CN117618538A - Stem cell enhanced type bi-component biogel for skin repair - Google Patents
Stem cell enhanced type bi-component biogel for skin repair Download PDFInfo
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Classifications
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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
- A61K38/18—Growth factors; Growth regulators
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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Abstract
The invention discloses a stem cell enhanced type bi-component biogel for skin repair, which relates to the technical field of skin repair gel, and comprises the following components: the invention provides a biological gel rich in bioactive factors, which is combined with activated collagen, hyaluronic acid, stem cell exosomes and growth factors in the pH responsive nano-carrier to ensure rapid wound healing, wherein the concentration of the activated collagen is 3-7% (w/v), the concentration of the hyaluronic acid is 1-3% (w/v), the concentration of the stem cell exosomes is 0.1-1.0mg/mL, the pH responsive PLGA-PEG-PLGA nano-carrier is coated with 0.5-1.5 mug/mL growth factors (aFGF), and the balance of water. The nano carrier ensures the sustained and targeted release of the growth factors and avoids the rapid degradation of the growth factors in vivo. Activating collagen enhances the stability and bioactivity of the gel.
Description
Technical Field
The invention relates to the technical field of skin repair gel, in particular to stem cell enhanced type bi-component biogel for skin repair.
Background
The skin is used as the largest organ of human body, and has the functions of protecting body, regulating body temperature and sensing external stimulus. The skin is susceptible to various injuries such as burns, wounds, post-operative wounds, etc. due to its direct contact with the external environment. With the development of medicine and biotechnology, wound repair and regeneration have been increasingly studied, especially in the combined application of biomaterials, stem cell technology and nanotechnology.
Currently, there are various bio-gels for skin repair on the market, and these gels mainly consist of collagen, hyaluronic acid and other biological materials. These biogels, while capable to some extent of promoting wound healing, still present some problems. For example, conventional biogels may lack sufficient bioactivity to sufficiently promote cell proliferation and migration, thereby affecting rapid healing of the wound. In addition, growth factors in some biogels may degrade rapidly in vivo, resulting in unstable therapeutic effects. Although researchers have attempted to introduce nanotechnology and stem cell technology into biogel, these attempts still suffer from stability, insufficient bioactivity, and high cost.
In view of the above-described deficiencies of the background art, the present invention is directed to a novel stem cell-enhanced two-component biogel for skin repair. The biogel combines activated collagen, hyaluronic acid, stem cell exosomes and a pH responsive PLGA-PEG-PLGA nano-carrier technology. Wherein, the pH responsive nano-carrier is used as an innovative technology, and can realize the sustained and targeted release of the growth factors, thereby improving the stability and the biological activity of the growth factors in vivo. In addition, the activated collagen further enhances the stability and bioactivity of the activated collagen by covalently binding to a growth factor; the stem cell exosomes provide abundant growth factors and cytokines for wounds, promoting wound healing. By these key technical means, the present invention aims to provide a more effective and economical skin repair method.
Disclosure of Invention
In order to achieve the above object, the present invention provides a stem cell-enhanced two-component biogel for skin repair, comprising: activated collagen with the concentration of 3-7% (w/v), hyaluronic acid with the concentration of 1-3% (w/v), stem cell exosomes with the concentration of 0.1-1.0mg/mL, pH responsive PLGA-PEG-PLGA nano-carriers coated with 0.5-1.5 mug/mL growth factors (aFGF), and the balance of water.
Preferably, the stem cell exosomes are extracted by the following method:
mesenchymal Stem Cells (MSCs) were selected as a source of stem cells;
culturing the mesenchymal stem cells in a serum-free medium for 24-48 hours;
collecting the culture medium, and removing cells and cell fragments therein by centrifugation;
further purifying the culture medium by using an exosome extraction kit to extract exosomes;
preferably, the collagen powder is dissolved in 0.01M acetic acid at a concentration of 5% (w/v) and stirred for 4 hours;
EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) and NHS (N-hydroxysuccinimide acid) were added to the collagen solution and the mixture was reacted for 2-4 hours with gentle stirring at room temperature. .
Preferably, the molar ratio of EDC to NHS is 1:1 and their total molar amount is 2-5 times based on the number of carboxyl groups in collagen.
Preferably, the hyaluronic acid is obtained by dissolving hyaluronic acid powder at a concentration of 2% (w/v) in physiological saline and stirring for 2 hours.
Preferably, the pH responsive PLGA-PEG-PLGA nano-carrier is prepared by the following method:
PLGA-PEG-PLGA copolymer was dissolved in dichloromethane at a concentration of 1% (w/v);
adding aFGF, PLGA-PEG-PLGA with the mass ratio of 0.01-0.015% into the solution, and fully mixing;
emulsifying by using an ultrasonic emulsifier to obtain nano emulsion;
and (3) rotary evaporation is carried out under the vacuum condition, and the organic solvent is removed, so that the pH responsive nano-carrier is obtained.
Preferably, the emulsification time is 3-5 minutes.
Preferably, the growth factor is an epidermal growth factor (aFGF).
Preferably, the biogel is prepared by the following method:
mixing the cultured stem cell exosomes, the nano-carrier, activated collagen and a hyaluronic acid solution according to the volume ratio of 1:1:1, adding a proper amount of water to adjust the viscosity to the required viscosity, and obtaining the stem cell enhanced bi-component biogel.
Hyaluronic acid: hyaluronic acid is a polysaccharide that can provide support and moisturization to cells while also attracting and retaining moisture, thereby helping to maintain the moisturization and elasticity of the skin.
Stem cell exosomes: stem cell exosomes are small vesicles secreted by cells containing a variety of bioactive molecules, such as proteins, RNAs, and lipids. These molecules can promote cell proliferation, migration and differentiation, thereby accelerating wound healing.
pH responsive PLGA-PEG-PLGA nanocarriers: the intelligent nano-carrier can change the structure and the property according to the pH value of the environment. Under normal physiological conditions, the nanocarrier is stable, but at the wound or inflammation site, due to the change in pH, it releases encapsulated growth factors, thereby achieving targeted therapy.
Epidermal growth factor (aFGF): aFGF is a protein that promotes cell proliferation and migration, thereby accelerating wound healing. In this regimen, aFGF is entrapped in a pH-responsive nanocarrier, ensuring a sustained and targeted release at the wound site.
Collagen is a major component of skin and is critical for skin repair. In this scheme, collagen is activated by EDC and NHS to be more easily combined with other components, thereby improving stability and bioactivity of the biogel.
Activation of the interaction between collagen and aFGF may also enhance the stability of the biogel. aFGF can stimulate cells to produce more collagen, thereby making the biogel more stable. The activated collagen can enhance the mechanical property of the biogel and make the biogel more durable
The biogel may provide a sustained therapeutic effect due to the interaction between the activated collagen and the aFGF. Activation of collagen provides a durable scaffold for cells, while aFGF provides a sustained proliferation signal to cells, thereby ensuring long-term in vivo action of the biogel.
The technical scheme of the invention has the beneficial effects that:
conventional skin repair materials may not provide sufficient bioactive factors to promote rapid wound healing. The invention provides an environment rich in bioactive factors for the wound by combining activated collagen, hyaluronic acid, stem cell exosomes and growth factors in the pH responsive nano-carrier, thereby accelerating wound healing.
Conventional biogel may not achieve sustained and targeted release of growth factors, resulting in rapid degradation or loss of growth factors in vivo. The pH responsive PLGA-PEG-PLGA nano carrier ensures the continuous and targeted release of the growth factors at the wound site, thereby realizing the efficient utilization of the growth factors.
Traditional biogels may degrade rapidly in vivo, resulting in a decrease in their biological activity. The invention enhances the stability and biological activity of the biological gel by activating the collagen, so that the biological gel has longer duration in vivo.
Many existing biogel preparation methods are complex and time consuming. The invention provides a simplified preparation method, which only needs to mix and adjust the components to the required viscosity according to a certain volume ratio, thereby simplifying the production process and reducing the production cost.
Drawings
Fig. 1 is a picture of wound healing in example 1.
Fig. 2 is a picture of wound healing of comparative example 1.
Fig. 3 is a picture of wound healing of comparative example 2.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. Of course, the specific embodiments described herein are for purposes of illustration only and are not intended to limit the invention.
Example 1
1. Composition of the biogel:
activation of collagen: 7% (w/v)
Hyaluronic acid: 3% (w/v)
Stem cell exosomes: 1.0mg/mL
pH responsive PLGA-PEG-PLGA nanocarriers: coated with 1.5. Mu.g/mL of epidermal growth factor (aFGF)
The rest is water.
2. Extraction of stem cell exosomes:
mesenchymal Stem Cells (MSCs) were selected as a source of stem cells;
MSCs were cultured in serum-free medium for 48 hours;
collecting the culture medium and removing cells and cell debris by centrifugation;
purifying by using an exosome extraction kit to obtain stem cell exosomes.
3. Preparation of activated collagen:
dissolving collagen powder in acetic acid with concentration of 7% (w/v) of 0.01M, and stirring for 4 hours;
EDC and NHS are added, the molar ratio is 1:1, and the total molar quantity is 5 times of the quantity of carboxyl groups in the collagen;
the reaction was stirred slightly at room temperature for 4 hours.
4. Preparation of hyaluronic acid:
the hyaluronic acid powder was dissolved in physiological saline at a concentration of 3% (w/v), and stirred for 2 hours.
Preparation of a ph-responsive nanocarrier:
PLGA-PEG-PLGA copolymer was dissolved in dichloromethane at a concentration of 1% (w/v);
adding aFGF, and fully mixing the mixture, wherein the mass ratio of the aFGF to the PLGA to the PEG-PLGA is 0.015%;
the aFGF is an epidermal growth factor produced by Dou Yunxi chemical industry Co., ltd.
Emulsifying for 5 minutes by using an ultrasonic emulsifier;
and (3) rotary evaporation is carried out under vacuum, and the organic solvent is removed, so that the nano-carrier is obtained.
6. Preparation of the biogel:
mixing stem cell exosomes, nano-carriers, activated collagen and hyaluronic acid solution according to the volume ratio of 1:1:1:1;
adding a proper amount of water to adjust the viscosity to be required;
obtaining the stem cell enhanced two-component biogel.
Example 2
The procedure was as in example 1, except that activated collagen was added at a concentration of 3% (w/v).
Example 3
The procedure was as in example 1, except that activated collagen was added at a concentration of 7% (w/v).
Example 4
The procedure was carried out as in example 1, except that the molar ratio of EDC to NHS was 1:1 and their total molar amount was 2 times based on the number of carboxyl groups in collagen.
Example 5
The procedure was carried out as in example 1, except that the molar ratio of EDC to NHS was 1:1 and their total molar amount was 5 times based on the number of carboxyl groups in collagen.
Example 6
The procedure was as in example 1, except that the pH-responsive PLGA-PEG-PLGA nanocarriers were coated with 0.5. Mu.g/mL growth factor (aFGF).
Example 7
The procedure was as in example 1, except that the pH-responsive PLGA-PEG-PLGA nanocarriers were coated with 1.5- μg/mL growth factor (aFGF).
Example 8
The procedure was as in example 1, except that the mass ratio of PLGA-PEG-PLGA was 0.01%.
Example 9
The method of implementation is the same as in example 1, except that,
1. composition of the biogel:
hyaluronic acid: 1% (w/v)
Stem cell exosomes: 0.1mg/mL
2. Extraction of stem cell exosomes:
MSCs were cultured in serum-free medium for 24 hours;
3. preparation of activated collagen:
the reaction was stirred slightly at room temperature for 2 hours.
Preparation of a ph-responsive nanocarrier:
emulsification was carried out using an ultrasonic emulsifier for 3 minutes.
Comparative example 1
The procedure was as in example 1, except that the collagen was not subjected to the activation treatment.
Comparative example 2
The procedure was as in example 1, except that no pH-responsive PLGA-PEG-PLGA nanocarrier was added.
1. Test of wound healing speed:
a. experiments in healthy rats were selected; 15 healthy rats were selected and had a body weight ranging from 200 to 250g.
Rats were randomly divided into three groups: experimental group, control group, 5 per group.
b. After anesthesia, standardized skin wounds were made on the back of the animals using a sterile scalpel;
c. selecting a wound to smear biological gel of the experiment;
d. covering the wound with sterile gauze, and fixing;
e. the size and healing of the wounds were observed and recorded daily;
f. photographs of the wounds were taken at 7 days, 14 days, and the unhealed wound area was measured using image analysis software.
2. Testing of growth factor release kinetics:
a. phosphate buffer solution with pH of 5.5 is prepared and put into a culture bottle;
b. adding experimental biogel into a buffer solution, and placing the container into an incubator at 37 ℃;
c. at 1 hour, 8 hours, 24 hours of buffer samples, the growth factor concentration in the samples was measured using an ELISA kit.
TABLE 1 wound closure rate
| Project | 7day | 14day |
| Example 1 | 81.25％ | 96.32％ |
| Comparative example 1 | 71.36％ | 85.68 |
| Comparative example 2 | 52.64％ | 74.23％ |
TABLE 2 test of growth factor release kinetics
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (9)
1. A stem cell-enhanced two-component biogel for skin repair, the biogel comprising: activated collagen with the concentration of 3-7% (w/v), hyaluronic acid with the concentration of 1-3% (w/v), stem cell exosomes with the concentration of 0.1-1.0mg/mL, pH responsive PLGA-PEG-PLGA nano-carriers coated with 0.5-1.5 mug/mL growth factors (aFGF), and the balance of water.
2. The biogel according to claim 1, wherein the stem cell exosomes are extracted by the following method:
mesenchymal Stem Cells (MSCs) were selected as a source of stem cells;
culturing the mesenchymal stem cells in a serum-free medium for 24-48 hours;
collecting the culture medium, and removing cells and cell fragments therein by centrifugation;
the culture medium is further purified by an exosome extraction kit to extract exosomes.
3. The biogel according to claim 1, wherein:
dissolving collagen powder in 0.01M acetic acid at a concentration of 5% (w/v), and stirring for 4 hr; EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) and NHS (N-hydroxysuccinimide acid) were added to the collagen solution and the mixture was reacted for 2-4 hours with gentle stirring at room temperature.
4. A biogel according to claim 3 wherein the molar ratio of EDC to NHS is 1:1 and their total molar amount is 2-5 times based on the number of carboxyl groups in the collagen.
5. The biogel according to claim 1, wherein the hyaluronic acid is obtained by dissolving hyaluronic acid powder at a concentration of 2% (w/v) in physiological saline and stirring for 2 hours.
6. The biogel of claim 1, wherein the pH responsive PLGA-PEG-PLGA nanocarrier is prepared by:
PLGA-PEG-PLGA copolymer was dissolved in dichloromethane at a concentration of 1% (w/v);
adding aFGF and PLGA-PEG-PLGA in the mass ratio of 0.01-0.015% into the solution, and mixing thoroughly;
emulsifying by using an ultrasonic emulsifier to obtain nano emulsion;
and (3) rotary evaporation is carried out under the vacuum condition, and the organic solvent is removed, so that the pH responsive nano-carrier is obtained.
7. The biogel of claim 6, wherein the emulsification time is 3 to 5 minutes.
8. The biogel of claim 1, wherein the growth factor is epidermal growth factor (aFGF).
9. The biogel according to claim 1, wherein the biogel is prepared by the process of:
mixing the cultured stem cell exosomes, the nano-carrier, activated collagen and a hyaluronic acid solution according to the volume ratio of 1:1:1, adding a proper amount of water to adjust the viscosity to the required viscosity, and obtaining the stem cell enhanced bi-component biogel.
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| JP2013226205A (en) * | 2012-04-24 | 2013-11-07 | Kaohsiung Medical Univ | Biological material for wound healing and preparation the same |
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| CN102389585A (en) * | 2011-07-07 | 2012-03-28 | 中山大学 | Method for loading active molecules on surface of biomedical material |
| JP2013226205A (en) * | 2012-04-24 | 2013-11-07 | Kaohsiung Medical Univ | Biological material for wound healing and preparation the same |
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