ratio of 1:20 and the extraction was continued for 2 days. After the extraction was completed, the material was centrifuged to take the supernatant, and 2.0M sodium chloride was added for salting out for 4 hours, stirred at 300rpm, and the temperature was 10°C, and then left to stand for 8 hours. The salt precipitation was obtained by centrifugation and dissolved in 0.4 M acetic acid. After being fully dissolved, the product was dialyzed against 0.1% acetic acid solution in a dialysis bag with a molecular weight of 8000-14000 Da for 4 days, with the solution changed 3 times a day. The conductivity of the dialyzed external fluid was finally measured to be 50-200 μs / cm. The dialyzed collagen solution was sterilized and frozen or freeze-dried for storage.

[0089] Embodiment 4:

[0090] Take the sterilized collagen solution and dilute it with a pH 2 HCl aqueous solution to a collagen concentration of 8 mg / mL, with a final total volume of 1 L. Slowly add NaCl crystals to the collagen solution to make the NaCl concentration of the system 200 mM, and stir in a low-temperature water bath at 4°C for 2 hours. After stirring, dilute the collagen concentration of the system to 2 mg / mL with water for injection, and add NaCl to make its concentration 100 mM. Slowly increase the pH value of the system to 3 with a 0.4M NaOH aqueous solution and stir in a water bath at 4°C for 2 hours. Then use a 0.05M NaOH aqueous solution to slowly increase the pH value to 4.5, while raising the water bath temperature to 8°C, slowing the rotation speed to 20 rpm, and stirring for 12 hours. After the system is stirred evenly and stably, adjust the pH to 6 with 0.05M NaOH aqueous solution, stir in a 10°C water bath for 2 hours, and finally, under negative pressure and aseptic conditions, fill the prepared collagen solution into a 3mL prefilled syringe to obtain a natural undenatured collagen solution for injection.

[0091] Embodiment 5:

[0092] Take the sterilized and frozen collagen solution prepared in Example 2, dilute it with an aqueous solution of HCl with a pH value of 2.5 to a collagen concentration of 9 mg / mL, and the final total volume is 1L. Slowly add NaCl crystals to the collagen solution to make the NaCl concentration of the system 300mM, and stir it in a low-temperature water bath at 7°C for 2h. After stirring, dilute the collagen concentration of the system to 4mg / mL with water for injection, and add NaCl to make its concentration 150mM. Slowly increase the pH value of the system to 3.4 with a 0.6M NaOH aqueous solution and stir it in a water bath at 7°C for 2h. Then use a 0.1M NaOH aqueous solution to slowly increase the pH value to 4.7, while raising the water bath temperature to 12°C, slowing the speed to 35rpm, and stirring for 12h. After the system is stirred evenly and stably, increase the pH value to 7.4 with a 0.1M NaOH aqueous solution and stir in a 15°C water bath for 3h. Finally, under negative pressure and aseptic conditions, the prepared collagen solution is bottled into a 3 mL pre-filled syringe to obtain a natural undenatured collagen solution for injection.

[0093] Embodiment 6:

[0094] Take the sterilized collagen solution and dilute it with a pH 3 HCl aqueous solution to a collagen concentration of 10 mg / mL, and the final total volume is 1L. Slowly add NaCl crystals to the collagen solution to make the NaCl concentration of the system 400mM, and stir it in a low-temperature water bath at 10°C for 2h. After stirring, dilute the collagen concentration of the system to 6mg / mL with water for injection, and add NaCl to make its concentration 200mM. Slowly increase the pH of the system to 3.8 with a 0.8M NaOH aqueous solution and stir it in a water bath at 10°C for 2h. Then use a 0.2M NaOH aqueous solution to slowly increase the pH value to 5.0, while raising the water bath temperature to 15°C, slowing the speed to 50rpm, and stirring for 12h. After the system is stirred evenly and stably, increase the pH value to 8.0 with a 0.1M NaOH aqueous solution and stir in a 20°C water bath for 4h. Finally, under negative pressure and aseptic conditions, the prepared collagen solution is bottled into a 3 mL pre-filled syringe to obtain a natural undenatured collagen solution for injection.

[0095] Comparative Example 1:

[0096] Compared with Example 5, the pH value of the final product of Comparative Example 1 is 4.7, which is controlled to be weakly acidic rather than neutral. The preparation process is as follows:

[0097] Take the sterilized and frozen collagen solution prepared in Example 2, dilute it with an aqueous HCl solution with a pH value of 2.5 to a collagen concentration of 9 mg / mL, and the final total volume is 1L. Slowly add NaCl crystals to the collagen solution to make the NaCl concentration of the system 300mM, and stir it at a low temperature water bath at 7°C for 2h. After stirring, dilute the collagen concentration of the system to 4mg / mL with water for injection, and add NaCl to make its concentration 150mM. Slowly increase the pH value of the system to 3.4 with a 0.6M NaOH aqueous solution and stir it at 7°C in a water bath for 2h. Then use a 0.1M NaOH aqueous solution to slowly increase the pH value to 4.7, while raising the water bath temperature to 12°C, slowing the speed to 35rpm, and stirring for 12h. Finally, under negative pressure and aseptic conditions, the prepared collagen solution is canned in a 3mL prefilled syringe to obtain a collagen solution for injection.

[0098] Comparative Example 2:

[0099] Compared with Example 5, in Comparative Example 2, when adjusting the pH value of the system from acidic to neutral, a one-time NaOH solution was used to directly and quickly adjust the pH value. The preparation process is as follows:

[0100] Take the sterilized and frozen collagen solution prepared in Example 2, dilute it with an aqueous HCl solution with a pH value of 2.5 to a collagen concentration of 9 mg / mL, and the final total volume is 1 L. Slowly add NaCl crystals to the collagen solution to make the NaCl concentration of the system 300 mM, and stir it in a low-temperature water bath at 7°C for 2 hours. After stirring, dilute the collagen concentration of the system to 4 mg / mL with water for injection, and add NaCl to make its concentration about 150 mM. Use 0.6M NaOH aqueous solution to adjust the pH value of the system to 7.4, and stir it in a water bath at 7°C for 12 hours. Finally, under negative pressure and aseptic conditions, the prepared collagen solution is canned in a 3mL prefilled syringe to obtain a collagen solution for injection.

[0101] Comparative Example 3:

[0102] Compared with Example 5, the final solvent system of Comparative Example 3 contains 0.01 M PBS, and the preparation process is as follows:

[0103] Take the sterilized and frozen collagen solution prepared in Example 2, dilute it with an aqueous solution of HCl with a pH value of 2.5 to a collagen concentration of 9 mg / mL, and the final total volume is 1L. Slowly add NaCl crystals, 4.4g disodium hydrogen phosphate dodecahydrate, and 0.4g sodium dihydrogen phosphate dihydrate to the collagen solution, and stir it in a low-temperature water bath at 7°C for 2h. After stirring, dilute the collagen concentration of the system to 4mg / mL with water for injection, and add NaCl to make the solvent system 0.01M PBS sodium chloride concentration of 150mM. Slowly adjust the pH value of the system to 3.4 with a 0.6M NaOH aqueous solution and stir it in a water bath at 7°C for 2h. Then use a 0.1M NaOH aqueous solution to slowly increase the pH value to 4.7, while raising the water bath temperature to 12°C, slowing the speed to 35rpm and stirring for 12h. After the system was stirred evenly and stably, the pH value was adjusted to 7.4 with 0.1M NaOH aqueous solution and stirred in a 15°C water bath for 3 hours. Finally, under negative pressure and aseptic conditions, the prepared collagen solution was filled into a 3mL pre-filled syringe to obtain a natural undenatured collagen solution for injection.

[0104] Comparative Example 4:

[0105] Compared with Example 5, the product of Comparative Example 4 was not stirred for 12 hours and was directly filled. The preparation process was as follows:

[0106] Take the sterilized and frozen collagen solution prepared in Example 2, dilute it with an aqueous HCl solution with a pH value of 2.5 to a collagen concentration of 9 mg / mL, and the final total volume is 1L. Slowly add NaCl crystals to the collagen solution to make the NaCl concentration of the system 300mM, and stir it in a low-temperature water bath at 7°C for 2h. After stirring, dilute the collagen concentration of the system to 4mg / mL with water for injection, and add NaCl to make its concentration 150mM. Slowly increase the pH of the system to 3.4 with a 0.6M NaOH aqueous solution and stir it in a water bath at 7°C for 2h. Then use a 0.1M NaOH aqueous solution to slowly increase the pH to 4.7. After the system is stirred evenly and stably, use a 0.1M NaOH aqueous solution to increase the pH to 7.4, and stir in a 15°C water bath for 3h. Finally, under negative pressure and aseptic conditions, the prepared collagen solution is canned in a 3mL pre-filled syringe to obtain a natural undenatured collagen solution for injection.

[0107] Test example

[0108] Electrophoresis analysis experiment

[0109] The natural undenatured collagen solution for injection of Example 5 was subjected to electrophoresis analysis. The electrophoresis results are as follows: Figure 1 As shown. Figure 1It can be seen from the electrophoresis results that the natural undenatured collagen solution for injection in Example 5 exhibits a characteristic triple helical structure band of collagen, with two α1 chains and one α2 chain around 100 kDa, and a total molecular weight of about 300 kDa.

[0110] Thermal stability test

[0111] The natural undenatured collagen solution for injection of Example 5 was subjected to a thermal stability test to obtain a DSC thermal denaturation temperature diagram, as shown in FIG. Figure 2 As shown. Figure 2 It can be seen from the DSC results that the denaturation temperature of collagen in the natural undenatured collagen solution for injection in Example 5 is about 39.7°C, which is consistent with the denaturation temperature of bovine skin collagen reported in the literature, indicating that it has high stability and a complete triple helix structure.

[0112] Push force test:

[0113] After centrifugation to remove bubbles, the collagen solutions of Example 5 and Comparative Examples 1-4 were filled into a 3 mL syringe and a 30G needle was used to measure the pushing force of the samples at a pushing speed of 20 mm / min using a tensile testing machine (equipped with a special kit for measuring pushing force). 15 seconds of the sample test process was taken as the object of investigation, and a curve of the change of the sample pushing force during the pushing process was drawn, as shown in FIG. Figure 3-Figure 7 As shown:

[0114] from Figure 3-7 According to analysis, the natural undenatured collagen solution for injection in Example 5 has a maximum pushing force of about 5N and an average pushing force of less than 5N, which meets the requirements. The collagen solution of Comparative Example 1 is an acidic sample, and the system is a uniform collagen solution without collagen aggregates, so the pushing force meets the requirements, but the product is acidic, which may have adverse effects; and there are no collagen aggregates in the system, and the maintenance time is short. When the pH of the collagen solution of Comparative Example 2 is adjusted to neutral, sodium hydroxide is used to increase it once, resulting in rapid and uneven precipitation of collagen. After the uneven collagen aggregates accumulate, the syringe needle is blocked, and the pushing force increases uniformly, with a maximum pushing force of about 45N. The final system of the collagen solution of Comparative Example 3 contains a phosphate system of 0.01M, which is not conducive to the dissolution of collagen molecules under neutral regulation. There are more collagen aggregates settling in the system, resulting in large fluctuations in the injection pushing force, and the maximum pushing force reaches about 37N. The collagen solution in Comparative Example 4 was not stirred for a long time, resulting in uneven sizes of collagen aggregates inside it, causing unstable final pushing force, with a maximum pushing force of about 25 N. Therefore, it can be known that the natural undenatured collagen solution for injection prepared by the method of the present invention has a small pushing force when injected with a 30G needle, which can meet the injection requirements. The collagen solutions in other Comparative Examples 2-4 have a large pushing force and are not suitable for injection.

[0115] Particle size analysis experiment:

[0116] The collagen solutions of Example 5 and Comparative Examples 1-4 were tested for molecular size distribution using dynamic light scattering. The testing method is as follows: all sample solutions were added to polystyrene cuvettes for testing. In the solution, collagen molecules will perform irregular Brownian motion. When dynamic light scattering is used to analyze the particle size distribution of collagen, the particles in the solution will scatter the light passing through. The mutual superposition of scattered light will cause the light intensity of the dark and bright areas of the particles to overlap or decrease, causing the light intensity to fluctuate. The collagen particles in the solution will also perform irregular motion after being continuously impacted by the Brownian motion of the solvent molecules around them. Under the irradiation of the laser, the scattered light intensity of the moving particles will also fluctuate. The frequency of the fluctuation is related to the size of the particles. The changes in the speed of ion fluctuations are recorded by dynamic light scattering to obtain the particle size distribution information of the particles in the solution. The results are as follows: Figure 8-12 shown.

[0117] from Figure 8 It can be seen that the particle size of the natural undenatured collagen solution for injection in Example 5 is concentrated in the range of 20-28nm and 600-850nm, which are two different particle size distributions unique to collagen molecules. There are about 15% collagen molecules at 20000-80000nm, which is the particle size distribution range of collagen aggregates. Therefore, the average particle size of the natural undenatured collagen solution for injection in Example 5 is 8035.55nm, and the maximum aggregate particle size is distributed in the range of 20000-80000nm (20-80μm). The inner diameter of a 30G needle is about 150μm, so the natural undenatured collagen solution for injection in Example 2 can be injected evenly; of which single molecule collagen accounts for about 85%, and collagen aggregates account for about 15%.

[0118] from Fig. 9 It can be seen that the collagen solution of Comparative Example 1 is an acidic solution, there is no collagen aggregate in the solution, uniform single molecule collagen, and the average particle size is 888.23 nm, which will result in a short injection maintenance time.

[0119] from Fig.10 It can be seen that when the pH of the collagen solution of Comparative Example 2 is adjusted to neutral, sodium hydroxide is used to adjust it once, and its average particle size is 113811.7nm, of which the particle size distribution of collagen aggregates is 102400~445890nm (102.4~445.89μm), accounting for about 66.6%. This shows that there are a large number of uneven aggregates in the sample, and the inner diameter of the 30G needle is about 150μm, so the collagen solution of Comparative Example 2 will cause needle blockage during injection.

[0120] from Fig.11It can be seen that the collagen solution of Comparative Example 3 is a phosphate system solution. Since phosphate will reduce the solubility of collagen under neutral conditions and cause collagen aggregation, the collagen solution of Comparative Example 3 has multiple groups of particle size distributions, among which aggregates in the range of 126870 to 201490 nm account for 29.47%, which will cause the sample to block the needle.

[0121] The collagen solution of Comparative Example 4 was not stirred for a long time. Fig.12 It can be seen that the collagen solution of Comparative Example 4 also has multiple groups of uneven particle size distribution ranges, and the maximum particle size distribution is 56980-254720 nm, and there is also a risk of needle clogging.

[0122] Therefore, from Figure 8-12 It can be known that the particle size and distribution of the collagen solution prepared by different preparation methods are different. Therefore, the natural undenatured collagen solution for injection prepared by the preparation method of the present invention has collagen particles within the inner diameter range of a 30G needle, with a small pushing force and no needle blockage. In addition to single-molecule collagen, it contains a small amount of collagen aggregates, which can increase the duration of the injection effect.

[0123] Cell culture experiments:

[0124] L929 cells were cultured in a high-glucose medium containing the natural undenatured injection collagen solution of Example 5, and the control group cultured in the same high-glucose medium without adding the natural undenatured injection collagen solution of Example 5. The culture results are as follows: Figure 13-14 shown.

[0125] contrast Figure 13-14 The culture medium results show that: Fig.13 The number and density of cells in Fig.14 ,illustrate Fig.13 In Example 5, the natural undenatured injectable collagen solution can serve as a scaffold for cell growth and attachment after being injected into the skin, induce the proliferation of fibroblasts, accelerate the repair of the skin at the cosmetic injection site, and improve the condition of the facial skin.

[0126] In summary, the method for preparing a natural undenatured collagen solution for injection of the present invention accurately controls the pH value, salt concentration and system temperature of the solution so that the solution is quickly and evenly dissolved under low temperature, low pH value and salt dissolution conditions to obtain a high-concentration collagen solution; a small amount of collagen molecules are formed into aggregates and stabilized under physiological salt concentration, weak acidity and elevated temperature; then the pH of the system is slowly increased to neutral to obtain a natural undenatured collagen solution in which a certain proportion of collagen molecule aggregates exist. The collagen solution is pushed at a speed of 20 mm / min and uses a 30G needle. The maximum pushing force is maintained below 20 N and the average pushing force is below 15 N. The endotoxin in the collagen solution is less than 20 EU / piece and is sterile, so the collagen solution can be used for injection, can be directly used for human tissue injection, and can increase the maintenance time of the injection effect.

[0127] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A method for preparing a natural undenatured collagen solution for injection, characterized in that: The following steps are involved: (1) Dissolve the collagen solution or freeze-dried collagen in a HCl aqueous solution with a pH value of 2 to 3 at low temperature; the concentration of the collagen after dissolution is 8 to 10 mg / mL; add NaCl to the solution to a concentration of 0.2 to 0.4 M during dissolution; the low temperature dissolution condition is stirring and dissolving at 4 to 10°C for 2 to 6 hours; (2) After the collagen solution is evenly dissolved, dilute the collagen concentration with water for injection, use NaOH to adjust the pH value of the collagen solution in stages, and slowly stir to obtain a stable collagen solution; the concentration of the diluted collagen is 2 to 6 mg / mL; after dilution, adjust the concentration of NaCl to 0.1 to 0.2 M; (3) using 0.05-0.2 M NaOH, adjusting the pH value of the stabilized collagen solution to 6-8, and stirring evenly to obtain the natural undenatured collagen solution for injection; The staged adjustment is as follows: in the first stage: using NaOH with a concentration of 0.4-0.8M to adjust the pH value of the collagen solution to 3-3.8, stirring for 1-3 hours; then performing the second stage: stirring for 12-20 hours at a temperature of 8-15°C and a stirring speed of 20-50rpm, and using 0.05-0.2M NaOH to adjust the pH value of the collagen solution to 4.5-5.

0.

2. The method for preparing a natural undenatured collagen solution for injection according to claim 1, characterized in that: The collagen solution or freeze-dried collagen is prepared by the following steps: (11) Soaking the animal tissue in an acid or alkaline solution and then washing it with water; (12) The cleaned animal tissue is placed in a weak organic acid and extracted by adding enzymes; (13) After the extraction is completed, the material is centrifuged to obtain the supernatant, and salt is added for salting out; (14) The salting-out precipitate is obtained by centrifugation, the precipitate is fully dissolved in acetic acid, and then dialyzed until the conductivity of the dialyzate external fluid is 50 to 200 μs / cm. The dialyzed collagen solution is frozen or freeze-dried to obtain the collagen solution or freeze-dried collagen.

3. The method for preparing a natural undenatured collagen solution for injection according to claim 2, characterized in that: The animal tissue is animal skin or Achilles tendon that has been defatted, dehaired, and deproteinized; before soaking in step (11), the animal tissue is rinsed with ethanol; the concentration of the ethanol is 70-80%; after soaking in step (11), the pH value of the solution system is adjusted to 6-8 and then washed with water.

4. The method for preparing a natural undenatured collagen solution for injection according to claim 3, characterized in that: The concentration of the acid or alkali solution in step (11) is 0.2-0.4M; the mass ratio of the material to the liquid during soaking is 1:40-80; and the soaking time is 4-6h.

5. A natural undenatured collagen solution for injection, characterized in that: The method is prepared by the method for preparing the natural undenatured collagen solution for injection according to any one of claims 1 to 4.

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