CN114652896A

CN114652896A - Preparation method of polysaccharide-active protein/polypeptide-based active hydrogel microspheres with high cell affinity

Info

Publication number: CN114652896A
Application number: CN202011527283.7A
Authority: CN
Inventors: 魏长征; 谈浩琪; 郭家宏; 余青青; 蒋丽霞
Original assignee: Shanghai Qisheng Biological Preparation Co ltd
Current assignee: Shanghai Qisheng Biological Preparation Co ltd
Priority date: 2020-12-22
Filing date: 2020-12-22
Publication date: 2022-06-24

Abstract

The invention relates to a preparation method of a polysaccharide-active protein/polypeptide-based active hydrogel microsphere with high cell affinity. Specifically, the hydrogel reactive microspheres adopt a two-component system, namely (1) water-soluble polysaccharide with a plurality of carboxyl functional groups; and (2) water-soluble protein or polypeptide chain segment with arginine-glycine-aspartic acid sequence (RGD peptide segment) and two or more lysines (amino functional groups) as cross-linking agent. By an emulsion method, according to different properties of a gel forming component solution, under the catalysis of a condensing agent such as 4- (4, 6-dimethoxytriazine-2-yl) -4-methylmorpholine hydrochloride (DMTMM) or 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS), the two components can be subjected to amidation covalent crosslinking (- (C ═ O) -N-) in an emulsion system to form hydrogel microspheres. The polysaccharide-active protein/polypeptide-based hydrogel disclosed by the invention has the functions of supporting cell growth, stimulating the fibroblast to secrete collagen to generate, promoting normal tissue generation and the like; the system material has simple preparation process and excellent biocompatibility, and can endow the material with multiple functions.

Description

Preparation method of high-cell-affinity polysaccharide-active protein/polypeptide-based active hydrogel microspheres

Technical Field

The invention belongs to the field of tissue filling and tissue engineering microcarrier materials, and particularly relates to a preparation method of polysaccharide-active protein/polypeptide-based active hydrogel microspheres with high cell affinity, and an obtained product and application thereof.

Background

With the development of socio-economic and the increasing consciousness of people against aging, the demand of people for cosmetic fillers is gradually increasing. In recent years, dermal fillers obtained by dispersing microspheres in a gel liquid matrix according to a certain proportion have attracted much attention, and the microspheres are gradually wrapped by fibroblasts in vivo, meanwhile, the fibroblasts are stimulated to secrete collagen, and then the generated collagen is filled in gaps caused by slow degradation of the microspheres. The filling effect of the microsphere dispersion is maintained for a long time because of the collagen stimulating effect. Representative polymer microspheres for stimulating collagen regeneration at present are polycaprolactone, hydroxyapatite and polyvinyl alcohol.

Polysaccharides are important bioactive components, including hyaluronic acid (sodium), alginic acid (sodium), heparin (sodium), carboxymethyl chitosan, carboxymethyl cellulose, and the like. The Hyaluronic Acid (HA) is also called hyaluronic acid, is the most popular injection cosmetic filler at the present stage, HAs the advantages of excellent biocompatibility, no immunogenicity, biodegradability and the like, and can also play a role in supporting and retaining water for the skin. With the improvement of the permeability of global medical and American projects, HA still HAs great development space in the future. While polysaccharide-based materials, including HA, are not covalently bound to proteins, numerous studies have shown that HA-based hydrogels and HA coatings are not conducive to cell and protein adhesion, but cell adhesion can affect subsequent cell proliferation and differentiation. In order to improve the cell affinity of polysaccharide materials, it is necessary to modify them.

Arg-Gly-Asp (RGD) is a tripeptide sequence consisting of arginine, glycine and aspartic acid, derived from fibronectin, present in a variety of extracellular matrices. RGD binds specifically to cell surface protein integrins and is an important recognition site for cell adhesion and migration. Research shows that RGD is introduced into a hydrogel structure, and can effectively promote the adhesion of cells to biological materials. Therefore, the active protein/polypeptide containing the RGD short peptide sequence is very suitable to be used as a material for improving the cell affinity.

In conclusion, the polysaccharide polymer is used as a substrate, the substrate of the active protein/polypeptide containing the RGD short peptide sequence is introduced, and additional grafting and attracting cell adhesion molecules are not needed, so that a filling agent which actively guides cell adhesion and has cell affinity is developed, and mechanical support is provided for the growth and migration of cells and further extension of tissues. The preparation process is innovative in concept, feasible in principle and expected to be applied to the field of tissue filling and tissue regeneration materials.

Disclosure of Invention

Aiming at the problem of insufficient cell affinity of polysaccharide materials, the invention provides a polysaccharide-active protein/polypeptide-based active hydrogel microsphere with high cell affinity and a preparation method thereof.

The invention provides a preparation method of a high-cell affinity polysaccharide-active protein/polypeptide-based active hydrogel microsphere, which adopts a two-component system, namely a water-soluble polysaccharide with a plurality of carboxyl functional groups and a water-soluble protein or polypeptide chain segment with RGD peptide segment and two or more lysine (amino functional groups) at the same time, and the two component systems are subjected to amidation covalent crosslinking in an emulsion system to form the hydrogel microsphere under the catalysis of a condensing agent through an emulsion method.

The specific technical scheme of the invention is as follows:

(a) preparing a gel forming buffer solution;

(b) dissolving polysaccharide with carboxyl group in gelling buffer to obtain solution (1);

(c) dissolving protein or polypeptide molecules with RGD peptide segments and multiple primary amino groups on the surfaces of the molecules by using a gelling buffer solution to obtain a solution (2);

(d) after the solution (1) and the solution (2) are mixed, a condensing agent is selected according to the pH value of the selected buffer solution, and is added into the solution to be dissolved and uniformly stirred to obtain a gel-forming solution (3) and the reaction is started;

(e) adding the emulsifier into the oil and uniformly stirring to obtain an oil phase;

(f) slowly dripping the gelling solution (3) into an oil phase in the stirring process, emulsifying and continuously reacting;

(h) after the reaction is finished, centrifuging to remove the supernatant oil phase, carrying out centrifugal washing by using a gradient ethanol water solution containing 30%, 50% and 70%, and finally carrying out suction filtration washing for multiple times by using a PBS buffer solution.

The polysaccharide-active protein/polypeptide-based active hydrogel microsphere with high cell affinity prepared by the method consists of biocompatible polysaccharide and protein or polypeptide chain segments with RGD active ingredients, has adjustable particle size, and has the functions of supporting cell growth, stimulating fibroblast to secrete collagen, promoting normal tissue generation and the like.

Description of the terms

Adhesive polypeptide RGD: the polypeptide with arginine-glycine-aspartic acid sequence has polypeptide chain segment combined specifically with integrin and has cell adhesion promoting effect.

Active protein: active protein is a complex organic compound. The compound can perform specific functions in vivo, such as promoting cell growth and proliferation, and maintaining normal vital activities of cells.

Cell affinity: the affinity of cells refers to the phenomenon of selective adhesion of cells.

Amidation reaction: the carboxyl functional group and the amino functional group react under the action of the condensing agent.

Condensing agent: the condensing agent is a reaction auxiliary agent added in the condensation reaction. There are generally catalytic condensing agents and condensing agents which bind to the separating atoms or radicals formed during condensation.

Emulsion method: under the action of emulsifier, the reaction monomer solution is dispersed into small milky liquid in oil phase via mechanical stirring and reacted continuously.

The invention has the advantages that:

(1) the invention starts from material composition, and introduces water-soluble protein or polypeptide chain segment simultaneously with RGD peptide segment and two or more than two lysines (amino functional groups) into water-soluble polysaccharide with a plurality of carboxyl functional groups, wherein the RGD peptide segment improves the cell affinity of the polysaccharide microsphere and is beneficial to the initial adhesion and the later proliferation and migration of cells.

(2) Besides the RGD peptide segment, the active protein also has two or more lysines (amino functional groups) at the same time, can be directly used as a cross-linking agent of hydrogel, avoids the toxic and side effects of a chemical cross-linking agent, and improves the biological safety of the microsphere.

(3) By adjusting and controlling the particle size, the hydrogel microsphere with cell affinity has the functions of stimulating fibroblasts to secrete collagen, promoting normal tissue generation and the like.

Drawings

FIG. 1 apparent size of gel spheres prepared in example 1;

FIG. 2 particle size distribution diagram of gel spheres prepared in example 1;

FIG. 3 is a diagram showing the cell morphology observed in a bright field of fibroblasts (L929) planted on the surface of gel spheres prepared in example 1;

FIG. 4 is a dead-live fluorescence staining pattern of fibroblasts (L929) seeded on the surface of gel beads prepared in example 1;

FIG. 5 apparent size of gel spheres prepared in example 5;

FIG. 6 particle size distribution diagram of gel spheres prepared in example 5;

FIG. 7 is a diagram showing the cell morphology observed in a bright field of fibroblasts (L929) planted on the surface of gel spheres prepared in example 5;

FIG. 8 is a dead-live fluorescence staining pattern of fibroblasts (L929) seeded on the surface of gel beads prepared in example 5;

Detailed Description

The features of the present invention and other related features are described in further detail below by way of examples to facilitate understanding by persons skilled in the art, but are not to be construed as limiting the invention in any way. Those skilled in the art should also realize that such changes, modifications, additions and substitutions can be made without departing from the spirit and scope of the invention.

Example 1: preparation of hyaluronic acid-lysozyme hydrogel microspheres

Solution 1: 0.12g of sodium hyaluronate powder (Mw 40kDa) was accurately weighed and dissolved in 2.5g of phosphate buffer (pH 7.2) and stirred until a clear and transparent solution was obtained.

Solution 2: 0.24g of lysozyme powder was accurately weighed and dissolved in 2.5g of phosphate buffer (pH 7.2) and stirred until a clear and transparent solution was obtained.

And mixing and stirring the solution 1 and the solution 2 to form a homogeneous phase, adding 0.02g of DMTMM powder into the homogeneous phase, and continuously stirring and uniformly mixing to obtain a colloid-forming solution 3.

Solution 4: 0.75g of surfactant Span-80 is added into 50g of dimethyl silicon oil, and the mixture is stirred evenly at room temperature until clear and transparent solution is obtained.

Dropwise adding the solution 3 into the solution 4 at room temperature, stirring at 400rpm, reacting at room temperature for 24h, and centrifuging to remove the supernatant oil phase. And gradient washing is carried out by using 30%, 50% and 70% ethanol solution, and finally, the gel spheres are subjected to suction filtration and washing for multiple times by using PBS buffer solution, wherein the appearance of the gel spheres is shown in figure 1.

Example 2: hyaluronic acid-lysozyme hydrogel microsphere particle size range test

The gel beads of example 1 were immersed in a phosphate buffer (pH 7.2) and the particle size distribution of the gel beads was measured by pipetting an appropriate amount. The size of the gel spheres is 20-100 μm, and the particle size is normally distributed, as shown in FIG. 2.

Example 3: observation of growth condition of L929 cells on surface of hyaluronic acid-lysozyme hydrogel microspheres

The gel spheres in example 1 were sterilized with 75% ethanol solution, fibroblasts were seeded on the surfaces of the gel spheres, and after 1 day, the cells attached on the surfaces of the materials grew, and as shown in fig. 3, the fibroblasts attached on the surfaces of the gel spheres had both fusiform and round shapes.

Example 4: l929 cell live and dead fluorescent staining attached to surface of hyaluronic acid-lysozyme hydrogel microsphere

Preparing a staining solution for cell death: weighing two staining reagents of Calcein-AM and PI according to a proportion, and dissolving in PBS buffer solution. After the gel beads with fibroblasts adhered to the surfaces in example 4 were washed with a PBS solution, a cell viability stain was added thereto, and after culturing for 15min in a cell incubator at 37 ℃, the cell viability was observed by fluorescence. As shown in FIG. 4, the fibroblasts adhered to the surface of the gel beads were all green viable cells, but the number of dead cells was small, indicating that the gel beads prepared in example 1 had excellent biocompatibility with cells.

Example 5: preparation of hyaluronic acid-collagen hydrogel microspheres

Solution 1: 0.10g of sodium hyaluronate powder (Mw 37kDa) was accurately weighed out and dissolved in morpholine ethanesulfonic acid buffer (pH 5.5) and stirred until a clear and transparent solution was obtained.

Solution 2: accurately weighed 0.075g of collagen was dissolved in 2.5g of morpholinoethanesulfonic acid buffer (pH 5.5) and stirred until a clear and transparent solution was obtained.

And mixing and stirring the solution 1 and the solution 2 to be homogeneous, adding 0.025g of EDC/NHS powder into the mixture, and continuously stirring and uniformly mixing the mixture to obtain a gel-forming solution 3.

Solution 4: adding 1.00g of surfactant Tween-80 into 50g of vegetable oil, and stirring uniformly at room temperature to obtain a clear and transparent solution.

Dropwise adding the solution 3 into the solution 4 at room temperature, controlling the stirring speed to be 300rpm, reacting at room temperature for 72 hours, and centrifuging to remove a supernatant oil phase. And gradient washing with 30%, 50%, 70% ethanol solution, and washing with PBS buffer solution by multiple suction filtration, wherein the hydrogel microspheres are shown in figure 5.

Example 6: hyaluronic acid-collagen hydrogel microsphere particle size range test

The gel beads of example 5 were immersed in a phosphate buffer (pH 7.2) and the particle size distribution of the gel beads was measured by pipetting an appropriate amount. The gel spheres are 50-100 μm in size and are in a positive distribution of particle sizes, as shown in FIG. 6.

Example 7: observation of growth condition of L929 cells on hyaluronic acid-collagen hydrogel microsphere surface

The gel spheres in example 5 were sterilized by 75% ethanol solution, fibroblasts were seeded on the surfaces of the gel spheres, and after 1 day, the cells attached on the surfaces of the materials grew, and as shown in fig. 7, the fibroblasts attached on the surfaces of the gel spheres had both fusiform and round shapes.

Example 8: l929 cell live and dead fluorescent staining attached to surface of hyaluronic acid-collagen hydrogel microsphere

Preparing a staining solution for cell death: weighing two staining reagents of Calcein-AM and PI according to a proportion, and dissolving in PBS buffer solution. The gel beads with fibroblasts adhered to the surfaces in example 6 were washed with a PBS solution, and then a cell viability stain was added thereto, and after culturing for 15min in a cell incubator at 37 ℃, the cell viability was observed by fluorescence. As shown in FIG. 8, the fibroblasts adhered to the surface of the gel beads were all green living cells, but the number of dead cells was small, indicating that the gel beads prepared in example 6 had excellent biocompatibility with cells.

Claims

1. A preparation method of a high-cell-affinity polysaccharide-active protein/polypeptide-based active hydrogel microsphere is characterized in that the hydrogel microsphere comprises:

(1) optionally a polysaccharide- (C ═ O) -N-protein;

(2) optionally a polysaccharide- (C ═ O) -N-polypeptide; and

(3) a gelling buffer solution;

wherein the optional polysaccharide is derived from a saccharide repeating unit containing carboxyl functional groups;

the protein contains RGD sequence and has two or more lysine structural units (namely active primary amino groups);

the polypeptide chain contains RGD sequence and has two or more lysine structural units.

2. The method for preparing the polysaccharide-active protein/polypeptide-based active hydrogel microspheres according to claim 1, wherein the polysaccharide component has one or more of the following characteristics:

(a) the polysaccharide repeating unit contains carboxyl or sodium carboxylate structure;

(b) the polysaccharide has good solubility in water, and is clear and transparent after being dissolved at 5% (wt/v) generally, and has no obvious particles or flocculent substances.

(b) Weight average molecular weight of 6X 10⁵Da or less;

(c) the purity is more than 99%.

3. The method for preparing the polysaccharide-active protein/polypeptide-based active hydrogel microspheres with high cell affinity according to claim 1, wherein the protein or the polypeptide has one or more of the following characteristics:

(a) the peptide chain structure contains one or more arginine-glycine-aspartic acid sequence (RGD peptide segment) units;

(b) the surface of the molecular structure contains two or more lysine structures;

(c) the purity is more than 99 percent;

(d) the protein or polypeptide has good solubility in water; generally 5% (wt/v%) is clear and transparent after dissolution, and no obvious particles or flocculent substances exist.

4. The method for preparing the high cell affinity polysaccharide-active protein/polypeptide-based active hydrogel microspheres according to claim 1, wherein the polysaccharide is selected from the group consisting of: hyaluronic acid (sodium), alginic acid (sodium), heparin (sodium), carboxymethyl chitosan, carboxymethyl cellulose, or a combination thereof, but is not limited thereto.

5. The method for preparing the polysaccharide-active protein/polypeptide-based active hydrogel microspheres with high cell affinity according to claim 1, wherein the active protein/polypeptide is selected from the group consisting of: collagen, gelatin, lysozyme, tussah silk, RGD-containing synthetic polypeptides, or combinations thereof, but is not limited thereto.

6. The method for preparing the polysaccharide-active protein/polypeptide-based active hydrogel microspheres with high cell affinity according to claim 1, wherein the gel-forming buffer is phosphate buffer (pH 6.0-8.0) or morpholine ethanesulfonic acid buffer (pH 5.0-6.0).

7. The method for preparing the high-cell-affinity polysaccharide-active protein/polypeptide-based active hydrogel microsphere as claimed in claim 1, wherein the hydrogel microsphere is uniformly dispersed in PBS and is spherical, and the particle size of the hydrogel microsphere is 20-100 μm.

8. The method for preparing the high cell affinity polysaccharide-active protein/polypeptide-based active hydrogel microsphere as claimed in claim 1, wherein the cells can be adhered and proliferated on the surface of the hydrogel microsphere, and the cells can include but are not limited to mesenchymal stem cells, fibroblasts, myoblasts, keratinocytes, etc.

9. A method for preparing the high cell affinity polysaccharide-active protein/polypeptide-based active hydrogel microspheres according to claim 1, comprising the steps of:

(a) preparing a gel forming buffer solution;

Thereby forming said hydrogel microsphere material.

10. The method of claim 9, wherein the method comprises one or more of the following features:

(a) the content of polysaccharide in the gelling reaction liquid is 1-3% (wt/v); the content of active protein/polypeptide is 0.3-3% (wt/v); the content of the condensing agent is 0.1-1% (wt/v);

(b) the content of the emulsifier in the oil phase is 1-2% (wt/v); wherein the oil is one of liquid paraffin, vegetable oil, and dimethyl silicon oil, but is not limited thereto; the surfactant is nonionic surfactant, usually span-20-80 and tween-20-80, but is not limited thereto;

(c) the volume ratio of the gelling solution to the oil phase is not more than 1: 10;

(d) mechanical stirring is adopted in the reaction process; the rotating speed is 300-600 rpm;

(e) the reaction gelling time is 24-72 hours.