CN117442549A - Preparation method of thermosensitive gel capable of carrying exosomes - Google Patents

Preparation method of thermosensitive gel capable of carrying exosomes Download PDF

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Publication number
CN117442549A
CN117442549A CN202311415044.6A CN202311415044A CN117442549A CN 117442549 A CN117442549 A CN 117442549A CN 202311415044 A CN202311415044 A CN 202311415044A CN 117442549 A CN117442549 A CN 117442549A
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temperature
collagen
sensitive gel
exosome
mesenchymal stem
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汪巍巍
石亚宁
王健
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Anling Shanghai Biotechnology Co ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

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Abstract

The invention provides a preparation method of a temperature-sensitive gel capable of carrying exosomes, wherein the temperature-sensitive gel carrying exosomes comprises hydroxybutyl chitosan, collagen and mesenchymal stem cell exosomes. The temperature-sensitive gel is prepared by crosslinking hydroxybutyl chitosan and collagen by a crosslinking agent, and the exosome is stored by freeze drying and is uniformly mixed with the temperature-sensitive gel before use, so that the stability of the exosome is fully ensured, and the exosome is convenient to store and transport. The invention provides a simple and effective exosome application mode, and improves the clinical application feasibility of the exosome.

Description

Preparation method of thermosensitive gel capable of carrying exosomes
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a preparation method of a thermosensitive gel capable of carrying exosomes.
Technical Field
Mesenchymal stem cells (MCSs) have the functions of multidirectional differentiation, regulation of body immunity, secretion of various bioactive substances and the like. MCSs can stably proliferate in vitro and have low decay rate, and simultaneously have the advantages of easily obtained materials, capability of obtaining a large number of stem cells by a small amount of tissues, suitability for large-scale culture, small damage to organisms and the like. The exosomes are used as main components of the MCSs, the paracrine of the MCSs is used for regulating the formation of new blood vessels, promoting cell proliferation, and the exosomes are widely researched in the fields of arthritis, cartilage repair and spinal cord injury repair, and have great application potential in the aspects of treatment of chronic wounds such as traumatic wounds, diabetic feet and the like, skin system diseases, skin rejuvenation and the like.
The action time of simple exosome (Exosomes) injection is too short, and the use mode has the defects of rapid dilution and rapid discharge, so that long-term effect cannot be achieved, and repeated injection is obviously not feasible in clinical treatment. To solve these problems, it is required to develop a sustained release system suitable for exosomes. The exosomes are combined with biological materials, such as chitosan hydrogel, chitosan/silk gel sponge, graphene nano materials and the like, and the biological materials can enable the exosomes to be continuously released for a long time and exert a lasting treatment effect, so that a better application effect is achieved. Based on this, the development of a simple temperature responsive biomaterial would further increase the clinical feasibility of exosomes.
The hydroxybutyl chitosan (HBC) is a derivative of chitosan, the HBC is easily dissolved in water at low temperature, the temperature is increased, the in-situ rapid gel formation can be realized, the high-porosity water-retaining property, the high elasticity, the high biocompatibility and the high degradability are realized, and the tissue engineering material has wide application prospect. The positively charged HBC can conveniently encapsulate electronegative protein drugs to protect the protein drugs from enzyme degradation and prolong the biological activity of the protein drugs in vivo.
Collagen (Collagen) is the most abundant and most widely distributed functional protein in mammals. The preparation has good biocompatibility, biodegradability and bioactivity, so that the preparation has wide application in the fields of food, medicine, tissue engineering and the like. The recombinant III type humanized collagen is produced by a biological fermentation technology, has single component, high purity and high safety, and is easy to absorb. HBC and recombinant humanized collagen are properly crosslinked, so that the recombinant humanized collagen still has better biocompatibility, the degradation time of the temperature-sensitive material in the body can be prolonged, and the release of exosomes can be effectively controlled. Different blending ratios of HBC and recombinant humanized collagen can meet the requirements of various fields such as tooth Zhou Xiufu, facial filling, tissue wound repair and the like.
The mesenchymal stem cell exosome (MCSs-Exo) freeze-dried preparation is used as a solid dry powder, can be stored for a long time at normal temperature, has stable performance and is convenient to transport. The combination of the MCSs-Exo and the degradable temperature sensitive material is a simple and effective exosome application mode, and provides more possibility for the clinical application of exosome.
Disclosure of Invention
Aiming at the defects of the application of the existing exosomes, the invention provides a preparation and use method of a thermosensitive gel capable of carrying the exosomes, wherein the biological material is hydroxybutyl chitosan and recombinant collagen which are crosslinked through a crosslinking agent.
Furthermore, the invention provides a preparation method of the temperature-sensitive gel capable of carrying the MCSs-Exo, and the cross-linking agent is genipin.
Further, the invention provides a preparation method of the thermosensitive gel capable of carrying MCSs-Exo, which comprises the following steps:
(1) Alkalizing chitosan, and then modifying the chitosan by taking 1, 2-epoxybutane as an etherifying agent to prepare the hydroxybutyl chitosan (HBC) with good water solubility and temperature sensitivity.
(2) Dispersing HBC and recombinant collagen in water solution respectively, stirring uniformly, and concentrating to 5-20g/L.
(3) HBC and recombinant collagen solution volume ratio 1:1, and stirring for 1h at 4 ℃ to obtain a mixed solution.
(4) According to HBC and genipin crosslinking agent 1: feeding in a proportion of 0.1-10, and regulating the pH value of the mixed solution to 6.4-7.4.
(5) Dialysis was performed with deionized water at 4℃for 48 hours, with water being changed every 12 hours.
(6) The mixed solution is injected into a penicillin bottle, 60 co-gamma rays (12 kGy) were sterilized by irradiation and stored in a refrigerator at 4 ℃.
Further, the invention provides a preparation method of the thermosensitive gel capable of carrying MCSs-Exo, which comprises the following steps:
(1) Mesenchymal Stem Cells (MSCs) were expanded into 5-layer T175 flasks and incubated at 37℃with 5% CO 2 Culturing in incubator until fusion degree is about 85%, collecting supernatant for exosome extraction.
(2) Centrifuging the supernatant at 300g and 4 ℃ for 5min; centrifuging again at 2000g and 4 ℃ for 10min; the supernatant was removed and filtered 0.22 μm.
(3) The filtrate was concentrated by ultrafiltration using a tangential flow filtration system (TFF), and the final product was sterile filtered using a 0.22 μm filter to give a suspension containing the exosomes and identified.
(4) Mixing the obtained exosome suspension with preservative 1:2, freeze drying, and preserving.
Further, the invention provides a preparation method of the thermosensitive gel containing the MCSs-Exo, which comprises the following steps: before use, the temperature-sensitive gel is injected into the exosome freeze-dried powder, and is gently mixed and shaken until the gel is completely dissolved.
Compared with the prior art, the invention has the advantages that:
(1) The invention provides a preparation method of hydroxybutyl chitosan/collagen crosslinking temperature-sensitive gel.
(2) The novel composite gel HBC-COL-Exo formed by compounding MCSs-Exo with hydroxybutyl chitosan/collagen serving as a carrier can still respond to temperature change.
(3) The invention provides an application method for combining exosomes and biological materials, wherein the exosomes and hydroxybutyl chitosan/collagen cross-linked gel are stored separately and used in combination, and in vitro experiments prove that the exosomes and the hydroxybutyl chitosan/collagen cross-linked gel have no potential cytotoxicity.
Drawings
FIG. 1 shows a preparation process flow;
FIG. 2 is a photograph of the status of temperature sensitive hydrogel samples at different temperatures;
FIG. 3L929 is a view of a cell microscope;
FIG. 4 in vitro cytotoxicity test of temperature sensitive gels of different concentrations of MCSs-Exo;
fig. 5 intradermal injection site profile.
Detailed description of the preferred embodiments
The gel system is obtained by crosslinking hydroxybutyl chitosan and collagen and carrying exosomes. The present invention will be further described in detail with reference to specific examples, which are not intended to limit the scope of the invention, for better understanding of the invention.
Example 1
1g of hydroxybutyl chitosan was weighed out and dispersed in 100ml of deionized water, and stirred under ice bath until dissolved.
0.5g of recombinant humanized collagen is weighed and dispersed in 100ml of deionized water under ice bath and stirred uniformly.
Mixing the hydroxybutyl chitosan solution and the collagen solution in a volume ratio of 1:1, and stirring for 1h to obtain a mixed solution.
Adding HBC and genipin cross-linking agent in a ratio of 1:0.1, adjusting the pH value of the mixed solution to 6.4, and stirring for 2 hours in an ice bath.
Dialysis was performed with deionized water at 4℃for 48 hours, with water being changed every 12 hours.
The hydroxybutyl chitosan/collagen is dialyzed after being crosslinked, and the mixed solution is injected into a penicillin bottle and is subjected to the following steps of 60 Co-gamma rays (12 kGy) were sterilized by irradiation and stored at 4 ℃.
Mesenchymal Stem Cells (MSCs) were expanded into 5-layer T175 flasks and incubated at 37℃with 5% CO 2 The culture was carried out in an incubator until the degree of fusion was about 85%, and the supernatant was collected.
Centrifuging the supernatant at 300g and 4 ℃ for 5min; centrifuging again at 2000g and 4 ℃ for 10min; the supernatant was filtered at 0.22. Mu.m.
The filtrate was concentrated by ultrafiltration using a tangential flow filtration system (TFF), and the final product was sterile filtered using a 0.22 μm filter to give a suspension containing the exosomes and identified.
Mixing the obtained exosome suspension with preservative 1:2, freeze drying, and preserving.
Preparing temperature-sensitive gel: adding appropriate amount of temperature-sensitive gel into exosome lyophilized powder, and regulating exosome concentration to 2×10 respectively 8 particles/ml、4*10 8 particles/ml、8*10 8 The parts/ml are used for preparing the mesenchymal stem cell exosome temperature-sensitive gel.
Example 2
Temperature sensitive gel was prepared as in example 1
Gel temperature experiment
Equal mass of hydroxybutyl chitosan/collagen gel (HBC-COL) and hydroxybutyl chitosan/collagen gel containing exosomes (HBC-COL-Exo) were weighed into a penicillin bottle, slowly warmed up from 25 ℃ to 40 ℃ at a rate of 1 ℃/min, observed once per minute, the temperature at the time of just-started solidification was recorded, defined as the gelation temperature, and each sample was repeated 3 times.
Table 1 determination of gelation temperature (n=3)
The result shows that the prepared gel has temperature-sensitive property, can be made into gel at the temperature of more than 32 ℃ (figure 2), and the exosome preservative takes trehalose as a main raw material and has little influence on the gel temperature, but the exosome has no obvious change on the temperature-sensitive property of the HBC-COL gel.
Example 3
Temperature sensitive gel was prepared as in example 1
In vitro cytotoxicity assay
0.2g of HBC-COL and HBC-COL-Exo gel solution is taken, 1ml of serum-containing medium is taken as extraction medium, and the mixture is soaked for 24 hours at 37 ℃ to prepare test liquid.
The L929 mouse fibroblast density was adjusted in centrifuge tubes with each test solution, 100. Mu.l of cell suspension was inoculated into 96-well plates, 5 duplicate wells per group, and the plates were incubatedThe incubator was pre-incubated for 1,5,7 days (37 ℃,5% CO) 2 Is under the condition of (2).
The medium was removed prior to detection and the cells were washed twice with medium, then fresh medium was added to remove the effect of the formulation contents, and 10 μl CCK-8 solution was added per well.
The plates were incubated in the incubator for 1 hour.
The absorbance at 450nm was measured with a microplate reader.
Each formulation test solution cultures L929 cells for CCK-8 experiments, and survival rates (%) of L929 cells at 1,5,7 days were calculated according to the formula:
a (experiment): absorbance of wells with cells, CCK-8 solution and experimental solution
A (blank): absorbance of wells with medium and CCK-8 solution without cells
A (0 dosing): absorbance of wells with cells, CCK-8 solution without drug solution
The results of in vitro cytotoxicity assays are shown in FIG. 2, where each formulation sample was not potentially cytotoxic and the presence of exosomes slightly promoted proliferation of L929 cells.
The L929 cells were observed under a microscope for 24 hours, most of the cells in the negative control group and the experimental group were normal in morphology, a small amount of cells were round-contracted, loose and adherent, no intracellular particles were present or morphological changes were shown, and the cell state of the experimental group was observed under a microscope as shown in FIG. 3.
Skin sensitization test
Hydroxybutyl chitosan/collagen gel (HBC-COL) is prepared by using a leaching solvent, the leaching solution is injected into the part of the inner side of the haired shoulder swelling bone of a guinea pig in a skin mode for intradermal induction and local induction is carried out through sealing application to generate sensitization, a control group animal is given a solvent control solution in the same method, after induction, test animals are subjected to application excitation by using a test solution, and skin reactions of all animal excitation parts are observed and graded 24h and 48h after the application is removed.
Sample preparation
(1) Polar leaching solution
The product is taken, 0.9 percent sodium chloride injection is taken as a leaching medium, and the test solution is prepared according to the leaching proportion of 0.2g HBC-COL to 1ml leaching solution, and the temperature is kept at 37 ℃ for 72 hours. The test liquid is colorless and transparent.
(2) Nonpolar leaching liquor
The sample is prepared by taking cottonseed oil as a leaching medium, and a test solution is prepared according to a leaching ratio of 0.2g sample to 1ml leaching solution and maintaining the temperature at 37 ℃ for 72 hours. The test liquid is pale yellow and transparent.
(3) Solvent control solution
The same batch of leaching solvent I (polar solvent and nonpolar solvent) was taken, no sample was added, and the same conditions were used for preparation. The polar solvent control liquid is a non-transparent liquid, and the nonpolar solvent control liquid is a pale yellow transparent liquid.
(4) Positive control solution
The same batch of leaching solvent (polar solvent and nonpolar solvent) is taken and added with 1-chloro-2, 4-dinitrobenzene to prepare positive control solution.
Test procedure
(1) Prior to the test, each animal was numbered and weighed and the test site was thoroughly shaved. Each animal was observed for health.
(2) Intradermal induction: as shown in FIG. 4, 0.1ml of the dehaired medial portion of the shoulder and spleen bones was injected intradermally in pairs into each animal.
Site A. Freund's complete adjuvant is injected with a stabilizing emulsifier mixed with the selected solvent in a 50:50 (volume ratio) ratio.
Site B, injection of test sample (undiluted extract) control animals were injected with the corresponding solvent only.
Site C, the test sample (concentration used in site B) was mixed with an emulsifier formulated with Freund's complete adjuvant and solvent (50%) in a 50:50 volume ratio and injected intradermally; the control group is injected with an emulsifying agent prepared by blank liquid and an adjuvant.
(3) Local induction: no stimulation reaction is generated after intradermal inductionShould be. On day 7 after intradermal induction, the test zone was pretreated with 10% sodium dodecyl sulfate and massage introduced into the skin with an area of 8cm on day 8 2 The patch (absorbent gauze) was impregnated with the extract, and applied to the inner side of the shoulder swelling bone of each guinea pig locally, covering the induction injection point. Fixing with a closed type binding belt, and removing the binding belt and the gauze piece after 48 hours. Animals of the control group were treated with the solvent control solution in the same manner.
(4) Excitation local: all test animals and control animals were challenged with the test samples 14 days after induction. The application patch was soaked in the test sample or the solvent control solution, and applied to the upper abdomen dehairing area (non-test area in the induction stage) of each animal. Secured with a closed wrap and the wrap and patch removed after 24 hours.
(5) Skin reactions at the sites of challenge were observed 24 and 48 hours after patch removal in the animals of the test and control groups, and skin erythema and edema reactions were described and graded for each site and for each time of observation according to Magnusson and Kligman grading standards.
TABLE 2 Magnusson and Kligman fractionation
Application test reaction Grade
No obvious change 0
Sporadic or spotted erythema 1
Moderate fusion erythema 2
Severe erythema/or edema 3
Test results
Under the test conditions, the skin of the animal excitation part of the two leaching solvents of the test sample has no erythema and edema, the grading is 0, and the skin sensitization reaction of guinea pigs is not caused.
The above is only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited by this, and any modification made on the basis of the technical scheme according to the technical idea of the present invention falls within the protection scope of the claims of the present invention.

Claims (10)

1. A temperature sensitive gel capable of carrying exosomes, the gel comprising hydroxybutyl chitosan, collagen and mesenchymal stem cell exosomes.
2. The method for preparing a temperature-sensitive gel capable of carrying an exosome according to claim 1, wherein the hydroxybutyl chitosan and collagen are crosslinked by a crosslinking agent, and are preserved in a penicillin bottle after sterilization.
3. The method for preparing a temperature-sensitive gel according to claim 2, wherein the concentration of the hydroxybutyl chitosan solution is 5-20g/L.
4. The method for preparing a thermosensitive gel according to claim 2, wherein the collagen is recombinant humanized collagen using an escherichia coli expression system, and the concentration of the solution is 5-20g/L.
5. The method for preparing a temperature-sensitive gel according to claim 2, wherein the hydroxybutyl chitosan and collagen are crosslinked by genipin, and dialyzed with deionized water at 4 ℃ for 48 hours.
6. The method for preparing a temperature-sensitive gel according to claim 2, wherein the hydroxybutyl chitosan solution is preferably at a concentration of 10g/L and the collagen solution is preferably at a concentration of 5g/L.
7. The method for preparing a temperature-sensitive gel according to claim 2, wherein the hydroxybutyl chitosan solution and the collagen solution are subjected to crosslinking and dialysis, then injected into a penicillin bottle, sterilized by irradiation, and stored at 4 ℃.
8. The method of claim 1, wherein the mesenchymal stem cells comprise adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, hair follicle mesenchymal stem cells, and engineering mesenchymal stem cell exosomes.
9. The preparation of the temperature-sensitive gel capable of carrying an exosome according to claim 1, wherein the temperature-sensitive gel is injected into the exosome freeze-dried powder before use, and is mixed and shaken uniformly.
10. The preparation of a temperature-sensitive gel capable of carrying exosomes according to claim 1, wherein the exosome concentration is 2 x 10 8 - 8*10 10 particles/ml。
CN202311415044.6A 2023-10-30 2023-10-30 Preparation method of thermosensitive gel capable of carrying exosomes Pending CN117442549A (en)

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