CN114129710A - Fibroblast growth factor hydrogel and preparation method thereof - Google Patents
Fibroblast growth factor hydrogel and preparation method thereof Download PDFInfo
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- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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Abstract
本发明涉及一种成纤维细胞生长因子水凝胶及其制备方法，所述水凝胶包括0.1‑0.3wt％成纤维细胞生长因子(bFGF)、3‑6wt％卡波姆、2‑4wt％透明质酸、4‑8wt％腺苷甲硫氨酸、适量pH调节剂和余量的去离子水。本发明通过bFGF与特定配比的卡姆波、透明质酸、腺苷甲硫氨酸物理混合技术，在一定程度上改善药物的稳定性，增强药物应用的定向效应，并一定程度上发挥了凝胶剂的可塑性和使用的适应性，为急性创伤性脑损伤的治疗提供了新的方案。
The present invention relates to a fibroblast growth factor hydrogel and a preparation method thereof. The hydrogel comprises 0.1-0.3wt% fibroblast growth factor (bFGF), 3-6wt% carbomer, 2-4wt% Hyaluronic acid, 4-8wt% adenosylmethionine, an appropriate amount of pH adjuster and the balance of deionized water. The present invention improves the stability of the drug to a certain extent, enhances the directional effect of the drug application, and exerts a certain degree of effect through the physical mixing technology of bFGF and a specific ratio of Cambo, hyaluronic acid and adenosylmethionine. The plasticity of the gel and the adaptability of its use provide a new solution for the treatment of acute traumatic brain injury.
Description
Technical Field
The invention belongs to the field of biomedical materials, and particularly relates to a fibroblast growth factor hydrogel and a preparation method thereof.
Background
With the continuous improvement of the economic level of modern society, the rapid development of high-speed traffic networks, and the active expansion of industries such as the construction industry, acute Traumatic Brain Injury (TBI) has gradually become an important problem threatening human health in the fields of clinical first aid and critical medicine. In developed countries dominated by europe and the united states, approximately 260 million people per year are covered in the shadow of acute traumatic brain injury, and in these patients, over 40% remain permanently disabled. As one of the main diseases accompanied by multiple injuries, the incidence rate of acute traumatic brain injury in China is increased sharply in recent years, and the mortality rate is between 2.7 and 21.8 percent. The high fatality rate and disability rate of the traditional Chinese medicine become main medical problems in the medical field and even the whole society.
At present, the clinical commonly used treatment means are acute-phase operation and convalescent-phase neurotrophic medicaments, the operation only takes life saving as the first aim, and the effects of the commonly used medicaments such as brain protein extract, oxiracetam and the like are not exact. For the key factors influencing prognosis and quality of life, such as recovery of nerve function, protection of blood brain barrier and improvement aiming at myelination of neurons, higher limitation exists, and no specific medicine exists clinically up to now. Therefore, the search for a candidate drug for improving nerve function and promoting nerve repair in the acute phase after acute traumatic brain injury has been a hot and key problem for the research and development of new drugs in the field for a long time. Growth factor-type drugs have long been considered to have good potential in treating nerve injury and promoting recovery of nerve function. A plurality of Fibroblast Growth Factor (FGF) products, such as Beifuji, Beifuxin and Chuangbi, which are independently developed by research teams of the applicant are applied to clinic, and proved for more than twenty years, the products show good safety and effectiveness, but are mainly used for wound repair, such as the fields of skin, corneal ulcer, refractory chronic wounds and the like. In the field of nerve injury and repair, Basic fibroblast growth factor (bFGF) and a signal path mediated by the bFGF are found to play an important role in the processes of nerve function recovery, blood brain barrier repair and the like after acute central nerve trauma according to the previous research results. However, because the protein activity is unstable, the degradation and inactivation are easy, the half-life period is short, and the like, the clinical application is limited, so that the development of a gel preparation for open nerve injury is hoped, the activity and stability of the drug protein are protected, the drug action time is prolonged, the gel preparation is better suitable for a defective focus, and the drug can play a role in promoting nerve repair at an injury part in the early stage of injury.
Chinese patent application CN113069533A discloses a long-acting fibroblast growth factor gel, which belongs to the field of fibroblast growth factors and solves the problem of poor effect of intramuscular injection of bFGF related drugs in the prior art, and the technical scheme for solving the problem is mainly that the long-acting fibroblast growth factor gel is formed by mixing polyethylene glycol diacrylate and rhbFGF stock solution with the purity of more than 99 percent, the concentration of the long-acting fibroblast growth factor gel is 500000 IU/mL-550000 IU/mL, and the viscosity of the long-acting fibroblast growth factor gel is 45-55 Pa.s. The application is mainly used for directly acting the long-acting fibroblast growth factor gel on the optic nerve fibers, and has the advantages of slow absorption, good biocompatibility, realization of slow release of the medicine and better treatment effect.
Chinese patent application CN108721136A discloses a repair gel and a preparation method and application thereof, relating to the technical field of skin care products, wherein the skin care gel comprises basic fibroblast growth factor bFGF and precursor sol; the mass ratio of the basic fibroblast growth factor bFGF to the precursor sol is 1-2: 1000000. the bFGF can keep better stability in the precursor sol, still has better activity after being stored for half a year at the normal temperature through detection, and effectively solves the problem that the existing skin care product containing the bFGF component is not well stored at the normal temperature. Meanwhile, the components in the precursor sol and the bFGF are matched for use, so that the original effects can be exerted respectively, the components can play a synergistic effect, the curative effect is further enhanced, and the skin care product is obviously better than the existing skin care products in relieving symptoms such as dry skin, aging, dark yellow skin, color spots, acnes, allergy and the like.
Although the prior art has disclosed bFGF gels, it would be desirable to be able to find a suitable carrier: the price is low and the raw materials are easy to obtain; has excellent biocompatibility; can be degraded by itself; can slowly and stably release bFGF, and prolongs the action time of bFGF.
Disclosure of Invention
Based on the above background art, the technical problem to be solved by the present invention is to provide a fibroblast growth factor hydrogel and a preparation method thereof, which can maintain the activity of fibroblast growth factor at room temperature and rapidly release active ingredients at elevated temperature (for example, when reaching normal body temperature). In order to realize the purpose of the invention, the following technical scheme is adopted:
the invention relates to a fibroblast growth factor hydrogel, which comprises 0.1-0.3 wt% of fibroblast growth factor (bFGF), 3-6 wt% of carbomer, 2-4 wt% of hyaluronic acid, 4-8 wt% of adenosylmethionine, a proper amount of pH regulator and the balance of deionized water.
In a preferred embodiment of the invention, the hydrogel is a film. The hydrogel is arranged into a film form, so that the hydrogel can be directly used as a dressing for a wound part of brain injury.
In a preferred embodiment of the present invention, the hydrogel has a pH of 6.5 to 7.5.
In a preferred embodiment of the invention, the amount of ademetionine is from 6 to 8% by weight. The present invention contributes to further improving the storage stability of fibroblast growth factor (bFGF) by the preferred amount of ademetionine.
In a preferred embodiment of the invention, the carbomer is selected from the group consisting of carbomer 941 and/or carbomer 942; carbomer 941 is preferred.
In a preferred embodiment of the present invention, the fibroblast growth factor hydrogel has a significantly changed release rate of fibroblast growth factor (bFGF) at 37.0 ℃.
The invention also relates to a preparation method of the fibroblast growth factor hydrogel, which comprises the following steps:
1. adding deionized water into a preparation tank, adding hyaluronic acid and methionine, heating to 50-70 ℃, and uniformly stirring for later use;
2. dissolving carbomer in the rest deionized water heated to 80-95 deg.C, cooling to room temperature, adding fibroblast growth factor (bFGF), and stirring;
3. and (3) mixing the dispersions obtained in the step (1) and the step (2), adjusting the pH, placing the mixture into a container with a plane bottom surface, and cooling at room temperature to obtain a gel film.
The invention also relates to application of the hydrogel, which is applied to rapid repair of acute traumatic brain injury.
Advantageous effects
The invention improves the stability of the medicament to a certain extent, enhances the directional effect of the medicament application, exerts the plasticity and the use adaptability of the gel to a certain extent and provides a new scheme for treating acute traumatic brain injury by adopting a physical mixing technology of bFGF and carmowu, hyaluronic acid and adenosylmethionine in a specific ratio.
Drawings
FIG. 1: the release rate of fibroblast growth factor at different temperatures.
Detailed Description
In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available.
Example 1: a preparation method of fibroblast growth factor hydrogel comprises the following steps:
the preparation method comprises the following steps:
1. adding deionized water of which the total amount is half of the total amount in a preparation tank, adding hyaluronic acid and methionine, heating to 60 ℃, and uniformly stirring for later use;
2. dissolving carbomer 941 in the rest deionized water heated to 90 ℃, cooling to room temperature, adding fibroblast growth factor (bFGF), and stirring uniformly for later use;
3. mixing the dispersion obtained in step 1 and step 2, adjusting pH to 7.0, placing in a culture dish, and standing at room temperature to obtain a gel film with a thickness of 2 mm.
Example 2: a preparation method of fibroblast growth factor hydrogel comprises the following steps:
a gel film having a thickness of 2mm was obtained in the same manner as in example 1.
Example 3: a preparation method of fibroblast growth factor hydrogel comprises the following steps:
a gel film having a thickness of 2mm was obtained in the same manner as in example 1.
Comparative example 1:
same as example 1, except that S-adenosylmethionine was not added.
Comparative example 2:
same as example 1, except that hyaluronic acid was not added.
Comparative example 3:
the same as example 1 except that without adding carbomer 941, fibroblast growth factor (bFGF) was directly dissolved in deionized water in step (2).
Example 4: stability evaluation test
The activity of the basic fibroblast growth factor in the newly prepared samples, stored at 4 ℃ for 2 months and stored at 25 ℃ for 2 months is respectively determined, and the specific experimental steps are as follows:
(1) the gel films to be tested are respectively taken, cut into the size of 2cm multiplied by 2cm, and placed in 4mL PBS buffer solution with the pH value of 7.4, the PBS buffer solution is kept at 37 ℃, and the gel films are released for 2 hours by shaking.
(2) Adding the PBS buffer solution obtained in the step (1) into micropores of an enzyme detection plate to perform ELISA content determination (refer to the technology of frugal, xu Xiao Peng, Wang hong, and the like. basic fibroblast growth factor monoclonal antibody ELISA microscale detection, the technology of journal of Chinese immunology, 2006, 22(4)), determining the optical density of bFGF at 450nm, and repeating the determination for 3 times.
(3) For ease of comparison, the assay results for each freshly prepared sample were normalized to 100%, and the activity of the stored sample was taken as its activity data as a percentage of the assay result relative to the results of the corresponding freshly prepared sample for that sample, and the experimental results are shown in table 1.
Table 1 stability evaluation test results
| Group of | Novel preparation | Storing at 4 deg.C for 2 months | Storing at 25 deg.C for 2 months |
| Example 1 | 100±5％ | 92±6％ | 82±7％ |
| Example 2 | 100±7％ | 87±5％ | 76±8％ |
| Example 3 | 100±6％ | 85±7％ | 74±6％ |
| Comparative example 1 | 100±5％ | 42±4％ | 31±3％ |
| Comparative example 2 | 100±7％ | 57±8％ | 45±5％ |
| Comparative example 3 | 100±8％ | 62±7％ | 48±6％ |
As can be seen from the above, when stored at both room temperature (25 ℃ C.) and 4 ℃ C, bFGF activity decreases in order with the storage time; the low temperature is beneficial to maintaining the bFGF activity, the loss of the bFGF activity is relatively quick when the bFGF is stored at the normal temperature, but the bFGF activity loss can be obviously reduced in the embodiment of the invention, and particularly, the formula in the embodiment 1 has the highest stability. In contrast, comparative example 1, which lacks S-adenosylmethionine, showed the most significant decrease in activity, which indicates that S-adenosylmethionine, carbomer and hyaluronic acid in a specific ratio is helpful for improving the long-term storage stability of fibroblast growth factor hydrogel.
Example 5: evaluation test of Release efficiency
The prepared gel films are respectively taken to be 2cm multiplied by 2cm and placed in PBS buffer solution with the pH value of 4ml and 7.4, and the gel films are completely immersed in the PBS solution. The temperature of PBS was controlled, respectively, the concentration of fibroblast growth factor (bFGF) in the PBS buffer solution was measured after 1 hour of immersion and the amount of fibroblast growth factor (bFGF) released from the gel film was estimated. The amount of released fibroblast growth factor (bFGF) of the gel film at different temperatures for 1 hour was determined based on the amount of original bFGF and the amount of released bFGF in the gel film.
Referring to fig. 1, it can be seen from fig. 1 that the gel films of examples 1 to 3 show a significant change in release rate at 37.5 ℃, thereby illustrating that the gel films of examples have temperature sensitivity around 37.5 ℃. Unlike the examples, comparative examples 1-3 showed less pronounced tendency of the release rate to change with temperature, and particularly comparative example 1, which lacks temperature sensitivity due to the absence of S-adenosylmethionine, although the release rate was higher.
The foregoing describes preferred embodiments of the present invention, but is not intended to limit the invention thereto. Modifications and variations of the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.
Claims (8)
1. A fibroblast growth factor hydrogel comprises 0.1-0.3 wt% of fibroblast growth factor bFGF, 3-6 wt% of carbomer, 2-4 wt% of hyaluronic acid, 4-8 wt% of adenosylmethionine, a proper amount of pH regulator and the balance of deionized water.
2. The hydrogel according to claim 1, which is a film, and the hydrogel is provided in the form of a film, so that the hydrogel can be directly used as a dressing for a wound part of brain injury.
3. The hydrogel according to claim 1, wherein the hydrogel has a pH of 6.5 to 7.5.
4. The hydrogel of claim 1, wherein said ademetionine is present in an amount of 6-8 wt%.
5. The hydrogel of claim 1, said carbomer being selected from the group consisting of carbomer 941 and/or carbomer 942; carbomer 941 is preferred.
6. The hydrogel of claim 1, wherein the fibroblast growth factor hydrogel has a significant change in the rate of release of fibroblast growth factor (bFGF) at 37.0 ℃.
7. A process for the preparation of a hydrogel according to any one of claims 1 to 6, which process comprises the following steps:
(1) adding deionized water into a preparation tank, adding hyaluronic acid and methionine, heating to 50-70 ℃, and uniformly stirring for later use;
(2) dissolving carbomer in the rest deionized water heated to 80-95 deg.C, cooling to room temperature, adding fibroblast growth factor bFGF, and stirring;
(3) and (3) mixing the dispersion obtained in the step (1) and the dispersion obtained in the step (2), adjusting the pH, placing the mixture in a container with a plane bottom surface, and cooling at room temperature to obtain a gel film.
8. Use of the hydrogel of any one of claims 1 to 6 for the rapid repair of acute traumatic brain injury.
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