CN108126186B - Hydrogel containing nerve growth factor and preparation method thereof - Google Patents
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Abstract
The invention discloses a hydrogel containing nerve growth factor and a preparation method thereof. Consists of carbomer 980, glycerol, propylene glycol, povidone K30, methylparaben, EDTA-2Na, azone, human serum albumin, mannitol and protein, and the balance of water. The hydrogel has the characteristics of simplicity, practicability, stability and good curative effect, and can be further applied to treatment of pressure ulcer and other refractory ulcers.
Description
Technical Field
The invention relates to the field of biological preparations, in particular to a preparation method of hydrogel containing nerve growth factors.
Background
Nerve Growth Factor (NGF) is widely used, with the beta subunit being the active region of Nerve Growth Factor. Currently commercialized m-beta NGF such as Enjinfu
Is obtained by separating and purifying submandibular gland of adult male mouse. Nerve growth factor is widely used for treating diseases such as nerve atrophy, neurodegeneration, traumatic repair and the like in ophthalmology, neurosurgery, orthopaedics, neurology, pediatrics and endocrinology, but no related product of the nerve growth factor for external use is on the market at present.
Pressure Ulcers (PU), also known as bedsores, are ischemic lesions of the skin and its underlying tissues caused by the obstruction of blood and lymph flow caused by external Pressure, and are one of the most common clinical complications. With the rapid development of the aging population of China, the incidence rate of various chronic diseases is increased, and in addition, the improvement of medical technology can cure a large number of critical patients, so that the number of patients accompanied with complications is increased, and the incidence rate of the pressure ulcer is increased day by day at present. Topical treatment of pressure ulcers is easier to apply and has fewer side effects than other routes of administration. In addition, topical drugs with shorter half-lives have better safety than other drugs.
Literature studies have demonstrated that topical administration of nerve growth factor can cause a gradual recovery of nerve function, such as a recurrence of pain sensitivity. There is also evidence that nerve growth factors stimulate blood vessel formation and keratinocyte growth and differentiation. Nerve growth factors may contribute to the recovery of pressure ulcer wounds by stimulating keratinocyte proliferation and neovascularization either directly or indirectly through the action of other neurotrophic factors, thereby accelerating wound healing.
Therefore, the nerve growth factor for external use has great potential value in treating refractory ulcers such as pressure ulcers. The development of external preparations such as nerve growth factor-containing hydrogel is of great significance.
Chinese patent CN200310120873.8 discloses a nerve growth factor external medicament, wherein the thickening agent of the gel is carbomer 940, the humectant is propylene glycol (or glycerol) used alone, and the main medicament is a venom nerve growth factor of the northeast Changbai mountain pallas pit viper. However, the source of the main drug of the gel is the venom of the east-north Changbai mountain pallas pit viper, the source is limited, and the potential immunogenicity of the snake venom nerve growth factor has certain clinical risk. Carbomer 940 used in the gel matrix has excessive benzene content and potential carcinogenic risk, does not meet the requirements of FDA and American national formulary, and needs to be replaced by safe and nontoxic new auxiliary materials. Literature studies have shown that the requirement for ensuring moist healing of the affected site such as ulcer is important for accelerating the healing of ulcer, and the use of a combination of various moisturizing agents enables the maintenance of the moisture retention of the external preparation under various conditions. However, the moisturizing effect of the gel by using propylene glycol alone or glycerol alone is not verified, and the requirement of the gel on moist healing of the affected part such as ulcer part cannot be met.
In addition, the biological activity of most proteins, including nerve growth factor, is rapidly attenuated in the absence or in the absence of protein protectants. The gel lacks necessary protein protective agent, which causes poor stability of biological activity of nerve growth factor protein in hydrogel. In addition, it is particularly noted that the structure of the thickening agent (e.g., carbomer 940 used in the hydrogel) in the gel is dense, making it difficult to release the pharmaceutically active molecules or proteins, resulting in poor therapeutic effect of the topical hydrogel formulation.
In conclusion, the traditional gel has defects in many aspects, and particularly, the nerve growth factor serving as a protein is difficult to release from hydrogel to an ulcer surface to exert curative effect, and the clinical curative effect is poor. And the maintenance of the long-term biological activity stability of the nerve growth factor-containing hydrogel is also one of the keys of industrialization.
Therefore, the emergence of novel nerve growth factor hydrogel with higher safety, more stable protein activity, more complete release and capability of ensuring wet healing is urgently needed clinically, and the novel nerve growth factor hydrogel has important clinical and industrial values.
Disclosure of Invention
The invention aims to provide a hydrogel agent which is simple, practical, stable and good in curative effect and a preparation method thereof, and is further applied to treatment of difficult-to-heal ulcers such as pressure ulcers.
In order to achieve the purpose, the invention provides a protein-containing hydrogel which is characterized by comprising carbomer 980, glycerol, propylene glycol, povidone K30, methyl paraben, EDTA-2Na, azone, human serum albumin, mannitol and protein, and the balance of water.
Further, the ratio of the povidone K30 to the carbomer 980 is 1:2-2: 1; preferably, the ratio of povidone K30 to carbomer 980 is 1: 1.
Further, the weight percentage composition of the oral liquid is 1% of carbomer 980, 20% of glycerol, 20% of propylene glycol, 1% of povidone K30, 0.15% of methyl paraben, 0.05% of EDTA-2Na, 1% of azone, 0.5% of human serum albumin, 0.5% of mannitol, 0.001-0.012% of protein and the balance of water.
Further, the protein is a nerve growth factor.
Further, the preparation method comprises the following steps of,
respectively taking glycerol and propylene glycol, adding water for injection, uniformly stirring, adding methyl paraben, EDTA-2Na and azone for dissolving, weighing carbomer 980, adding the carbomer 980, swelling overnight to obtain a mixed solution, slowly dripping an NaOH aqueous solution into the mixed solution, uniformly stirring and mixing to obtain a viscous gel, adjusting the pH value to be 6.5-7.5, sterilizing at high temperature, cooling to obtain the hydrogel, adding povidone K30, and supplementing the hydrogel with the water for injection to obtain the hydrogel matrix.
Adding nerve growth factor solution into the hydrogel matrix at 2-8 deg.C, slowly stirring to avoid bubble generation, and obtaining hydrogel preparation containing nerve growth factor; preferably, the hydrogel preparation containing the nerve growth factor is continuously centrifuged, and then subpackaged and stored.
Further, the preparation method comprises the following steps of,
respectively taking 5-20g of glycerol and 5-20g of propylene glycol, adding 30-80ml of water for injection, uniformly stirring, adding 0.15g of methylparaben, 0.05g of EDTA-2Na and 1g of azone, dissolving, weighing 0.4-1.6g of carbomer 980(Carbopol 980, auxiliary materials are shown in the formula F20130033, Luborun advanced materials Co.), adding the materials into the solution overnight, swelling to obtain a mixed solution, slowly dripping 0.5-1.2ml of 50% (w/w) NaOH aqueous solution into the mixed solution, uniformly stirring and mixing to obtain viscous gel, adjusting the pH to 6.5-7.5, sterilizing at high temperature of 0.1MPa and 121 ℃ for 20min, cooling to 4 ℃, adding 5-20ml of 10% povidone K30 into the viscous gel, and supplementing the total weight of the water for injection to 95g to obtain 95g of hydrogel matrix;
adding 5ml of nerve growth factor solution into the hydrogel matrix at 4 ℃, slowly stirring uniformly to avoid bubbles, and obtaining 100g of hydrogel preparation containing nerve growth factor; preferably, the nerve growth factor-containing hydrogel preparation is centrifuged at 12000rpm for 5 minutes, and then stored in separate containers.
Further, the nerve growth factor solution contains mouse nerve growth factor stock solution for injection, human albumin and mannitol.
Furthermore, 5ml of the nerve growth factor solution contains 1-12mg of stock solution of mouse nerve growth factor for injection, 0.5g of human serum albumin, 0.5g of mannitol and the balance of water.
The invention provides a preparation method of the hydrogel containing the protein, which is characterized in that,
respectively taking glycerol and propylene glycol, adding water for injection, uniformly stirring, adding methyl paraben, EDTA-2Na and azone to dissolve, weighing carbomer 980, adding the carbomer 980, swelling overnight to obtain a mixed solution, slowly dripping NaOH aqueous solution into the mixed solution, uniformly stirring and mixing to obtain viscous gel, adjusting the pH to 6.5-7.5, sterilizing at high temperature, cooling to 4 ℃, adding povidone K30, and supplementing the injection water; obtaining the hydrogel matrix;
adding nerve growth factor solution into the hydrogel matrix at 2-8 deg.C, slowly stirring to avoid bubble generation, and obtaining hydrogel preparation containing nerve growth factor; preferably, the hydrogel preparation containing the nerve growth factor is continuously centrifuged, and then subpackaged and stored;
more preferably, 5-20g of glycerol and 5-20g of propylene glycol are respectively taken, 30-80ml of water for injection is added and uniformly stirred, 0.15g of methyl paraben, 0.05g of EDTA-2Na and 1g of azone are added to dissolve the methyl paraben, 0.4-1.6g of carbomer 980(Carbopol 980, an auxiliary material is shown in the formula F20133, Luobu advanced materials) is added to the mixture and swelled overnight to obtain a mixed solution, then 0.5-1.2ml of 50% (w/w) NaOH aqueous solution is slowly dripped into the mixed solution, the mixed solution is uniformly stirred and mixed to form viscous gel, the pH is adjusted to 6.5-7.5, the gel is sterilized at high temperature of 0.1MPa and 121 ℃ for 20min, the mixed solution is cooled to 4 ℃, 5-20ml of 10% polyvidone K30 is added to the mixed solution, and the total weight of the gel is supplemented with the water for injection to 95g, so that 95g of the hydrogel is obtained;
adding 5ml of nerve growth factor solution into the hydrogel matrix at 4 ℃, slowly stirring uniformly to avoid bubbles, and obtaining 100g of hydrogel preparation containing nerve growth factor; most preferably, the nerve growth factor-containing hydrogel preparation is centrifuged at 12000rpm for 5 minutes and then stored in separate containers;
optionally, the nerve growth factor solution contains rat nerve growth factor stock solution for injection, human albumin and mannitol;
optionally, 5ml of the nerve growth factor solution contains 1-12mg of mouse nerve growth factor stock solution for injection, 0.5g of human serum albumin, 0.5g of mannitol and the balance of water.
The preparation of the hydrogel matrix of the invention is that sodium hydroxide (strong base) is added to adjust the pH value, so that the hydrogel is changed into gel from liquid state, and the strong base can denature and inactivate protein, so that the hydrogel matrix is firstly added with water, and then the single nerve growth factor solution is added.
Carbomer 980 in the hydrogel matrix is used as a thickening agent, and povidone K30 is used as a gel pore structure regulator so as to regulate the lubricity, viscosity and release performance of nerve growth factors of the hydrogel; the glycerol and the propylene glycol are mainly used as humectants to meet the requirements of wet healing of action parts such as ulcer parts and the like; the azone is a transdermal absorbent, and the glycerol and the propylene glycol also have the function of the transdermal absorbent, so that the transdermal absorption of the nerve growth factor after external use is ensured, and the curative effect is improved; EDTA-2Na is a metal chelator which chelates a variety of metal ions as a coenzyme, thereby inhibiting enzymatic protein degradation and maintaining the stability of nerve growth factor; the methyl paraben is a preservative, can prevent the growth of microorganisms and meets the aseptic requirement of the hydrogel containing the nerve growth factor. The nerve growth factor in the nerve growth factor solution is used as a pharmaceutical active ingredient, and has the effects of promoting wound healing and ulcer healing and accelerating the formation of new blood vessels; human serum albumin and mannitol are used as protein protective agents to maintain the long-term stability of biological activity of nerve growth factor.
The invention has the following advantages:
1. the invention adopts carbomer 980 as a matrix material to prepare the hydrogel, the hydrogel has good lubricity and viscosity, has the characteristics of good ductility, easy smearing and attachment on skin, good biocompatibility, no irritation to skin, capability of absorbing tissue penetrating fluid and the like, and does not have the condition that the traditional carbomer 940 contains carcinogenic substance benzene.
2. The invention adopts the optimized humectant (propylene glycol, glycerol) combination formula, can ensure the wet healing requirements of the acting parts such as wounds, ulcer parts and the like, and is beneficial to accelerating the healing of the ulcer.
3. The nerve growth factor-containing hydrogel prepared by the invention has good stability, and the biological activity of the nerve growth factor in the gel is kept stable for a long time by adding protein protective agents (human serum albumin, mannitol) and metal chelating agents (EDTA-2 Na).
4. The hydrogel containing the nerve growth factor breaks through the technical difficulty that the nerve growth factor in the hydrogel is difficult to release by adding the gel pore structure regulator (povidone K30), greatly improves the transdermal release amount of the nerve growth factor in the hydrogel, and ensures that the nerve growth factor can reach the ulcer surface to exert curative effect.
Drawings
FIG. 1 is a photograph of a pathological section of the skin of a pressure ulcer wound on 15 days after administration to rats in group G1. Wherein (A) is the result of wound surface 1 magnification of 2X and magnification of 10X of G1 group No. 1 rat (total number 1), and (B) is the result of wound surface 2 magnification of 2X and magnification of 10X of G1 group No. 1 rat (total number 1).
FIG. 2 is a photograph of a pathological section of the skin of a pressure ulcer wound on 15 days after administration to rats in group G2. Wherein (A) is the result of wound surface 1 magnification of 2X and magnification of 10X of G2 group No. 1 rat (total number 13), and (B) is the result of wound surface 2 magnification of 2X and magnification of 10X of G2 group No. 1 rat (total number 13).
FIG. 3 is a photograph of a pathological section of the skin of a pressure ulcer wound on 15 days after administration to rats in group G3. Wherein (A) is the result of wound 1 magnification of 2X and magnification of 10X of G3 group No. 2 rat (total number 26), and (B) is the result of wound 2 magnification of 2X and magnification of 10X of G3 group No. 2 rat (total number 26).
FIG. 4 is a photograph of a pathological section of the skin of a pressure ulcer wound on 15 days after administration to rats in group G4. Wherein (A) is the result of wound surface 1 magnification of 2X and magnification of 10X of G4 group No. 5 rat (total number 41), and (B) is the result of wound surface 2 magnification of 2X and magnification of 10X of G4 group No. 5 rat (total number 41).
FIG. 5 is a photograph of a pathological section of the skin of a pressure ulcer wound on 15 days after administration to rats in group G5. Wherein (A) results of wound surface 1 magnification factor 2X and magnification factor 10X for G5 group No. 4 rat (total number 52) are shown, and (B) results of wound surface 2 magnification factor 2X and magnification factor 10X for G5 group No. 4 rat (total number 52) are shown.
FIG. 6 is a photograph of a pathological section of the skin of a pressure ulcer wound on 15 days after administration to rats in group G6. Wherein (A) is the result of wound 1 magnification of 2X and magnification of 10X of G6 group No. 2 rat (total number 62), and (B) is the result of wound 2 magnification of 2X and magnification of 10X of G6 group No. 2 rat (total number 62).
FIG. 7 is a photograph of a pathological section of the skin of a pressure ulcer wound on 15 days after administration to rats in group G7. Wherein (A) results of wound surface 1 magnification of 2X and magnification of 10X for G7 group No. 6 rat (total number 78) are shown, and (B) results of wound surface 2 magnification of 2X and magnification of 10X for G7 group No. 6 rat (total number 78) are shown.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
EXAMPLE 1 preparation of hydrogel containing nerve growth factor
Respectively taking 5-20g of glycerol and 5-20g of propylene glycol, adding 30-80ml of water for injection, uniformly stirring, adding 0.15g of methylparaben, 0.05g of EDTA-2Na and 1g of azone, dissolving, weighing 0.4-1.6g of carbomer 980(Carbopol 980, auxiliary materials are shown in the formula F20130033, Luborun advanced materials Co.), adding the materials into the solution overnight, swelling to obtain a mixed solution, slowly dripping 0.5-1.2ml of 50% (w/w) NaOH aqueous solution into the mixed solution, uniformly stirring and mixing to obtain viscous gel, adjusting the pH to 6.5-7.5, sterilizing at the high temperature of 0.1MPa and 121 ℃ for 20min, and cooling to 4 ℃. 5-20ml of 10% povidone K30 solution (PVPK30, supplementary material F20120023, BASF (China) Co., Ltd.) is added into the gel, so that the content ratio of povidone K30 and carbomer 980 contained in the 10% povidone K30 solution is 1:2-2:1, and the total weight is 95g by supplementing water for injection. 95g of hydrogel matrix is obtained.
Adding 5ml nerve growth factor solution (containing rat nerve growth factor stock solution for injection, human albumin and mannitol) into the hydrogel matrix at 4 deg.C, slowly stirring to avoid bubble generation, and obtaining 100g hydrogel preparation containing nerve growth factor. The nerve growth factor-containing hydrogel preparation was centrifuged at 12000rpm for 5 minutes, and then packed in a sterile aluminum tube and stored at 4 ℃.
The preparation method of the nerve growth factor solution comprises the steps of adding a prescription dose of nerve growth factor stock solution (rat nerve growth factor stock solution for injection, batch number L015, not known as biological medicine company Limited), human serum albumin and mannitol into water for injection, fully mixing uniformly to ensure that the concentrations of the nerve growth factor stock solution, the human serum albumin and the mannitol are 0.2-2.4mg/ml, 100mg/ml and 100mg/ml respectively, filtering and sterilizing by using a 0.22 mu m filter membrane under the aseptic condition, and keeping the mixture at 4 ℃ for later use.
Example 2 homogeneous design experiment of hydrogel matrix
The hydrogel matrix was prepared as in example 1.
The total weight of the hydrogel is 100g, the addition amount of the 10% povidone K30 solution is fixed to be 10ml, the dosage of other components is fixed according to the proportion in the example 1, only the dosage of carbomer 980, glycerol and propylene glycol is adjusted, and finally the total weight of the hydrogel matrix is 95g by adding water for injection. The concentration of the fixed nerve growth factor solution is 0.2mg/ml, and the addition amount is 5 ml. According to the results of preliminary experiments, the amounts of carbomer 980(X1), glycerol (X2), and propylene glycol (X3) were selected as variable factors to be uniformly designed. The carbomer 980 is set to be 0.4-1.6g (i.e., 0.4% -1.6% (w/w)), the glycerol is set to be 5-20g (i.e., 5% -20% (w/w)), and the propylene glycol is set to be 5-20g (i.e., 5% -20% (w/w)).
Look up uniform design table, U5(53) The variation (D) in uniformity of the scribing performed in the 3-factor test was 0.4570, U6 *(64) The D for the 3-factor test was 0.2656, U7(74) The D for the 3-factor test was 0.3721, U7 *(74) The D for the 3-factor test was 0.2132, U8 *(85) D for performing 3-factor test is 0.2000, and U is selected by comprehensively considering test times and uniformity7 *(74) 3-factor tests were performed, each factor was set to 7 levels, the test design factors and levels are shown in table 1, the scoring criteria are shown in table 2, and the results of optimum ratios of carbomer 980, glycerol, and propylene glycol are shown in table 3.
Table 1 homogeneous design factor and leveling of hydrogel matrices
The uniform design test investigation indexes comprise character uniformity (Y1), lubricity (Y2), viscosity (Y3) and moisture retention rate (Y4), wherein 3 technicians with related experience are selected to observe the character uniformity such as uniformity, fineness, clarity and the like of the hydrogel, meanwhile, a coating test is carried out to investigate the lubricity and viscosity of the hydrogel, the average values are obtained after the evaluation is carried out respectively, and the evaluation standards are shown in a table 2.
TABLE 2 Scoring standard table for different indexes of uniform design test
Moisture retention test method: gel matrices were accurately weighed (to the nearest 0.0001g) in 5g each, coated on a 7.5cm × 7.5cm glass plate with 3M adhesive tape, placed in a drug stability test chamber at 25 ℃ and 80% relative humidity for 12h and weighed, and the moisture retention rate was calculated (moisture retention rate% (% gel weight-weight after 12h gel)/original gel weight 100%), and each gel was subjected to 3 tests and averaged. The uniform design test is shown in Table 3.
Table 3 table of test results of different indexes of uniform design test
The 7 formulations with uniform design can pass through the centrifugal resistance and the cold and heat resistance.
Further using origin9.0 software for property uniformity, lubricity, viscosity in table 3And the moisture retention rate result is analyzed, and the result shows that: the straight line equation between hydrogel property uniformity (Y1) and carbomer 980(X1) has the highest degree of fitting, Y1 is 11.678-4.821X1, and R2The equation is very significant when the ratio is 0.829, F30.124 and P0.003, and shows that the hydrogel property uniformity (Y1) is inversely proportional to the amount of carbomer 980(X1) in the range of 0.4% to 1.6% (w/w), and is not much related to the amounts of glycerol (X2) and propylene glycol (X3).
The straight line equation between the lubricating property (Y2) and glycerol (X2) and propylene glycol (X3) has the highest fitting degree, wherein Y2 is 0.286+0.300X1+0.100X2, and R is2The equation is significant when 0.717, 8.580 for F and 0.035 for P, indicating that lubricity (Y2) is directly proportional to the amounts of glycerol (X2) and propylene glycol (X3) in the range of 5% to 20% (w/w), and is not as great a function of carbomer 980.
The quadratic equation between viscosity (Y3) and carbomer 980(X1) has the highest degree of fit, Y3 ═ 27.679X1-13.393X126.214, 0.762 for R2, 76.657 for F, 0.001 for P, which is very significant, and illustrates that the viscosity (Y3) tends to be better and worse with the amount of carbomer 980(X1) in the range of 0.4% to 1.6% (w/w), while the amount of glycerol (X2) and propylene glycol (X3) is less relevant.
The straight line equation between the moisture retention rate (Y4) and the glycerol (X2) and the propylene glycol (X3) has the highest fitting degree, wherein Y2 is 0.148+0.017X1+0.010X2, and R is2The equation is very significant when 0.787, 12.087, 0.020 and P, indicating that the moisture retention rate (Y4) is proportional to the amounts of glycerol (X2) and propylene glycol (X3) in the range of 5% to 20% (w/w), and is not much related to the amount of carbomer 980.
By comprehensively analyzing the uniform design results of the character uniformity (Y1), the lubricity (Y2), the viscosity (Y3) and the moisture retention rate (Y4) and the industrial operability of the hydrogel preparation process, the optimal formula of the hydrogel matrix is determined to be carbomer 980(X1) 1% (w/w), glycerol (X2) 20% (w/w) and propylene glycol (X3) 20% (w/w).
Example 3 in vitro transdermal assay with optimized addition of Povidone K30
The hydrogel matrix was prepared as in example 1.
Calculated by taking the total weight of the hydrogel as 100g, the adding amount of carbomer 980 is fixed to be 1g, the adding amounts of glycerol and propylene glycol are both fixed to be 20g, the adding amounts of other components are fixed, the adding amounts of 10% povidone K30 solution are respectively adjusted to be 5ml, 10ml and 20ml, and the total weight of the hydrogel matrix is supplemented with injection water to be 95 g. The concentration of the fixed nerve growth factor solution is 0.2mg/ml, and the addition amount is 5 ml. The adding amount of the povidone K30 and the ratio of the povidone K30 to the carbomer are respectively 1:2, 1:1 and 2:1, water for injection is used for replacing the 10% povidone K30 solution as a blank control, hydrogels with different formulas are respectively prepared, and an in-vitro transdermal experiment is carried out to detect the release of the nerve growth factor.
In vitro transdermal assay methods: the method comprises fixing pretreated rabbit skin stratum corneum layer downwards between a supply tank and a receiving tank by adopting a drug transdermal diffusion experimental apparatus (JRYJ-6A, Shanghai yellow sea drug testing apparatus, Ltd.), adding 1.0g of gel into the supply tank, adding 6.6ml of normal saline as receiving medium into the receiving tank, stirring at 300 r/min with a magnetic stirrer, and having a diffusion area of 2.54cm2And is externally arranged in a constant temperature water bath (32.0 +/-1.0 ℃).
The method for treating the pretreated rabbit skin comprises the following steps: taking healthy rabbits with the weight of about 3kg, killing and peeling off the skin, taking the abdominal skin, depilating with 10% sodium sulfide, removing subcutaneous fat, repeatedly washing with purified water until no turbidity exists, soaking in physiological saline, and freezing and storing in a refrigerator for later use. Before each experiment, the rabbit skin was thawed at room temperature and carefully checked for integrity without any damage.
After the in vitro transdermal experiment is carried out for 12 hours, all the receiving medium is extracted from the oblique opening of the receiving pool, the concentration of the nerve growth factor in the receiving medium is detected by an ELISA method (CYT304 nerve growth factor ELISA detection kit, Mercury Micheobo (China) Co., Ltd., LOT2513960), the transdermal release amount of the nerve growth factor (the percentage of the nerve growth factor content in the receiving medium to the total nerve growth factor content in the gel) for 12 hours is calculated, each group of samples is respectively carried out for 3 times of experiments, the results are described by means of the mean number plus or minus standard deviation, t test is carried out, and the difference P <0.05 has statistical significance.
In vitro transdermal experiments tested the effect of povidone K30 and carbomer 980 on the 12h nerve growth factor transdermal release, and the results are shown in table 4.
TABLE 4 influence of Povidone K30 on carbomer 980 on the transdermal delivery of nerve growth factor for 12h
| Ratio of Povidone K30 to carbomer 980 | Blank control | 1:2 | 1:1 | 2:1 |
| 12h transdermal delivery of nerve growth factor | 36.5±8.6 | 53.6±7.3* | 56.2±7.9* | 41.8±9.5 |
Note:*the difference was statistically significant (P) compared to the control group<0.05)。
From table 4, it can be seen that the drug release rate of the nerve growth factor-containing hydrogel added with povidone K30 is greatly improved, and the drug release rate of each group added with different proportions is obviously higher than that of the blank control group without added povidone K30. When the ratio of the povidone K30 to the carbomer 980 is 1:1, the transdermal release amount of the nerve growth factor is the largest within 12 hours, which is remarkably higher than that of a blank control group (P is less than 0.05) and reaches more than 1.5 times of that of the blank control group; however, when the ratio of the povidone K30 to the carbomer 980 is increased to 2:1, the 12h nerve growth factor transdermal release amount is reduced, and the difference is not statistically significant compared with a blank control group (P is more than 0.05); the preferred ratio of povidone K30 to carbomer 980 is illustrated as 1: 1.
Therefore, the povidone K30 and the carbomer 980 are added into the hydrogel gel matrix containing the nerve growth factors to be matched, an unexpected technical effect is achieved, and the transdermal release amount of the nerve growth factors in the hydrogel is greatly improved. The reason for this is that povidone K30 has water-swelling property, can be used as a regulator of the pore structure of the hydrogel, and when it is used in combination with carbomer 980, the network structure of the hydrogel is relatively loose, and the pore channels are enlarged, thereby increasing the release of nerve growth factors in the hydrogel. EXAMPLE 4 preparation of nerve growth factor-containing hydrogel of the present invention
The preparation of the nerve growth factor-containing hydrogel of the present invention is shown in Table 5, wherein example 4 is a blank hydrogel, and examples 5 to 8 are hydrogels containing 10. mu.g/g to 120. mu.g/g of nerve growth factor.
Table 5 examples 4-8 prescription scale
The preparation method is the same as example 1.
EXAMPLE 5 preparation of nerve growth factor-containing hydrogel of the present invention
The prescription is shown in table 5;
the preparation method is the same as example 1.
EXAMPLE 6 preparation of nerve growth factor-containing hydrogel of the present invention
The prescription is shown in table 5;
the preparation method is the same as example 1.
EXAMPLE 7 preparation of nerve growth factor-containing hydrogel of the present invention
The prescription is shown in table 5;
the preparation method is the same as example 1.
EXAMPLE 8 preparation of nerve growth factor-containing hydrogel of the present invention
The prescription is shown in table 5;
the preparation method is the same as example 1.
Comparative example 9 preparation of hydrogel containing nerve growth factor
The preparation is carried out according to the prescription of the nerve growth factor gel in Chinese patent CN200310120873.8, and the prescription is as follows: 9400.6-0.8g carbomer; 3.5-6g of triethanolamine; 2-4g of azone; 9-12g of propylene glycol; 0.6-1.0g of ethylparaben; nerve growth factor 1 mg.
Dispersing carbomer 940 in proper amount of hot water for injection, standing at 45-58 deg.C for 24h for self-dissolving, adjusting pH to 6.5-7.5 with triethanolamine, adding nerve growth factor dissolved in small amount of double distilled water, ethylparaben, propylene glycol and azone in prescription amount into gel, mixing, and adding the rest water (total mass of gel is 100g) to obtain nerve growth factor gel.
EXAMPLE 10 nerve growth factor-containing hydrogel Activity assay
The hydrogel activity assay of the nerve growth factor refers to the 2015 edition Chinese pharmacopoeia' biological activity assay of the mouse nerve growth factor, and the second method: TF-1 cells/MTS colorimetry. The specific implementation steps are as follows:
in this example, the basic culture solution is 90% RMPI 1640+ 10% fetal bovine serum, the complete culture solution is 90% RMPI 1640+ 10% fetal bovine serum +25ng/ml nerve growth factor, both RMPI 1640 (11875-.
Diluting the nerve growth factor activity international standard (96/366, NISBC, 10000U/branch) to 100U/ml (dilution of each step can not be more than 10 times) by using a basic culture medium in a gradient manner to obtain a standard solution.
Dissolving a proper amount of hydrogel containing nerve growth factors in a basic culture medium, and performing gradient dilution to 100U/ml (the dilution in each step is not more than 10 times) by using the basic culture medium to obtain a test solution.
TF-1 cell line was cultured in 5% carbon dioxide at 37 ℃ using the whole culture medium, and the cell concentration was controlled to 1.0X 10 per 1ml5～7.0×105And (4) carrying out biological activity determination on the individual cells 24-36h after passage. The solution used for the test was pre-warmed to 37 ℃. Collecting sufficient TF-1 cell culture, centrifuging to collect TF-1 cells, washing with RPMI 1640 culture medium for 3 times, and resuspending in basic culture medium to obtain a culture medium containing 5.0 × 10/1 ml4The cell suspension of each cell was kept at 37 ℃ for further use.
A96-well cell culture plate containing 100. mu.l of the standard solution and the test solution was incubated at 37 ℃ for 72 hours with 5% carbon dioxide by adding 100. mu.l of the cell suspension to each well. Mu.l of MTS solution was added to each well and incubated at 37 ℃ for 3 hours under 5% carbon dioxide. The above operations are carried out under aseptic conditions. Placing 96-well cell culture plate into enzyme labeling instrument, measuring absorbance at 490nm with 550nm as reference wavelength, recording measurement result, and adopting test data
Pro 5Software data analysis Software for processing and programming the results as follows:
in the formula: prIs the biological activity of a standard substance, U/ml; dsPre-diluting for the sample; drPre-diluting the standard substance by multiple times; esThe dilution factor of the sample corresponding to half effective amount of the standard sample is adopted; erIs the dilution multiple of half effective amount of the standard.
Specific activity (U/g) of hydrogel test sample containing nerve growth factor (biological activity/gel mass) of test sample
After the preparation of the gel was completed, the nerve growth factor-containing hydrogels prepared in examples 4 to 8 and comparative example 9 were measured for activity, and the results are shown in Table 6.
TABLE 6 biological Activity of nerve growth factor-containing hydrogels prepared in examples 4-8 and comparative example 9
| Hydrogels | Example 4 | Example 5 | Example 6 | Example 7 | Example 8 | Comparative example 9 |
| Biological Activity (U/g) | 0 | 10929 | 21145 | 61112 | 123045 | 10067 |
As can be seen from Table 6, the blank gel prepared in example 4 was not interfered with the activity test, and the activities of the nerve growth factor-containing hydrogels of examples 5 to 8 and comparative example 9 were in proportion to the amount of the nerve growth factor added.
EXAMPLE 11 evaluation of nerve growth factor-containing hydrogel Properties
The nerve growth factor-containing hydrogels prepared in examples 4 to 8 and comparative example 9 were evaluated for their properties according to the method of example 2, and the results are shown in Table 7.
TABLE 7 evaluation tables of nerve growth factor-containing hydrogels prepared in examples 4 to 8 and comparative example 9
As can be seen from Table 7, the hydrogel containing nerve growth factor prepared in examples 4-8 has substantially the same properties, the nerve growth factor with different content has no influence on the gel properties, the property uniformity scores of examples 4-8 are not much different from those of comparative example 9, but the lubricity and viscosity scores of examples 4-8 are obviously higher than those of comparative example 9. In addition, the 12-hour moisturizing rates of the hydrogels containing nerve growth factors prepared in examples 4-8 are all obviously higher than that of comparative example 9 and are all more than 2.5 times of that of comparative example 9. The hydrogel containing the nerve growth factor has uniform properties, proper lubricity and viscosity and good moisturizing effect, can meet the requirements of the treatment of skin ulcer and the like on moist healing, and promotes the repair and healing of skin wounds.
EXAMPLE 12 evaluation of stability of hydrogel containing nerve growth factor
The nerve growth factor-containing hydrogels prepared in examples 4 to 8 and comparative example 9 were filled in an internally coated sealed aluminum tube, sealed and placed at 4 ℃, and the appearance, filling amount, pH, biological activity, sterility test, and other property evaluations of the gels were performed at 0 month, 1 month, 3 months, 6 months, and 12 months, respectively, wherein the biological activity was measured according to example 9, and the other items were measured according to the "chinese pharmacopoeia" 2015 edition, and the results are shown in tables 8 to 13.
Table 8 evaluation table of stability of hydrogel containing nerve growth factor prepared in example 4
TABLE 9 evaluation table of stability of hydrogel containing nerve growth factor prepared in example 5
TABLE 10 evaluation table of stability of hydrogel containing nerve growth factor prepared in example 6
TABLE 11 stability evaluation Table for nerve growth factor-containing hydrogel prepared in example 7
TABLE 12 evaluation table of stability of hydrogel containing nerve growth factor prepared in example 8
TABLE 13 evaluation table of stability of hydrogel containing nerve growth factor prepared in comparative example 9
As can be seen from tables 8 to 13, the nerve growth factor-containing hydrogels of the present invention (examples 4 to 8) satisfy the specifications for 12 months, in which the biological activities of the nerve growth factor-containing hydrogels prepared in examples 5 to 8 were stable. In contrast, in comparative example 9, the hydrogel containing nerve growth factor has other indexes meeting the specification, but the biological activity of the hydrogel is obviously reduced. The hydrogel containing the nerve growth factor has extremely high stability, the biological activity of the hydrogel in 3-12 months is obviously higher than that of the comparative example, the hydrogel has obvious advantages in clinical application, and the hydrogel containing the nerve growth factor is beneficial to industrial production and application. EXAMPLE 13 determination of nerve growth factor-containing hydrogel nerve growth factor transdermal Release Performance of the present invention
The nerve growth factor-containing nerve growth factor transdermal delivery properties prepared in examples 4 to 8 and comparative example 9 were measured as in example 3, and the results are shown in Table 14.
TABLE 14 nerve growth factor-containing hydrogel prepared in examples 4 to 8 and comparative example 9 12h nerve growth factor
Transdermal delivery scale
| Hydrogels | Example 4 | Example 4 | Example 5 | Example 6 | Example 7 | Comparative example 9 |
| Transdermal delivery amount% | 0 | 54.7±8.1* | 56.1±10.1* | 56.3±9.2* | 56.8±7.5* | 35.9±7.8 |
Note:*the difference is statistically significant (P) compared to comparative example 9<0.05)。
As can be seen from Table 14, the blank gel prepared in example 4 has no interference to the nerve growth factor transdermal release test, the nerve growth factor transdermal release amounts (%) of 12h of the nerve growth factor-containing hydrogels prepared in examples 5-8 are substantially consistent, and the nerve growth factor transdermal release amount of 12h is significantly higher than that of comparative example 9(P < 0.05). The hydrogel containing the nerve growth factor can obviously improve the transdermal release performance of the nerve growth factor, thereby improving the drug concentration of the nerve growth factor, further improving the clinical treatment effect, reducing the drug administration frequency and having extremely high clinical value.
EXAMPLE 14 therapeutic Effect of nerve growth factor-containing hydrogel of the present invention on pressure ulcer of rat skin
An experimental method for treating pressure ulcer of rat skin by hydrogel containing nerve growth factor comprises the following steps:
after 3 days of adaptive feeding of purchased SPF-grade SD male rats (Shanghai Si Laike laboratory animal, Limited liability company, qualification No. 2015000506695), fasting is performed on the same day of the experiment, 10% chloral hydrate is used for intraperitoneal injection anesthesia, the backs of the rats are depilated by depilatory cream, after 24 hours, the skin at the middle part of the backs of the rats is pulled up, two disc-shaped magnets (with the diameter of 20mm, the thickness of 4mm and the average magnetic force of about 2500 Gauss) are clamped on the skin, the centers of the two magnets are spaced by about 3cm, and after 16 hours, the magnets are taken off to perform 1-cycle molding. Rats were fed free food and water during ischemia reperfusion, and were housed individually in stainless steel caged cages. Animals were randomly divided into 7 groups (10 x2 ulcer wounds per group depending on ulcer area uniformity) by ulcer area, ulcer score and animal weight: g1, normal saline; g2 blank gel (blank gel prepared in example 4); g3: 10. mu.g/g nerve growth factor-containing hydrogel (gel prepared in comparative example 9); g4, 10 mug/G nerveGrowth factor hydrogel (gel prepared in example 5); g5: 20. mu.g/G nerve growth factor-containing hydrogel (gel prepared in example 6); g6: 60. mu.g/G nerve growth factor-containing hydrogel (gel prepared in example 7); g7: 120. mu.g/G nerve growth factor-containing hydrogel (gel prepared in example 8). According to the area of pressure sores when grouped according to 100mg/cm2D applying the test substance in fixed amounts each time, 1 time per day. The gel spreadability and the toxic and irritant reaction of the rat skin were observed during the experiment, with an ulcer area (cm) of 1 time per week2) Observation, pathological section of the skin and analysis of the results were carried out after 15 days (FIG. 1). Statistical analysis was performed using SPSS19.0 software. Ulcer areas are described as mean ± standard deviation (x ± s), and t-test is performed. With P<A difference of 0.05 is statistically significant.
During the experiment, the smearing performance of G2 and G4-G7 is better, and the ulcer tissue penetrating fluid is absorbed, so that the effect is better than that of G3; all rats showed no skin toxicity and irritation; the comparison of the areas of pressure ulcers in the groups G1-G7 is shown in Table 15; the pathological sections of the pressure ulcer wound skin at 15 days after administration of the rats in each group of G1-G7 are shown in figures 1-7. FIG. 1 is a photograph of a pathological section of the skin of a pressure ulcer wound on 15 days after administration to rats in group G1. Wherein (A) is the result of wound surface 1 magnification of 2X and magnification of 10X of G1 group No. 1 rat (total number 1), and (B) is the result of wound surface 2 magnification of 2X and magnification of 10X of G1 group No. 1 rat (total number 1). As can be seen in FIG. 1, the epithelium of the rats in group G1 appeared to be keratinized, crusted, infiltrated with inflammatory cells, and the connective tissue in the dermis was slightly to moderately fibrovitrified. FIG. 2 is a photograph of a pathological section of the skin of a pressure ulcer wound on 15 days after administration to rats in group G2. Wherein (A) is the result of wound surface 1 magnification of 2X and magnification of 10X of G2 group No. 1 rat (total number 13), and (B) is the result of wound surface 2 magnification of 2X and magnification of 10X of G2 group No. 1 rat (total number 13). As can be seen in FIG. 2, the epithelium of the rats in group G2 appeared to be cornified, crusted, infiltrated with inflammatory cells, and the connective tissue in the dermis was slightly to moderately fibrovitrified. FIG. 3 is a photograph of a pathological section of the skin of a pressure ulcer wound on 15 days after administration to rats in group G3. Wherein (A) is the result of wound 1 magnification of 2X and magnification of 10X of G3 group No. 2 rat (total number 26), and (B) is the result of wound 2 magnification of 2X and magnification of 10X of G3 group No. 2 rat (total number 26). As can be seen in FIG. 3, the epithelium of the rats in group G3 appeared to be keratinized, crusted, infiltrated with inflammatory cells, and the connective tissue in the dermis was slightly to moderately fibrovitrified. FIG. 4 is a photograph of a pathological section of the skin of a pressure ulcer wound on 15 days after administration to rats in group G4. Wherein (A) is the result of wound surface 1 magnification of 2X and magnification of 10X of G4 group No. 5 rat (total number 41), and (B) is the result of wound surface 2 magnification of 2X and magnification of 10X of G4 group No. 5 rat (total number 41). As can be seen in FIG. 4, the epithelium of the rats in group G4 appeared to be keratinized, crusted, infiltrated with inflammatory cells, and the connective tissue in the dermis was slightly to moderately fibrovitrified. FIG. 5 is a photograph of a pathological section of the skin of a pressure ulcer wound on 15 days after administration to rats in group G5. Wherein (A) results of wound surface 1 magnification factor 2X and magnification factor 10X for G5 group No. 4 rat (total number 52) are shown, and (B) results of wound surface 2 magnification factor 2X and magnification factor 10X for G5 group No. 4 rat (total number 52) are shown. As can be seen in FIG. 5, the epithelium of the rats in group G5 appeared to be cornified, crusted, and infiltrated with inflammatory cells, and the connective tissue in the dermis was apparently fibrovitrified. FIG. 6 is a photograph of a pathological section of the skin of a pressure ulcer wound on 15 days after administration to rats in group G6. Wherein (A) is the result of wound 1 magnification of 2X and magnification of 10X of G6 group No. 2 rat (total number 62), and (B) is the result of wound 2 magnification of 2X and magnification of 10X of G6 group No. 2 rat (total number 62). As can be seen in FIG. 6, the epithelium of the rats in group G6 appeared to be cornified, crusted, and infiltrated with inflammatory cells, and the connective tissue in the dermis was apparently fibrovitrified. FIG. 7 is a photograph of a pathological section of the skin of a pressure ulcer wound on 15 days after administration to rats in group G7. Wherein (A) results of wound surface 1 magnification of 2X and magnification of 10X for G7 group No. 6 rat (total number 78) are shown, and (B) results of wound surface 2 magnification of 2X and magnification of 10X for G7 group No. 6 rat (total number 78) are shown. As can be seen from FIG. 7, the epithelium of the rats in group G7 appeared to be cornified, crusted, and infiltrated with inflammatory cells, and the connective tissue in the dermis was apparently fibrovitrified.
TABLE 15 comparison of areas of pressure ulcers for each group of G1-G7
Note:①the differences were statistically significant (P) compared to G1, physiological saline<0.05)；②The difference was statistically significant (P) compared to G2, blank gel (gel prepared in example 4)<0.05)；③And G3: the difference was statistically significant (P) in comparison with the nerve growth factor-containing hydrogel (gel prepared in comparative example 9) at 10. mu.g/g<0.05)。
As is apparent from Table 15, at 15 days, the ulcer areas of the groups of examples 4 to 8(G4 to G7) and comparative example 9(G3) were significantly lower than that of the normal saline group (G1); wherein the ulcer areas of the groups of examples 5-8(G5-G7) and comparative example 9(G3) are all significantly lower than those of example 4 (G2); and the ulcer areas of the examples 5-8(G5-G7) are all obviously lower than that of the comparative example 9 (G3); the differences were all statistically significant (P < 0.05).
The purpose of the experiment is to detect the reaction of the pressure ulcer wound surface formed after being pressed by two magnets with the average magnetic force of about 2500 gauss after 15 days of treatment of the hydrogel containing the nerve growth factor after the normal skin of the rat bears the pressure, and observe the change condition of the ulcer surface so as to judge the treatment effect of the hydrogel containing the nerve growth factor. From the results of the pathological section of the skin of fig. 1, it can be seen that: the change in the subcutaneous tissues of each group G1-G7 is basically similar, and the epithelial keratinization, scabbing and inflammatory cell infiltration of each group have no significant difference, wherein the epithelia of the groups G4, G5 and G6 are proliferated and thickened; the connective tissue in the dermis of each group has a tendency to vitrify the fibers due to the effect of pressure, with slight to moderate presentation in groups G1, G2, G3 and G4, and more pronounced changes in groups G5, G6 and G7. These pathological section changes of the skin are mainly in direct proportion to newly generated microvessels, i.e., the greater the number of blood vessels, the greater the vitrified area, and the better the ulcer healing progresses.
The blank gel prepared in example 4(G2) has better therapeutic effect on pressure ulcer of rat skin, which shows that the blank hydrogel matrix prepared by the invention can meet the requirement of moist healing of wound and accelerate the healing of ulcer.
The ulcer areas of the groups of examples 5-8(G5-G7) and comparative example 9(G3) are all significantly lower than those of example 4, which shows that the nerve growth factor has significant curative effect on the pressure ulcer of the skin of the rat, and the hydrogel containing the nerve growth factor has important potential in treating the pressure ulcer of the skin of the rat.
Whereas, the ulcer areas of examples 5 to 8(G5 to G7) were all significantly lower than those of comparative example 9, indicating that the therapeutic effect of the nerve growth factor-containing hydrogel prepared according to the present invention was significantly better than that of comparative example 9(G3), which is probably due to: (1) comparative example 9(G3) prepared a gel matrix using carbomer 940, which had inferior lubricity and viscosity compared to hydrogels prepared from carbomer 980, and which may affect ulcer healing; (2) the gel prepared in comparative example 9(G3) had not been verified in terms of moisturizing properties and could not ensure the requirement for wet healing of the affected sites such as wounds, ulcers, etc.; (3) the gel prepared in comparative example 9(G3) has relatively poor stability, and may cause instability and reduction of the biological activity of nerve growth factor in the gel; 4) the gel prepared in the comparative example 9(G3) has a low transdermal release amount of nerve growth factor, and is obviously different from the nerve growth factor-containing hydrogel prepared in the invention, so that the nerve growth factor is retained in the gel and cannot exert a curative effect on an ulcer surface.
Therefore, the hydrogel containing the nerve growth factor has a good technical effect in the treatment of pressure ulcer, is simple to prepare, uniform in texture, easy to smear, free of skin irritation, good in moisture retention and tissue penetrating fluid absorption performance, breaks through the important technical problems of transdermal release and activity maintenance of the nerve growth factor in a hydrogel external preparation and has high industrialization potential.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made in the above embodiments by those of ordinary skill in the art without departing from the principle and spirit of the present invention.
Claims (11)
1. A hydrogel containing protein is characterized in that the hydrogel comprises 1% of carbomer 980, 20% of glycerol, 20% of propylene glycol, 1% of povidone K30, 0.15% of methyl paraben, 0.05% of EDTA-2Na, 1% of azone, 0.5% of human serum albumin, 0.5% of mannitol, 0.001-0.012% of protein and the balance of water in percentage by weight; the protein is nerve growth factor; the nerve growth factor is murine nerve growth factor.
2. The protein-containing hydrogel according to claim 1, which is prepared by,
respectively taking glycerol and propylene glycol, adding water for injection, uniformly stirring, adding methyl paraben, EDTA-2Na and azone for dissolving, weighing carbomer 980, adding the carbomer 980, swelling overnight to obtain a mixed solution, slowly dripping an NaOH aqueous solution into the mixed solution, uniformly stirring and mixing to form viscous gel, adjusting the pH =6.5-7.5, sterilizing at high temperature, cooling, adding polyvidone K30, and supplementing the injection water to obtain a hydrogel matrix;
adding nerve growth factor solution into the hydrogel matrix at 2-8 deg.C, slowly stirring to avoid bubble generation, and obtaining hydrogel preparation containing nerve growth factor; the nerve growth factor solution contains rat nerve growth factor stock solution for injection, human albumin and mannitol.
3. The protein-containing hydrogel according to claim 2, wherein the nerve growth factor-containing hydrogel preparation is further centrifuged and stored separately.
4. The protein-containing hydrogel according to claim 2, which is prepared by,
respectively taking 20g of glycerol and 20g of propylene glycol, adding 30-80ml of water for injection, uniformly stirring, adding 0.15g of methyl paraben, 0.05g of EDTA-2Na and 1g of azone, dissolving, weighing 9801.0 g of carbomer, adding the carbomer into the solution for overnight swelling to obtain a mixed solution, slowly dripping 0.5-1.2ml of 50% NaOH aqueous solution into the mixed solution, uniformly stirring and mixing to obtain a viscous gel, adjusting the pH to be =6.5-7.5, sterilizing at the conditions of 0.1MPa and 121 ℃ for 20min, cooling to 4 ℃, adding 10ml of 10% povidone K30 into the viscous gel, supplementing the water for injection to the total weight of 95g, and obtaining 95g of hydrogel matrix;
adding 5ml nerve growth factor solution into the hydrogel matrix at 4 deg.C, slowly stirring to avoid bubble generation, and obtaining 100g hydrogel preparation containing nerve growth factor.
5. The protein-containing hydrogel according to claim 4, wherein the nerve growth factor-containing hydrogel preparation is centrifuged at 12000rpm for 5 minutes and then stored in a packed state.
6. The protein-containing hydrogel according to claim 4, wherein 5ml of the nerve growth factor solution contains 1 to 12mg of mouse nerve growth factor stock solution for injection, 0.5g of human serum albumin, 0.5g of mannitol, and the balance of water.
7. A method for producing the protein-containing hydrogel according to any one of claims 1 to 6,
respectively taking glycerol and propylene glycol, adding water for injection, uniformly stirring, adding methyl paraben, EDTA-2Na and azone to dissolve, weighing carbomer 980, adding the carbomer 980, swelling overnight to obtain a mixed solution, slowly dripping NaOH aqueous solution into the mixed solution, uniformly stirring and mixing to form viscous gel, adjusting the pH =6.5-7.5, sterilizing at high temperature, cooling to 4 ℃, adding polyvidone K30, and supplementing with the water for injection; obtaining the hydrogel matrix;
adding nerve growth factor solution into the hydrogel matrix at 2-8 deg.C, slowly stirring to avoid bubble generation, and obtaining hydrogel preparation containing nerve growth factor; the nerve growth factor solution contains rat nerve growth factor stock solution for injection, human albumin and mannitol.
8. The method for preparing protein-containing hydrogel according to claim 7, wherein 5ml of the nerve growth factor solution contains 1 to 12mg of mouse nerve growth factor stock solution for injection, 0.5g of human serum albumin, 0.5g of mannitol, and the balance of water.
9. The method for producing a protein-containing hydrogel according to claim 7, wherein the nerve growth factor-containing hydrogel preparation is further centrifuged and stored separately.
10. The method for preparing the protein-containing hydrogel according to claim 7, wherein 20g of glycerol and 20g of propylene glycol are respectively added into 30-80ml of water for injection and stirred uniformly, 0.15g of methylparaben, 0.05g of EDTA-2Na and 1g of azone are added to dissolve the water, 9801.0 g of carbomer is weighed and added into the water for injection and swelled overnight to obtain a mixed solution, 0.5-1.2ml of 50% (w/w) NaOH aqueous solution is slowly added into the mixed solution, the mixed solution is stirred and mixed uniformly to form a viscous gel, the pH =6.5-7.5 is adjusted, the mixed solution is sterilized at high temperature of 0.1MPa and 121 ℃ for 20min, the mixed solution is cooled to 4 ℃, 10ml of 10% povidone K30 is added into the mixed solution, and the water for injection is supplemented to make the total weight of 95g, so as to obtain 95g of the hydrogel matrix;
adding 5ml nerve growth factor solution into the hydrogel matrix at 4 deg.C, slowly stirring to avoid bubble generation, and obtaining 100g hydrogel preparation containing nerve growth factor.
11. The method for producing a protein-containing hydrogel according to claim 10, wherein the nerve growth factor-containing hydrogel preparation is centrifuged at 12000rpm for 5 minutes, and then stored in a packed state.
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