Background
The skin can be gradually aged with time under the influence of external environmental factors such as endogenous aging of the organism, ultraviolet rays, environmental pollution and the like. The appearance of skin aging is manifested by thinning, dryness, wrinkles, sagging, pigmentation, etc. With age, collagen, elastin and hyaluronic acid synthesis ability in skin decreases. Skin aging is largely attributable to dermal fibroblast dysfunction and a decrease in its biosynthetic activity. Therefore, by injecting substances such as sodium hyaluronate, collagen, antioxidants and the like, better microenvironment and oxidation stress resistance are provided for fibroblasts, so that the skin state is improved, and the skin aging is delayed.
The products in the market at present mainly comprise sodium hyaluronate. Hyaluronic acid is a non-sulfated glycosaminoglycan long-chain polymer, has no species specificity, does not need skin test before injection, has the characteristics of softness, stability, good biocompatibility and the like, is easy to inject and maintain the form, does not combine with local cells and has no inflammatory reaction. The hyaluronic acid has extremely strong water locking function, can be combined with water with the weight of about 1000 times, and can quickly fill skin tissues when being injected into skin dermis layers, thereby producing a cosmetic effect. But the half-life of hyaluronic acid in the skin is very short. Under the action of hyaluronidase, long-chain hyaluronic acid is rapidly degraded into small molecule fragments, and then enters the circulatory system to be metabolically absorbed. Hyaluronan degradation is related to hyaluronidase, temperature, or oxygen radicals. By chemical crosslinking, rapid degradation of hyaluronic acid can be avoided, thereby prolonging the duration of action. Among the products on the market, 1, 4-butanediol diglycidyl ether (BDDE) and divinyl sulfone (DVS) are mainly used as crosslinking agents. Both crosslinking agents have biotoxicity, and the incomplete removal of the crosslinking agents can cause red swelling, allergy and other adverse reactions. In addition, sodium hyaluronate crosslinked by the chemical crosslinking agent has certain propping property, is not easy to diffuse at an injection position, and can form bulges. Collagen can promote proliferation and repair of fibroblast, and has good biocompatibility, biodegradability and bioactivity. The intradermal injection of collagen can supplement collagen in the skin in situ, improve skin quality, reduce static lines, improve skin relaxation and enhance skin elasticity; simultaneously, the synthesis of new collagen and the repair and remodelling of tissues are stimulated; amino acids after metabolism of the material can also be used as nutritional ingredients to continuously stimulate fibroblasts. The collagen products on the market at present are mainly animal-derived collagen. For example, "double skin-beautifying soft skin" is collagen extracted from pig skin, and "French" is bovine collagen. Animal-derived collagen is extracted from bovine Achilles tendon, pig skin, fish skin and other tissues, and there is a risk of immune reaction and virus carrying. In recent years, researchers have prepared recombinant collagen by genetic engineering techniques. The recombinant collagen is prepared by adopting recombinant DNA technology to carry out genetic manipulation and/or modification on genes encoding required human collagen, taking target genes into proper host cells (cells, yeast or other eukaryotic cells and the like) by using plasmids or viral vectors, expressing and translating the target genes into collagen or polypeptides similar to the collagen, extracting and purifying the target genes and the polypeptides. Compared with animal-derived collagen, the recombinant collagen has the advantages of good water solubility, low immunogenicity, no risk of cross infection and the like. Therefore, developing a recombinant III type humanized collagen and crosslinked sodium hyaluronate composite gel for injection and a preparation method thereof is a technical problem to be solved urgently in the field
Disclosure of Invention
(One) solving the technical problems
Aiming at the defects of the prior art, the invention provides recombinant III type humanized collagen and crosslinked sodium hyaluronate composite gel for injection and a preparation method thereof, and solves the problems.
(II) technical scheme
In order to achieve the above purpose, the present invention provides the following technical solutions: the recombinant III type humanized collagen and crosslinked sodium hyaluronate composite gel for injection is characterized by comprising the following raw materials: collagen 15 mg/mL; hyaluronic acid 15 mg/mL; mannitol 12 mg/mL; lidocaine 3 mg/mL
The preparation method of the recombinant III type humanized collagen and crosslinked sodium hyaluronate composite gel for injection is characterized by comprising the following steps of:
1) Preparing medical crosslinked gel, namely dissolving sodium hyaluronate in an alkaline solution, stirring to completely dissolve the sodium hyaluronate, and enabling the mass concentration of the hyaluronic acid in a mixed solution to be 6% -20%;
2) Dialyzing;
3) Sieving to obtain gel A, adding into phosphate buffer solution, stirring for dissolving, adding antioxidant, stirring for dissolving completely, adding pH regulator to adjust pH to 6-8, and preparing into sodium hyaluronate-recombinant collagen mixed solution;
4) Preparing a collagen solution B, adding the collagen solution B into a phosphate buffer solution, stirring and dissolving uniformly, adding an antioxidant, stirring until the antioxidant is completely dissolved, and then adding a pH regulator to regulate the pH to 6-8 to prepare a sodium hyaluronate-recombinant collagen mixed solution;
5) Adding the solution A into the solution B, uniformly stirring, fully absorbing, reacting for 2-24 hours at room temperature to obtain micro-crosslinked sodium hyaluronate-recombinant collagen composite gel, wherein the concentration of sodium hyaluronate in the micro-crosslinked sodium hyaluronate-recombinant collagen composite gel is 1-100 mg/mL, the concentration of recombinant collagen is 0.1-100 mg/mL, the concentration of enzyme inhibitor is 0.01-10 mg/mL, and the concentration of antioxidant is 0.1-10 mg/mL; the stirring is carried out uniformly, the stirring is carried out,
6) Vacuum drying, dewatering, and increasing gel viscosity to obtain gel C, wherein the bioactivity of the gel is improved along with the increase of viscosity;
7) Preparing trehalose solution D;
8) Adding HA dry powder into the solution D, shaking uniformly, and E;
9) Mixing E with C to obtain gel F (E is coated on the surface of C), wherein trehalose can improve heat resistance and increase stability of protein under hot environment;
10 Vacuum drying gel F, removing a part of water, and changing the viscosity of the gel again, thereby making the gel have higher bioactivity and heat resistance.
Preferably, the composite gel of the recombinant III-type humanized collagen and the crosslinked sodium hyaluronate has the appearance of colorless, transparent and free of any macroscopic foreign matter.
Preferably, the pH value of the recombinant III type humanized collagen and crosslinked sodium hyaluronate composite gel is 6.8-7.6.
Preferably, the composite gel osmotic pressure (mOsmol/L) of the recombinant type III humanized collagen and the crosslinked sodium hyaluronate is 270-350.
Preferably, the heavy metal content in the recombinant III-type humanized collagen and crosslinked sodium hyaluronate composite gel is less than 5 mug/g.
Preferably, the detection result of the hemolytic streptolysin in the recombinant III-type humanized collagen and crosslinked sodium hyaluronate composite gel is that no hemolytic ring exists.
Preferably, the bacterial endotoxin in the recombinant III-type humanized collagen and crosslinked sodium hyaluronate composite gel is less than 1.0EU/mL,
Preferably, the recombinant III humanized collagen and crosslinked sodium hyaluronate composite gel is used for performing a skin sensitization test on Dunkin Hartley guinea pigs, wherein the incidence rate of positive excitation results of the guinea pigs is 0%, and skin sensitization reaction is not caused.
Preferably, the recombinant type III humanized collagen and crosslinked sodium hyaluronate composite gel for injection is used for carrying out cytotoxicity test on mammalian cells (mouse fibroblasts), wherein the relative activity of the cells is more than 70%, and the composite gel has no potential cytotoxicity.
(III) beneficial effects
Compared with the prior art, the invention provides the recombinant III type humanized collagen and crosslinked sodium hyaluronate composite gel for injection and the preparation method thereof, and the recombinant III type humanized collagen and crosslinked sodium hyaluronate composite gel has the following beneficial effects:
1. The recombinant III-type humanized collagen and crosslinked sodium hyaluronate composite gel for injection and the preparation method thereof have the effects of improving periorbital, filling recesses, smoothing wrinkles and improving skin firmness and plumpness.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Embodiment one:
1) Preparing medical crosslinked gel, namely dissolving sodium hyaluronate in an alkaline solution, stirring to completely dissolve the sodium hyaluronate, and enabling the mass concentration of the hyaluronic acid in a mixed solution to be 10%;
2) Dialyzing;
3) Sieving to obtain gel A, adding into phosphate buffer solution, stirring for dissolving, adding antioxidant, stirring for dissolving completely, and adding pH regulator to adjust pH to 7.0 to obtain sodium hyaluronate-recombinant collagen mixed solution;
4) Preparing a collagen solution B, adding the collagen solution B into a phosphate buffer solution, stirring and dissolving uniformly, adding an antioxidant, stirring until the antioxidant is completely dissolved, and then adding a pH regulator to regulate the pH to 7.0 to prepare a sodium hyaluronate-recombinant collagen mixed solution;
5) Adding the solution A into the solution B, uniformly stirring, fully absorbing, reacting for 8 hours at room temperature to obtain micro-crosslinked sodium hyaluronate-recombinant collagen composite gel, wherein in the micro-crosslinked sodium hyaluronate-recombinant collagen composite gel, the concentration of sodium hyaluronate is 50g/mL, the concentration of recombinant collagen is 10mg/mL, the concentration of enzyme inhibitor is 50mg/mL, the concentration of antioxidant is 50mg/mL, and uniformly stirring;
6) Vacuum drying, dewatering, and increasing gel viscosity to obtain gel C, wherein the bioactivity of gel increases with viscosity.
7) Preparing trehalose solution D;
8) Adding HA dry powder into the solution D, shaking uniformly, and E;
9) Mixing E with C to obtain gel F (E is coated on the surface of C), and trehalose can improve heat resistance and increase protein stability under hot environment.
10 Vacuum drying gel F, removing a part of water, and changing the viscosity of the gel again, thereby making the gel have higher bioactivity and heat resistance.
Embodiment two:
1) Preparing medical crosslinked gel, namely dissolving sodium hyaluronate in an alkaline solution, stirring to completely dissolve the sodium hyaluronate, and enabling the mass concentration of the hyaluronic acid in a mixed solution to be 10%;
2) Dialyzing;
3) Sieving to obtain gel A, adding into phosphate buffer solution, stirring for dissolving, adding antioxidant, stirring for dissolving completely, and adding pH regulator to adjust pH to 7.0 to obtain sodium hyaluronate-recombinant collagen mixed solution;
4) Preparing a collagen solution B, adding the collagen solution B into a phosphate buffer solution, stirring and dissolving uniformly, adding an antioxidant, stirring until the antioxidant is completely dissolved, and then adding a pH regulator to regulate the pH to 7.0 to prepare a sodium hyaluronate-recombinant collagen mixed solution;
5) Adding the solution A into the solution B, stirring uniformly, fully absorbing, reacting for 16 hours at room temperature to obtain micro-crosslinked sodium hyaluronate-recombinant collagen composite gel, wherein in the micro-crosslinked sodium hyaluronate-recombinant collagen composite gel, the concentration of sodium hyaluronate is 50mg/mL, the concentration of recombinant collagen is 10mg/mL, the concentration of enzyme inhibitor is 50mg/mL, the concentration of antioxidant is 50mg/mL, and stirring uniformly;
6) Vacuum drying, dewatering, and increasing gel viscosity to obtain gel C, wherein the bioactivity of the gel is improved along with the increase of viscosity;
7) Preparing trehalose solution D;
8) Adding HA dry powder into the solution D, shaking uniformly, and E;
9) Mixing E with C to obtain gel F (E is coated on the surface of C), and trehalose can improve heat resistance and increase protein stability under hot environment.
10 Vacuum drying gel F, removing a part of water, and changing the viscosity of the gel again, thereby making the gel have higher bioactivity and heat resistance.
Embodiment III:
1) Preparing medical crosslinked gel, namely dissolving sodium hyaluronate in an alkaline solution, stirring to completely dissolve the sodium hyaluronate, and enabling the mass concentration of the hyaluronic acid in a mixed solution to be 10%;
2) Dialyzing;
3) Sieving to obtain gel A, adding into phosphate buffer solution, stirring for dissolving, adding antioxidant, stirring for dissolving completely, and adding pH regulator to adjust pH to 6 to obtain sodium hyaluronate-recombinant collagen mixed solution;
4) Preparing a collagen solution B, adding the collagen solution B into a phosphate buffer solution, stirring and dissolving uniformly, adding an antioxidant, stirring until the antioxidant is completely dissolved, and then adding a pH regulator to regulate the pH to 6 to prepare a sodium hyaluronate-recombinant collagen mixed solution;
5) Adding the solution A into the solution B, uniformly stirring, fully absorbing, reacting for 8 hours at room temperature to obtain micro-crosslinked sodium hyaluronate-recombinant collagen composite gel, wherein in the micro-crosslinked sodium hyaluronate-recombinant collagen composite gel, the concentration of sodium hyaluronate is 50mg/mL, the concentration of recombinant collagen is 10mg/mL, the concentration of enzyme inhibitor is 50mg/mL, the concentration of antioxidant is 50mg/mL, and uniformly stirring;
6) Vacuum drying, dewatering, and increasing gel viscosity to obtain gel C, wherein the bioactivity of the gel is improved along with the increase of viscosity;
7) Preparing trehalose solution D;
8) Adding HA dry powder into the solution D, shaking uniformly, and E;
9) Mixing E with C to obtain gel F (E is coated on the surface of C), wherein trehalose can improve heat resistance and increase stability of protein under hot environment;
10 Vacuum drying gel F, removing a part of water, and changing the viscosity of the gel again, thereby making the gel have higher bioactivity and heat resistance.
Embodiment four:
1) Preparing medical crosslinked gel, namely dissolving sodium hyaluronate in an alkaline solution, stirring to completely dissolve the sodium hyaluronate, and enabling the mass concentration of the hyaluronic acid in a mixed solution to be 10%;
2) Dialyzing;
3) Sieving to obtain gel A, adding into phosphate buffer solution, stirring for dissolving, adding antioxidant, stirring for dissolving completely, and adding pH regulator to adjust pH to 6 to obtain sodium hyaluronate-recombinant collagen mixed solution;
4) Preparing a collagen solution B, adding the collagen solution B into a phosphate buffer solution, stirring and dissolving uniformly, adding an antioxidant, stirring until the antioxidant is completely dissolved, and then adding a pH regulator to regulate the pH to 6 to prepare a sodium hyaluronate-recombinant collagen mixed solution;
5) Adding the solution A into the solution B, stirring uniformly, fully absorbing, reacting for 16 hours at room temperature to obtain micro-crosslinked sodium hyaluronate-recombinant collagen composite gel, wherein in the micro-crosslinked sodium hyaluronate-recombinant collagen composite gel, the concentration of sodium hyaluronate is 50mg/mL, the concentration of recombinant collagen is 10mg/mL, the concentration of enzyme inhibitor is 50mg/mL, the concentration of antioxidant is 50mg/mL, and stirring uniformly;
6) Vacuum drying, dewatering, and increasing gel viscosity to obtain gel C, wherein the bioactivity of gel increases with viscosity.
7) Preparing trehalose solution D
8) Adding HA dry powder into D, shaking, E
9) Mixing E with C to obtain gel F (E is coated on the surface of C), and trehalose can improve heat resistance and increase protein stability under hot environment.
10 Vacuum drying gel F, removing a part of water, and changing the viscosity of the gel again, thereby making the gel have higher bioactivity and heat resistance.
Judgment standard: in the first, second, third and fourth examples, it can be known that under the same process conditions, the preparation time has little difference in particle size distribution of the recombinant type iii humanized collagen and the crosslinked sodium hyaluronate composite gel, the difference in content of hyaluronic acid in the product is also little, and under the same process conditions, the solution ph value has much difference in particle size distribution of the recombinant type iii humanized collagen and the crosslinked sodium hyaluronate composite gel and the content of hyaluronic acid in the product, which indicates that the preparation effect is relatively higher in the environment with larger alkalinity of the recombinant type iii humanized collagen and the crosslinked sodium hyaluronate composite gel.
The beneficial effects of the invention are as follows: the recombinant III-type humanized collagen and crosslinked sodium hyaluronate composite gel for injection and the preparation method thereof have the effects of improving periorbital, filling recesses, smoothing wrinkles and improving skin firmness and plumpness.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.