CN114939098B

CN114939098B - Exosome-loaded hydrogel and preparation method and application thereof

Info

Publication number: CN114939098B
Application number: CN202210557830.9A
Authority: CN
Inventors: 魏玉君; 许尧
Original assignee: Mindenanga Chengdu Biotechnology Co ltd
Current assignee: Mindenanga Chengdu Biotechnology Co ltd
Priority date: 2022-05-19
Filing date: 2022-05-19
Publication date: 2023-09-26
Anticipated expiration: 2042-05-19
Also published as: CN114939098A

Abstract

The application discloses an exosome-loaded hydrogel and a preparation method and application thereof. The hydrogel loaded with the exosomes comprises a gel substrate and the exosomes loaded on the gel substrate, wherein the mass ratio of the exosomes to the gel substrate is 0.5-5:100000. The preparation method of the hydrogel is simple, quick and high in repeatability, and is suitable for industrial production. The prepared exosome-loaded composite hydrogel has the advantages of local high concentration and low toxicity of the whole body, and has the characteristics of skin permeability, long residence time and the like. In addition, the exosome-loaded composite hydrogel also has injectable, biodegradable and slow-release properties. In addition, exosomes are natural content substances, have low immunogenicity, are not sensitized, and have strong capability of transmitting information among cells. The slow release and local administration can be realized by matching with the release of the gel substrate, and the physical barrier is given to the skin, so that the regeneration of skin tissues can be realized, and the scar tissue generation and skin pigmentation can be effectively improved.

Description

Exosome-loaded hydrogel and preparation method and application thereof

Technical Field

The application belongs to the technical field of technical gel, and particularly relates to an exosome-loaded hydrogel, and a preparation method and application thereof.

Background

Exosomes are hot research hotspots on the prior art, but gel products of exosomes used in the anti-aging field are rarely reported. The exosomes derived from mesenchymal stem cells are the main source of their therapeutic effects. The exosomes are used as endogenous substances for mutually transmitting signals among cells, so that the risks of immunogenicity, sensitization, tumorigenicity and the like in the traditional cell preparation treatment process can be avoided, and the safety degree is improved. The exosome has stable property, easy control of dosage and concentration, and easy homing and storage. Many advantages have made exosomes of recent interest. The exosomes are membrane lipid vesicles with average diameters of 40-150 nm, and the content of the exosomes contains mRNA and miRNA, and also contains a large amount of cytokines, ceramide, metalloproteinases and the like. Especially, the content protein and RNA component contained in the exosome of the mesenchymal stem cells can be communicated with the damaged cells, can carry out biological control on the target cells, stimulates tissue regeneration and effectively repairs the damaged parts of the skin. The mesenchymal stem cell exosome has dual regulation functions of promoting wound healing and inhibiting scar formation on the regulation function of skin fibroblasts. Namely, the repair speed is improved by promoting the synthesis of collagen in the early stage of wound healing, and the formation of scar tissues is inhibited by reducing the synthesis of collagen in the later stage, so that the repair effect is improved. However, exosomes lack tissue specificity, so topical administration forms are ideal options.

Gel has been widely used in the fields of drug release and tissue engineering as a drug delivery platform. The hydrogel has high biological safety, good biocompatibility and biodegradability, and can be combined with exosomes in a physical or chemical adsorption mode to prepare corresponding dosage forms. The active ingredient is introduced into the human body through diffusion, osmosis and the like, so that the active ingredient is continuously and slowly released in the human body at a stable and controllable rate with proper concentration. The method has the advantages of effectively solving the problems of overlarge concentration of local active substances in human body, low bioavailability, strong toxic and side effects, short half-life of exosome and the like caused by the traditional mode. A common aqueous gel matrix is carbomer, which is a high molecular polymer of acrylic acid bonded allyl sucrose or pentaerythritol allyl ether. Carbomers and their decomposition products are non-toxic and harmless, giving the skin barrier effect and preventing microbial invasion. The gel containing carbomer has the advantages of better adhesiveness, water retention, slow release property and the like, and can exert ideal therapeutic effect on local parts. However, the existing carbomer-based hydrogel has no injectability or poor injectability, cannot effectively convey active ingredients to specific sites, and the existing older carbomer model uses benzene as solvent for extraction, so that the carbomer-based hydrogel has high benzene toxicity and is not easy to metabolize, and has residual potential risks. The old type carbomer has poor ion resistance and cannot be compatible with other substances such as ionic polysaccharide. These substances can disrupt the formed carbomer network, resulting in a decrease in gel strength.

Disclosure of Invention

Aiming at the prior art, the application provides the hydrogel loaded with the exosomes, and the preparation method and the application thereof, so as to obtain the hydrogel with excellent physical and chemical properties and good beautifying effect.

In order to achieve the above purpose, the technical scheme adopted by the application is as follows: providing an exosome-loaded hydrogel, comprising a gel substrate and exosome loaded on the gel substrate, wherein the mass ratio of the exosome to the gel substrate is 0.5-5:100000; the gel substrate is prepared from the following raw materials in parts by mass:

1 to 6 parts of gel matrix, 0.1 to 1 part of active sugar, 0.1 to 2 parts of citric acid, 1 to 5 parts of acidity regulator, 2 to 30 parts of humectant, 0.1 to 0.5 part of auxiliary agent and 55 to 95 parts of purified water.

The beneficial effects of the application adopting the technical scheme are as follows: according to the application, the exosomes are loaded on the gel substrate, the exosomes take the gel substrate as a carrier, so that the exosomes are not only beneficial to the preservation and transportation of the exosomes, but also are more diversified in use, the gel loaded with the exosomes can be transported to a specific position in a mode of injection and the like, and the slow release of the exosomes is realized under the action of the gel.

The gel substrate is prepared from components such as gel matrix, active sugar, citric acid, acidity regulator, humectant, auxiliary agent and purified water, wherein the components have synergistic effect, so that the gel substrate has a richer space network structure, the gel substrate structure is more stable, and the stable loading of exosomes on the gel substrate is facilitated.

On the basis of the technical scheme, the application can be improved as follows.

Further, the gel matrix is a mixture of carbomer, sodium alginate and carboxymethyl chitosan according to the mass ratio of 5-10:2-4:1.

Further, the active sugar is at least one of aloe active polysaccharide, fructo-oligosaccharide, galacto-oligosaccharide, raffinose, stachyose and xylo-oligosaccharide.

Further, the acidity regulator is triethanolamine or sodium hydroxide.

Further, the humectant is at least one of 1, 3-propanediol, 1, 2-propanediol and glycerin.

Further, the auxiliary agent is a mixture of phenoxyethanol, benzyl alcohol and butyl oxybenzene according to the mass ratio of 2-5:1-3:0.1.

Further, the gel substrate is prepared by the following steps:

and (S1) S: dissolving citric acid in 1/10 volume of purified water, adding a gel matrix, and swelling for 16-24 hours to obtain a gel substrate;

s2: dissolving active sugar in 1/10 volume of purified water to obtain active ingredient solution; dissolving the auxiliary agent in 1/20 volume of purified water, and adding the humectant to obtain a moisturizing liquid;

s3: adding an acidity regulator, an active ingredient solution, a moisturizing solution and the rest of purified water into a gel base material, stirring and reacting for 1-2 h at 35-45 ℃, and then centrifugally degassing for 20-30 min to obtain the product.

Further, exosomes are derived from umbilical cord mesenchymal stem cells.

The preparation method of the exosome-loaded hydrogel comprises the following steps:

s1: re-suspending the exosomes in 10mM PBS solution to obtain exosome suspension;

s2: adding the exosome suspension into the gel substrate, and stirring at the stirring speed of 600rpm/min for 5-10 min at the temperature of 4 ℃ to obtain the gel.

The beneficial effects of the application are as follows:

the preparation method of the hydrogel is simple, quick and high in repeatability, and is suitable for industrial production. The prepared exosome-loaded composite hydrogel has the advantages of local high concentration and low toxicity of the whole body, and has skin permeability and long residence time. In addition, the exosome-loaded composite hydrogel also has injectability, biodegradability and slow release performance. In addition, the exosomes are taken as natural content substances, have the advantages of low immunogenicity, no sensitization, strong capability of transmitting intercellular information, high safety and the like, can effectively realize the purposes of slow release and local administration by being matched with the release of a gel substrate, can give a physical barrier to skin, realize the regeneration of skin tissues and can effectively improve scar tissues and skin pigmentation.

Drawings

FIG. 1 is an injectability test chart of hydrogels obtained in examples and comparative examples 1 and 2.

Detailed Description

1. Extraction and purification of exosomes

Collecting supernatant of umbilical cord mesenchymal stem cells, sequentially performing ultracentrifugation and ultrafiltration on the supernatant, and separating to obtain exosomes from the supernatant. The method comprises the following specific steps:

ultracentrifugation: the low-temperature centrifuge is started in advance, and the temperature is set to be 4 ℃ for precooling. Adding the collected umbilical cord mesenchymal stem cell culture supernatant into a sterile centrifuge tube, placing 50mL of the culture supernatant into a centrifuge, setting the temperature to 4 ℃ and the time to 10 minutes, centrifuging, and discarding the precipitate to obtain supernatant;

transferring the supernatant to a new sterile centrifuge tube, setting the temperature to 4 ℃ and the time to 10 minutes, centrifuging, and discarding the precipitate to obtain the supernatant;

transferring the supernatant into a new sterile centrifuge tube, setting the temperature to 4 ℃ and the time to 30 minutes, centrifuging, and discarding the precipitate to obtain the supernatant;

sucking the supernatant by using a sterile syringe, filtering to a new sterile centrifuge tube by using a 0.22 mu m filter, and retaining filtrate;

opening an ultracentrifuge, precooling at 4 ℃, and putting a centrifugal rotor into a refrigerator at 4 ℃ in advance for precooling. And placing the filtrate into an ultracentrifugation adaptive centrifuge tube, balancing the corresponding centrifuge tube by using a balance, wherein the error is not more than 0.1g. Centrifuge parameters were set at 100000g, temperature at 4 ℃, time at 70 min centrifugation.

After centrifugation, the cell supernatant was gently aspirated using a pipette, taking care not to touch the bottom pellet. Then adding a proper amount of PBS, fully mixing, and transferring to a centrifuge tube by using a pipette.

Ultrafiltration: the centrifuge was started in advance and the temperature was set to 4 ℃ pre-chilled. The method comprises the steps that a liquid transferring gun sucks deionized water or buffer solution and drops the deionized water or buffer solution on the surface of a ultrafiltration tube filter membrane to enable the deionized water or buffer solution to infiltrate the whole membrane surface, the parameters of a centrifugal machine are set to be 1000g, the temperature is set to be 4 ℃, the time is set to be 10 minutes, and the lower liquid is discarded after centrifugation so as to wash superfluous glycerol or sodium azide on the membrane surface;

adding 15mL of heavy suspension into an upper layer tube of a ultrafilter tube, setting the parameters of a centrifuge to 3000g, setting the temperature to 4 ℃ and the time to 20 minutes, and retaining upper layer liquid after centrifugation;

the liquid in the upper tube of the ultrafiltration tube was carefully aspirated using a pipette and transferred to a fresh centrifuge tube, 10mL of PBS was added and thoroughly mixed, and added to the upper tube of the fresh ultrafiltration tube, the centrifuge parameters were set at 3000g, the temperature was set at 4℃and the time was set at 30 minutes. After centrifugation, the pipette aspirates the supernatant (about 200-500. Mu.L) and places it in a 2mL sterile centrifuge tube and stores it in a refrigerator at-80 ℃.

2. Preparation of gel substrates

The gel substrate is prepared from gel matrix, active sugar, citric acid, acidity regulator, humectant, auxiliary agent, purified water and the like. Wherein, the liquid crystal display device comprises a liquid crystal display device,

the gel matrix is a mixture of carbomer, sodium alginate and carboxymethyl chitosan according to the mass ratio of 5-10:2-4:1;

the active sugar is at least one of aloe active polysaccharide, fructo-oligosaccharide, galacto-oligosaccharide, raffinose, stachyose and xylo-oligosaccharide;

the acidity regulator is triethanolamine or sodium hydroxide;

the humectant is 1, 3-propylene glycol, 1, 2-propylene glycol or glycerol;

the auxiliary agent is a mixture of phenoxyethanol, benzyl alcohol and butyl oxybenzene according to the mass ratio of 2-5:1-3:0.1.

The preparation method of the gel substrate comprises the following steps:

The following describes the present application in detail with reference to examples.

Example 1

An exosome-loaded hydrogel comprises a gel substrate and exosomes loaded on the gel substrate, wherein the mass ratio of the exosomes to the gel substrate is 1:100000.

The adhesive substrate in this embodiment is prepared from the following raw materials in parts by mass:

1.5 parts of gel matrix, 0.5 part of active sugar, 0.5 part of citric acid, 1 part of acidity regulator, 20 parts of humectant, 0.5 part of auxiliary agent and 76 parts of purified water; wherein the gel matrix is a mixture of carbomer U30, sodium alginate and carboxymethyl chitosan according to the mass ratio of 8:3:1; the active sugar is aloe polysaccharide; an acidity regulator triethanolamine; the humectant is a mixture of 1, 3-propylene glycol, glycerol and the like; phenoxyethanol, benzyl alcohol and butyl oxybenzene are mixed according to a mass ratio of 3:2:0.1. The gel substrate is prepared through the following steps:

and (S1) S: dissolving citric acid in 1/10 volume of purified water, adding a gel matrix, and swelling for 20 hours to obtain a gel substrate;

s3: adding acidity regulator, active ingredient solution, moisturizing liquid and the rest purified water into gel base material, stirring at 40deg.C for reaction for 1 hr, and centrifuging and degassing for 25 min.

The exosomes in this example were derived from umbilical cord mesenchymal stem cells, and the extraction method was as described above.

The exosome-loaded hydrogel in this example was prepared by the following steps:

s2: adding the exosome suspension into gel substrate, and stirring at 4deg.C for 8min at 600 rpm/min.

Example 2

An exosome-loaded hydrogel comprises a gel substrate and exosomes loaded on the gel substrate, wherein the mass ratio of the exosomes to the gel substrate is 5:100000.

1 part of gel matrix, 1 part of active sugar, 0.1 part of citric acid, 1 part of acidity regulator, 2 parts of humectant, 0.1 part of auxiliary agent and 60 parts of purified water; wherein the gel matrix is a mixture of carbomer U30, sodium alginate and carboxymethyl chitosan according to the mass ratio of 5:4:1; the active sugar is aloe polysaccharide; acidity regulator sodium hydroxide; the humectant is a mixture of 1, 2-propylene glycol, glycerol and the like; the auxiliary agent is a mixture of phenoxyethanol, benzyl alcohol and butyl oxybenzene according to the mass ratio of 5:1:0.1. The gel substrate is prepared through the following steps:

and (S1) S: dissolving citric acid in 1/10 volume of purified water, adding a gel matrix, and swelling for 16 hours to obtain a gel substrate;

s3: adding acidity regulator, active ingredient solution, moisturizing liquid and the rest purified water into gel base material, stirring at 35deg.C for reacting for 2 hr, and centrifuging and degassing for 20 min.

s2: adding the exosome suspension into gel substrate, and stirring at 4deg.C for 5min at 600 rpm/min.

Example 3

6 parts of gel matrix, 0.1 part of active sugar, 2 parts of citric acid, 5 parts of acidity regulator, 30 parts of humectant, 0.5 part of auxiliary agent and 90 parts of purified water; wherein the gel matrix is a mixture of carbomer U30, sodium alginate and carboxymethyl chitosan according to the mass ratio of 10:2:1; the active sugar is aloe polysaccharide; acidity regulator sodium hydroxide; the humectant is a mixture of 1, 2-propylene glycol, glycerol and the like; the auxiliary agent is a mixture of phenoxyethanol, benzyl alcohol and butyl oxybenzene according to the mass ratio of 2:1:0.1. The gel substrate is prepared through the following steps:

and (S1) S: dissolving citric acid in 1/10 volume of purified water, adding a gel matrix, and swelling for 24 hours to obtain a gel substrate;

s3: adding acidity regulator, active ingredient solution, moisturizing liquid and the rest purified water into gel base material, stirring at 45deg.C for reaction for 1 hr, and centrifuging and degassing for 30 min.

s2: adding the exosome suspension into gel substrate, and stirring at 4deg.C for 10min at 600 rpm/min.

Comparative example 1

An exosome-loaded hydrogel. In comparison with example 1, the gel matrix included only carbomer U30 in the raw materials for preparing the gel matrix, and the remaining components and preparation methods were exactly the same as those of example.

Comparative example 2

An exosome-loaded hydrogel. In comparison with example 1, the gel substrate was prepared from the raw materials including only butyl oxybenzene and the rest of components and preparation method were exactly the same as those of example.

Analysis of results

Since the exosome-loaded hydrogels prepared in the three examples had similar properties, but the appearance conditions of example 1 were better, the properties of the hydrogels prepared in example 1 were described as an example.

The injectability of the hydrogels prepared in example 1 and comparative examples 1 and 2 was measured by syringe injection, and the results are shown in fig. 1, wherein (a) is an injectability measurement chart of the hydrogel of example 1, (b) is an injectability measurement chart of the hydrogel of comparative example 1, and (c) is an injectability measurement chart of the hydrogel of comparative example 2. As can be seen from FIG. 1, the hydrogel prepared by the raw materials and the method provided by the application is easier to flow after injection, which shows that the hydrogel has better injection performance, probably because the simple carbomer U30 has stronger adhesion, the sodium alginate and the carboxymethyl chitosan have an adjusting effect on the adhesion performance of the carbomer, and the phenoxyethanol, the benzyl alcohol and the butyl hydroxy benzoate in the auxiliary agent act together to adjust the adhesion of the carbomer U30, so that the adhesion of the carbomer U30 is improved, and the hydrogel with excellent injection performance is obtained.

In addition, the scavenging effect of the hydrogel on free radicals is verified by DPPH free radical scavenging experiments. The detection process comprises the following steps:

(1) Preparing a sample solution: accurately sucking 100 mu L of the prepared hydrogel, adding an equal volume of purified water for dilution, sucking 100 mu L of the diluted hydrogel solution into a 96-well plate, and adding 100 mu L of DPPH-ethanol solution with the concentration of 0.2 mmol/mL.

(2) Preparing a control solution: accurately sucking 100 mu L of the prepared hydrogel, adding an equal volume of purified water for dilution, then sucking 100 mu L of the diluted hydrogel solution into a 96-well plate, and then adding 100 mu L of ethanol.

(3) Preparing a blank solution: 100. Mu.L of distilled water was accurately aspirated into the 96-well plate, and 100. Mu.L of DPPH-ethanol solution at a concentration of 0.2mmol/mL was added.

After shaking up the sample solution, the control solution and the blank solution, respectively, the sample solution, the control solution and the blank solution are reacted for 20min in dark, the absorbance A is measured at 517nm, three groups of parallel tests are simultaneously carried out, and the results are averaged. Radical scavenging was calculated using the following formula.

DPPH radical clearance (%) =1- (a) _{Sample of} -A _Control )*100％/A _{Blank space}

In the formula: a is that _{Sample of} : absorbance of DPPH-sample solution; a is that _Control : absorbance of the control solution; a is that _{Blank space} : absorbance of the blank solution.

The radical scavenging rate of the hydrogels prepared in example 1 and comparative examples 1 and 2 was determined by the above detection method. The results are shown in Table 1.

TABLE 1 radical scavenging rate of hydrogels

	Example 1	Comparative example 1	Comparative example 2
				Radical scavenging rate (%)	43	31	33

As can be seen from Table 1, the hydrogel prepared by the raw materials and the method has better clearance rate to free radicals, probably because the gel substrate space network structure formed by simple carbomer U30 is poor, the loading rate of the exosomes on the gel substrate is low, the free radicals cannot be effectively cleared, the space network structure of the gel substrate can be effectively improved by regulating and controlling the compositions of the gel substrate and the auxiliary agent, the loading amount and the loading stability of the exosomes on the gel substrate are greatly improved, and the clearance rate to the free radicals is higher.

The raw materials for preparing the hydrogel are nontoxic and harmless, are friendly to human bodies, can effectively eliminate free radicals, can effectively slow down aging, and can be used as the raw materials for preparing cosmetic preparations.

While specific embodiments of the application have been described in detail in connection with the examples, it should not be construed as limiting the scope of protection of the patent. Various modifications and variations which may be made by those skilled in the art without the creative effort are within the scope of the patent described in the claims.

Claims

1. An exosome-loaded hydrogel, characterized by: comprises a gel substrate and an exosome supported on the gel substrate, wherein the mass ratio of the exosome to the gel substrate is 1:100000; the gel substrate is prepared from the following raw materials in parts by mass:

1-6 parts of gel matrix, 0.1-1 part of active sugar, 0.1-2 parts of citric acid, 1-5 parts of acidity regulator, 2-30 parts of humectant, 0.1-0.5 part of auxiliary agent and 55-95 parts of purified water; the gel substrate is prepared through the following steps:

s3: adding acidity regulator, active ingredient solution, moisturizing liquid and the rest purified water into gel base material, stirring at 40deg.C for reaction 1h, and centrifuging and degassing for 25min to obtain the final product;

the gel matrix is a mixture of carbomer, sodium alginate and carboxymethyl chitosan mixed according to the mass ratio of 8:3:1;

the auxiliary agent is a mixture of phenoxyethanol, benzyl alcohol and butyl oxybenzene in a mass ratio of 3:2:0.1.

2. The exosome-loaded hydrogel of claim 1, wherein: the active sugar is at least one of aloe polysaccharide, fructo-oligosaccharide, galacto-oligosaccharide, raffinose, stachyose and xylo-oligosaccharide.

3. The exosome-loaded hydrogel of claim 1, wherein: the acidity regulator is triethanolamine or sodium hydroxide.

4. The exosome-loaded hydrogel of claim 1, wherein: the humectant is at least one of 1, 3-propylene glycol, 1, 2-propylene glycol and glycerin.

5. The exosome-loaded hydrogel of claim 1, wherein: the exosomes are derived from umbilical cord mesenchymal stem cells.

6. The method for preparing the exosome-loaded hydrogel according to any one of claims 1 to 5, comprising the steps of:

s2: adding the exosome suspension into a gel substrate, and stirring at the stirring speed of 600rpm/min for 5-10 min at the temperature of 4 ℃ to obtain the gel.

7. Use of the exosome-loaded hydrogel according to any one of claims 1 to 5 in the preparation of a cosmetic formulation.