CN116622615A

CN116622615A - Soluble microcarriers, methods of making and methods of using the same

Info

Publication number: CN116622615A
Application number: CN202210127504.4A
Authority: CN
Inventors: 蔡协致; 林宣因; 杨铭乾; 黄俊强; 林佑玹
Original assignee: Individual
Current assignee: Individual
Priority date: 2022-02-11
Filing date: 2022-02-11
Publication date: 2023-08-22

Abstract

The present disclosure provides a dissolvable microcarrier, a method of making and methods of using the same, comprising a dissolvable polymer that bonds multiple dissolvable monomers to one another with a reducing crosslinker. The dissolvable microcarriers of the present disclosure facilitate cell attachment and ease cell detachment using reducing agents.

Description

Soluble microcarriers, methods of making and methods of using the same

Technical Field

The present disclosure relates to a microcarrier, a preparation method and a use method thereof, and more particularly, to a soluble microcarrier, a preparation method and a use method thereof.

Background

Microcarriers are considered to be the best technique for achieving high density stem cell expansion and for regenerative medicine. Microcarriers typically range in diameter from 100 micrometers (μm) to 400 μm, providing high surface area for cell expansion. Traditional microcarrier designs target high cell attachment and proliferation rates, however, the cell manufacturing process becomes difficult due to low cell recovery. It follows that culturing cells with microcarriers is technically challenging in the cell collection step.

Recently, corning Corp LtdA soluble microcarrier was developed which was crosslinked with calcium ions and coated with +.>II matrix. Cell harvesting of the dissolvable microcarriers can be achieved by adding ethylenediamine tetraacetic acid (ethylenediaminetetraacetic acid, EDTA), pectinase (pectinase) and trypsin (trypsin).

In addition, the use of non-enzymatic methods can avoid cell damage and immune type changes. Wherein the temperature-induced desorption is a non-invasive behavior, and the surface of the temperature-sensitive material is grafted on a culture dish or modified on the surface of the microcarrier. Adsorbable cells are hydrophobic in nature when the temperature is above the minimum critical solution temperature (lower critical solution temperature, LCST); when the control temperature is lower than the LCST, the hydrophobic property is changed into the hydrophilic property and the irregular curl phenomenon is caused, so that the cells are desorbed. Thermal induction has been used for two-dimensional cell culture using sensitive materials including pluronic, methylcellulose (MC) and poly (N-isopropyranylamide) (PNIPAM). However, this desorption method is time consuming or inefficient compared to the enzyme desorption method.

Thus, based on the above-mentioned drawbacks, the prior art is in need of improvement.

Disclosure of Invention

One embodiment of the present disclosure provides a dissolvable microcarrier comprising a dissolvable polymer that bonds multiple dissolvable monomers to one another with a reducing crosslinker.

In some embodiments, the reduced crosslinker comprises a linkage to a hydroxyl, amine, thiol, or carboxylic acid group of the dissolved polymer.

In some embodiments, the reduced crosslinking agent comprises a disulfide crosslinking agent, or a diselenide crosslinking agent.

In some embodiments, the disulfide cross-linking agent comprises bis (N-hydroxysuccinimide) 3,3' -dithiodipropionic acid di (N-hydroxysuccinimide ester), DTSP), 3' -dithiobis (sulfosuccinimidyl propionate) (3, 3' -Dithiobis (DTSSP), cystine (cysine), or dimercaptodisuccinimidyl propionic acid (dithiobis (succinimidyl propionate), DSP).

In some embodiments, the diselenide crosslinking agent comprises DSeDPA-NHS (3, 3' -diselenide dipropionate bis (N-hydroxysuccinimide ester; 3,3' -Dithiodipropionic acid di (N-hydroxysuccinimide ester)), 3' -diselenide dipropionate (3, 3' -diselanediyldipropionic acid), 2' -diselenide diethylamine (ethane-1-amine)), 2' -diselenide diethanol (2, 2' -diselenide diethylbis (ethane-1-ol)), or a combination thereof.

In some embodiments, the dissolution polymer comprises cellulose, collagen, gelatin, sodium alginate, chitosan, hyaluronic acid, fruit acid, or a combination thereof.

In some embodiments, the weight ratio of dissolved polymer to the reduced crosslinker is 1:0.08 to 1:0.8.

in some embodiments, the microcarrier further comprises a temperature-sensitive polymer, encapsulating the dissolved polymer.

In some embodiments, the temperature sensitive polymer comprises poly (N-isopropylacrylamide), PNIPAM, PNIPA, PNIPAAm, NIPA, or PNIPAA), poly (N, N-diethylacrylamide), PDEAAM, poly (N-vinylcaprolactam), PVCL, poly (2-isopropyl-2-oxazoline), PIOZ), poloxamer (poloxamer), or a combination thereof.

In some embodiments, the temperature-sensitive polymer further comprises Acrylic Acid (AAC), acrylamide (ALA), acrylamide (AAm), 2- (Methacryloyloxy) ethyl ] dimethyl- (3-sulfopropyl) ammonium hydroxide ([ 2- (methacryl) ethyl ] dimethyl- (3-sulfopropyl) ammonium hydroxide, DMAPS), diethylaminoethyl methacrylate (2- (diethyl amine) ethyl methacrylate, DEAEMA), hydroxyethyl methacrylate (2-Hydroxyethyl methacrylate, HEMA), or a combination thereof.

In some embodiments, the temperature sensitive polymer is poly (N-isopropylacrylamide-co-acrylic acid) (P (NIPAM-co-ALA)) or a combination thereof.

In some embodiments, acrylic acid comprises 1% to 15% by weight of the poly (N-isopropylacrylamide-co-acrylic acid).

In some embodiments, the temperature sensitive polymer is bound outside the dissolved polymer with the reduced crosslinker.

In some embodiments, the temperature sensitive polymer is physically bound to the outside of the degradable polymer.

An embodiment of the present disclosure further provides a method for preparing a soluble microcarrier, comprising the steps of: providing a dissolved polymer; and mixing the dissolved polymer with a reducing cross-linking agent, wherein the dissolved polymer is cross-linked when contacting with the reducing cross-linking agent, so as to obtain the soluble microcarrier.

In some embodiments, the step of providing the dissolved polymer comprises: heating the plurality of dissolved monomers to a liquid state; mixing an oil with a surfactant to obtain a mixed solution; mixing the mixed solution with the dissolved monomers to obtain a water-in-oil emulsion; and cooling the water-in-oil emulsion to set to obtain the dissolved polymer.

In some embodiments, the method further comprises providing a temperature-sensitive polymer; and mixing the soluble microcarrier with the temperature-sensitive polymer to obtain the soluble temperature-sensitive microcarrier.

In some embodiments, the oil comprises mineral oil (mineral oil), stearic acid, cottonseed oil, oleyl alcohol, white waxy oil, or a combination thereof.

In some embodiments, the surfactant comprises sorbitol monooleate 80Sorbitan Monooleate (span 80), hydroxylated lanolin (hydroxylated lanolin), polyoxyethylene sorbitol beeswax derivative (polyoxythylene sorbitol beeswax derivative), propylene glycol fatty acid ester (porpylene glycol fatty acid ester), propylene glycol monolaurate (propylene glycol monolaurate), diethylene glycol monooleate (di (ethylene glycol) monoleate), polyoxyethylene oleyl ether (Sodium lauryl ether sulfate,2 EO), polyoxyethylene sorbitol beeswax derivative (polyoxythylene sorbitol beeswax derivative), diethylene glycol distearate (diethylene glycol distearate), or a combination thereof.

In some embodiments, the step of mixing the mixed solution with the dissolved monomers comprises dropping the mixed solution into the dissolved monomers.

In some embodiments, the dissolved monomers comprise cellulose, collagen, gelatin, sodium alginate, chitosan, hyaluronic acid, fruit acid, or a combination thereof.

In some embodiments, the mixing process comprises a microchannel, titration, electrospinning, emulsion polymerization, thin film emulsification, or a combination thereof.

In another embodiment, the present disclosure provides a method of using a soluble microcarrier, wherein the soluble microcarrier disintegrates when the soluble microcarrier contacts a reducing agent.

In some embodiments, the reducing agent comprises Dithiothreitol (DTT), β -mercaptoethanol (β -mercaptoethanol), glutathione (GSH), cysteine (cysteine), 2-mercaptoethanol β -mercaptoethanol (β -ME), tris (2-carboxyethyl) phosphine Tris (2-carboxyyl) phosphine (TCEP), or a combination thereof.

In some embodiments, the concentration of the reducing agent is between 1mM and 50mM.

In another embodiment, the present disclosure provides a method of using a dissolvable microcarrier, wherein the dissolvable microcarrier disintegrates when the dissolvable microcarrier is contacted with a reducing agent, contacted with a lower critical solution temperature (lower critical solution temperature, LCST), contacted with a reducing agent followed by a lower critical solution temperature, or contacted with a lower critical solution temperature followed by a reducing agent.

Drawings

The various aspects of the present disclosure will be best understood when read in conjunction with the following detailed description. It should be noted that the various features may not be drawn to scale according to industry standard operating procedures. In fact, the dimensions of the various features may be arbitrarily increased or reduced for clarity of discussion. The foregoing and other objects, features, advantages and embodiments of the present disclosure will be apparent from the following description of the drawings in which:

FIG. 1 is a schematic diagram of microcarrier preparation according to an embodiment of the present disclosure;

FIG. 2 shows a temperature-sensitive polymer according to an embodiment of the present disclosure ¹ H-NMR spectrum;

FIG. 3 is a plot of the lowest critical line of temperature sensitive polymers at different ALA ratios for different temperatures according to one embodiment of the present disclosure;

FIG. 4 is a bar graph showing water contact angles of temperature-sensitive polymers with different ALA ratios at different temperatures according to one embodiment of the present disclosure;

FIG. 5 shows a DSeDPA diselenide crosslinking agent according to an embodiment of the present disclosure ¹ H-NMR spectrum;

FIG. 6 shows a DSeDPA-NHS diselenide cross-linker of an embodiment of the present disclosure ¹ H-NMR spectrum;

FIG. 7 is a Raman spectrum of a diselenide-bonded cross-linker according to an embodiment of the disclosure;

FIG. 8 is a Fourier transform infrared (FT-IR) spectrum of a diselenide-bonded cross-linking agent according to an embodiment of the disclosure;

FIG. 9 is a Raman spectrum of a plurality of soluble microspheres according to an embodiment of the disclosure;

FIG. 10 is a Raman spectrum of a plurality of soluble reduced microspheres according to an embodiment of the present disclosure;

FIG. 11 is a chart showing the Fourier transform infrared spectrum of the microsphere (Gms) soluble before and after crosslinking according to one embodiment of the present disclosure;

FIG. 12 is a chart showing the Fourier transform infrared spectrum of a plurality of soluble microspheres according to an embodiment of the present disclosure;

FIG. 13 is a chart showing the Fourier transform infrared spectra of various soluble reduced microspheres according to an embodiment of the present disclosure;

FIGS. 14 a-14 i are image views of a scanning electron microscope (scanning electron microscope, SEM) of a plurality of microspheres according to one embodiment of the present disclosure; the scale bars are all 100 micrometers (mum), the proportion of figure 14a, b, c, d, h, i is SEI 15.0kV, 150 times, WD 11.4-12.2, the proportion of figure 14d, e and f is SEI 5.0kV 50 times, WD 10.6-11.7;

FIG. 15 is a graph showing particle size distribution curves of swollen soluble reduced microspheres (Gms-DTSP) with different cross-linking agent concentrations according to an embodiment of the disclosure;

FIG. 16 is a graph showing expansion ratio lines after swelling of different microspheres according to an embodiment of the present disclosure;

FIG. 17 is a bar graph showing cell viability of a culture of canine kidney epithelial cells (MDCK cell) with 1% ALA-based polymer in accordance with one embodiment of the present disclosure; n=8;

FIG. 18 is a bar graph showing cell viability of a 3% ALA-containing temperature-sensitive polymer and canine kidney epithelial cell culture in accordance with one embodiment of the present disclosure; n=8;

FIG. 19 is a bar graph showing cell viability of a culture of canine kidney epithelial cells with 5% ALA-containing thermosensitive polymer according to one embodiment of the present disclosure; n=8; .

FIG. 20 is a bar graph showing cell viability of dissolvable and temperature sensitive microspheres according to one embodiment of the present disclosure;

FIG. 21 is a bar graph showing cell viability of the reduced soluble and temperature sensitive microspheres according to an embodiment of the present disclosure;

FIG. 22 is a graph showing fluorescent staining of a canine kidney epithelial cell culture with different proportions of ALA-based temperature sensitive polymer according to one embodiment of the present disclosure;

FIG. 23 is a graph showing the adhesion rate of MDCK cells to soluble and temperature-sensitive microspheres according to an embodiment of the disclosure;

FIG. 24 is a schematic diagram showing an image of a soluble reduced microsphere and a temperature sensitive microsphere according to an embodiment of the disclosure when the microsphere is disintegrated by GSH; the scale bar is 100 micrometers;

FIG. 25 is a diagram showing the image of the dissolution of the temperature sensitive microsphere and the dissolution of the temperature sensitive microsphere with L-cysteine according to one embodiment of the present disclosure; the scale bar is 100 micrometers;

FIG. 26 is a schematic diagram showing an image of a soluble reduced microsphere and a temperature sensitive microsphere according to an embodiment of the disclosure when they are broken down by DTT; the scale bar is 100 micrometers;

fig. 27 is a graph showing the adhesion rate of MDCK cells in a soluble reduction type and temperature-sensitive microspheres according to an embodiment of the disclosure.

Detailed Description

To make the description of the present disclosure more detailed and complete, the following illustrative descriptions are provided with respect to the embodiments and examples of the present disclosure, but are not intended to be the only forms of implementing or utilizing the embodiments of the present disclosure. The embodiments disclosed below may be combined with or substituted for each other as desired, and other embodiments may be added to one embodiment without further description or illustration. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the embodiments below. However, embodiments of the disclosure may be practiced without these specific details.

In addition, spatially relative terms, such as "lower," "upper," and the like, may be used for convenience in describing the relative relationship of one element or feature to another element or feature in the drawings. These spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. The device may be otherwise positioned (e.g., rotated 90 degrees or other orientations) and the spatially relative descriptors used herein interpreted accordingly.

In this document, the terms "a" and "an" may refer generally to one or more unless the context clearly dictates otherwise. It will be further understood that the terms "comprises," "comprising," "includes," and/or "having," when used herein, specify the presence of stated features, regions, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and/or groups thereof.

In some embodiments, preparing a soluble reduced microcarrier or a soluble microcarrier comprises a mixing process of a soluble polymer with a cross-linking agent that cross-links when the soluble polymer contacts the cross-linking agent to obtain a soluble reduced microcarrier or a soluble microcarrier. In one embodiment, the microcarrier may take the form of a microsphere including, but not limited to, an approximately spherical microsphere. In some embodiments, microspheres having a particle size of between 100 and 300 micrometers (μm) in the dry state are suitable for adherent cell growth, e.g., 110 micrometers, 120 micrometers, 140 micrometers, 200 micrometers, 220 micrometers, 250 micrometers, 280 micrometers, or any value in between these values. In some embodiments, the microcarriers are spherical after swelling in the medium, and have a particle size of between 150 and 400 microns, such as 160 microns, 170 microns, 180 microns, 190 microns, 200 microns, 250 microns, 300 microns, 310 microns, 320 microns, 330 microns, 340 microns, 350 microns, 370 microns, or any value in between these values.

The charge and hydrophilic nature of the microcarrier surface affects cell attachment behaviour, with positively charged chemical groups, such as amine groups (-NH) ₂ ) Has better cell adhesion than the surface of carboxylic acid group (-COOH) with negative electricity. In addition, the surface is slightly hydrophilic, compared with the hydrophobicity (water contact angle>90 DEG) and superhydrophobicity (water contact angle>150 deg.) has better protein adsorption characteristics.

In one embodiment, the dissolution polymer comprises cellulose, collagen, gelatin, sodium alginate, chitosan, hyaluronic acid, fruit acid, or a combination thereof. In one embodiment, the gelatin consists of 85% to 92% protein, mineral salt and water, a water-soluble mixture extracted from extracellular matrix collagen in animal skin, bone and connective tissue, a macromolecule which is highly biocompatible and biodegradable and nontoxic, consists of 300-4000 amino acid group heterogeneous single-chain and multi-chain polypeptides, and is prepared by acidic hydrolysis of pork skin to form A gelatin (pH 3.8-6.0; isoelectric point 6-8); basic animal bone and skin hydrolyzed gelatin type B (pH 5.0-7.4; isoelectric point 4.7-5.3). Gelatin has a unique amino acid sequence and consists of three parallel L-alpha chains, each chain consists of repeated amino acid sequences Gly-Xaa-Yaa (Gly: glycine, xaa: proline, yaa: hydroxyproline), and the gelatin is easily dissolved in a high-temperature aqueous solvent to form gel after cooling, and the sol-gel phase transition can occur at the highest critical solution temperature (UCST) of 30-35 ℃, and belongs to the reversible gelation phenomenon. In one embodiment, collagen is the major structural protein present in the extracellular matrix of numerous tissues, and is rich in arginine-glycine-aspartic acid (RGD) sequences, promoting cell adhesion and proliferation.

In one embodiment, the oil is mixed with the surfactant to form a mixed solution. In an embodiment, the oil comprises mineral oil, stearic acid, cottonseed oil, oleyl alcohol, white waxy oil, or a combination thereof. In one embodiment, the surfactant comprises sorbitol monooleate 80, hydroxylated lanolin, polyoxyethylene sorbitol beeswax derivative, propylene glycol fatty acid ester, sorbitan monooleate, propylene glycol monolaurate, diethylene glycol monooleate, polyoxyethylene oleyl ether, polyoxyethylene sorbitol beeswax derivative, diethylene glycol fatty acid ester, or a combination thereof. In one embodiment, a hydrophile-lipophile balance (HLB) of 0 indicates a fully lipophilic molecule, and a greater value indicates more hydrophilic. In some embodiments, the gelatin water-in-oil emulsification system selects the appropriate surfactant according to HLB, ranging from 3 to 5, e.g., 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, or any value in between these values; wherein, the HLB of the sorbitol monooleate 80 is 4.3, which is relatively lipophilic, is suitable for dispersing in an oil phase and avoids the liquid drops in an aqueous phase from merging together, thereby improving the stability of the emulsion.

In one embodiment, the dissolved polymer is then heated with water to form an aqueous solution of the dissolved polymer, and the aqueous solution of the dissolved polymer is then slowly dripped into the mixed solution to obtain a water-in-oil (W/O) emulsion (oil: water v/v from 10:1 to 4:1, including but not limited to 9:1, 8:1, 7:1, 6:1, 5:1 or any value in between these values). The oil quantity is not too low, so that bad molding effect is avoided. In one embodiment, the soluble polymer is heated with water to render the plurality of soluble polymers or monomers thereof liquid, including, but not limited to, 30 ℃ to 90 ℃, such as 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, or any value in between.

In one embodiment, the water-in-oil emulsion is cooled to cool the microspheres for shaping, and then a cross-linking agent is added and stirred for cross-linking reaction until the microspheres solidify. Herein, "shaping" refers to stabilizing the water in oil. Herein, "curing" means that the form of the water droplet is not changed; the emulsified material is modified in trace surface and property, and the molding shape of the material is not changed according to the modified property. In one embodiment, the water-in-oil emulsion is cooled as described herein to a temperature less than the heating temperature described above. In one embodiment, the soluble reduced microcarrier is prepared with the cross-linking agent being a reduced cross-linking agent that is bonded to the hydroxyl, amine, thiol, or carboxylic acid groups of the soluble polymer. In some embodiments, the reducing crosslinking agent includes, but is not limited to, a disulfide crosslinking agent, or a diselenide crosslinking agent. Disulfide cross-linking agents include, but are not limited to, bis (N-hydroxysuccinimide ester) 3,3' -Dithiodipropionate (DTSP), bis (sulfosuccinimidyl propionate) (DTSSP), cystine, or dimercaptodisuccinimidyl propionic acid (DSP). The diselenide crosslinking agent includes, but is not limited to, DSeDPA-NHS, 3' -dielanediyldipropeical, 2' -dielanediylbis (ethane-1-amine), 2' -dielanediylbis (ethane-1-ol), or combinations thereof. In another embodiment, the crosslinker comprises a zero length crosslinker and a non-zero length crosslinker in preparing the dissolvable microcarrier. So-called zero length cross-linking agents, which are removed after the cross-linking of the catalytically degradable polymer is completed; zero-length crosslinkers include, but are not limited to, carbodiimide (EDC), N' -Dicyclohexylcarbodiimide (DSC), N-Dicyclohexylcarbodiimide (DCC), or combinations thereof. Non-zero length cross-linking agents, including but not limited to formaldehyde, glutaraldehyde, acrylamide, isocyanate, gardenin, DTSP, DSeDPA-NHS, BSSS, DSG, sulfo-EGS, DSS, EGS, BS2G, DTSSP, DST, BSOCOES, DPDPB, sulfo DST, or DSP, will eventually be incorporated into the polymer network, with the cross-linking agent reacting with the degradable polymer to form covalent bonds between the degradable polymer.

In one embodiment, the microspheres are then cured, crosslinked, filtered to remove the oil phase and washed, and the microspheres are dried to provide either soluble reduced microspheres (Gms-DTSP) or soluble microspheres (Gms). In some embodiments, after the crosslinking reaction is completed, the oil phase is removed with a suction filtration device, washed several times with acetone/water solution (v/v, 5:1-1:1, e.g., 4:1, 3:1, or 2:1), and finally the microspheres are freeze-dried. In some embodiments, the acetone/water ratio may be fixed or decreasing in sequence, and the oil may be removed rapidly under high concentration acetone/water, but may not be cleaned with acetone, which may damage the microsphere surface or morphology.

In one embodiment, the weight ratio of the dissolved polymer to the reduced crosslinker is 1:0.08 to 1:0.8.

in one embodiment, the weight ratio of the dissolved polymer to the reduced crosslinker is 1:0.32 to 1:0.8.

in some embodiments, the weight ratio of gelatin to DTSP in the dried soluble reduced microspheres (Gms-DTSP) is about 99:1 to 1.86:1, e.g., about 90:1 to 2:1, 80:1 to 2:1, 70:1 to 2:1, 60:1 to 2:1, 50:1 to 2:1, 40:1 to 2:1, 30:1 to 2:1, 20:1 to 2:1, 10:1 to 2:1, 7:1 to 2:1, 5.67:1 to 1.86:1, 5:1 to 1.86:1, 4:1 to 1.86:1, 3:1 to 1.86:1, or any value in between any two of these values, as measured by an elemental analyzer).

In some embodiments, preparing the temperature-sensitive polymer comprises: and polymerizing the temperature-sensitive polymer and the hydrophilic monomer by a free radical polymerization method to obtain the temperature-sensitive polymer. In one embodiment, the hydrophilic monomer includes, but is not limited to, acrylic Acid (AAC), acrylamide (ALA), acrylamide (AAm), 2- (methacryloyloxy) ethyl ] dimethyl- (3-sulfopropyl) ammonium hydroxide (DMAPS), diethylaminoethyl methacrylate (DEAEMA), hydroxyethyl methacrylate (HEMA) or a combination thereof in one embodiment, the free radical polymerization method includes, but is not limited to, stable Free Radical Polymerization (SFRP), atom Transfer Radical Polymerization (ATRP), or reversible addition-fragmentation chain transfer polymerization (RAFT) in one embodiment, hydrophilic monomers include, but are not limited to, acrylic acid, acrylamide [2- (methacryloyloxy) ethyl ] dimethyl- (3-sulfopropyl) ammonium hydroxide (DMAPS), diethylaminoethyl methacrylate (DEAEMA), hydroxyethyl methacrylate (HEMA) or a combination thereof in one embodiment, the free radical polymerization method includes, but is not limited to, stable Free Radical Polymerization (SFRP), atom transfer radical polymerization (RARP), or reversible addition-fragmentation chain transfer polymerization (RAFT) in one embodiment, hydrophilic monomers include, but are not limited to, acrylic acid, acrylamide [2- (methacryloyloxy) ethyl ] dimethyl- (3-sulfopropyl) ammonium hydroxide (DMAC), a combination thereof in one embodiment, or a combination thereof with (AEMA) of some of methacrylic acid and hydroxyethyl methacrylate (HEMA) in one embodiment, wherein the allylamine comprises 1% to 5%, for example 2%, 3%, 4%, or any value in between these values, by weight of the temperature sensitive polymer.

In one embodiment, the temperature sensitive polymer, hydrophilic monomer, initiator and chain transfer agent are dissolved in an organic solvent by a reversible addition fragmentation chain transfer polymerization method, and a mixture is obtained by placing an ultrasonic oscillator for dissolution. After the mixture was charged into a reaction flask and purged with nitrogen, the polymerization was performed with heating and continuous stirring. The condensing tube is arranged above the reaction bottle to maintain the reflux of the system, so that the loss of the reactant caused by the heated volatilization of the reactant with overhigh temperature is avoided. After the viscosity of the mixture after the heating of the reaction flask was no longer thickened, the reaction flask was put into liquid nitrogen to terminate the reaction, to obtain a polymerization solution. In some embodiments, the temperature of heating may be between 50 ℃ and 90 ℃, such as 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃, 85 ℃, or any value in between these values. In some embodiments, the reaction time is from 4 hours to 48 hours, such as 6 hours, 8 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, or any value in between these values. In some embodiments, typical chain transfer agents include mercaptans, dodecyl mercaptan DDM, or haloalkanes, such as carbon tetrachloride. Chain transfer agents are also known as modifiers and control agents.

In one embodiment, the polymerization solution is reprecipitated in cold diethyl ether to ensure removal of unreacted monomers and chain transfer agent. The solid was obtained after precipitation and dried to remove diethyl ether. And then, a dialysis membrane is used for purification, the dried solid is dialyzed and purified in water to obtain a semi-finished product, and the semi-finished product is dehydrated to obtain the dried temperature-sensitive polymer or temperature-sensitive block copolymer.

In some embodiments, the physical preparation of a microsphere of the soluble reduced temperature-sensitive type (Gms-DTPS-pnipam) or a microsphere of the soluble temperature-sensitive type (Gms-pnipam) comprises: mixing the soluble reduced microcarrier with the temperature-sensitive polymer to obtain the soluble reduced temperature-sensitive microcarrier; or mixing the soluble microcarrier with the temperature-sensitive polymer to obtain the soluble temperature-sensitive microcarrier. In one embodiment, after preparing the aqueous solution of the temperature-sensitive polymer, the temperature-sensitive polymer is coated on the surface of the soluble-reduced-type microspheres (Gms-DTSP) or the soluble-type microspheres (Gms) by stirring at a low temperature. Washing the modified microspheres to remove residual temperature-sensitive polymer, and drying to remove water to obtain the soluble reduced temperature-sensitive microspheres (Gms-DTPS-pnipam) or soluble temperature-sensitive microspheres (Gms-pnipam). In some embodiments, the microspheres are stirred at a low temperature of 0 ℃ to 15 ℃, e.g., 1 ℃, 2 ℃, 3 ℃, 4 ℃, 5 ℃, 6 ℃, 10 ℃, 12 ℃, 14 ℃, or any value in between these values. In some embodiments, the microspheres are freeze-dried to remove moisture. In one embodiment, the temperature-sensitive polymer is physically coated on the surface of the microcarrier by intermolecular forces, such as Van der Waals (Van der Waals force), secondary bonds including but not limited to hydrogen bonds, etc.

In some embodiments, a diselenide-bond crosslinker is preparedComprising the following steps: 10 milliMoles (mmol) of selenium (Se) powder was placed in 3 ml of water and kept in a nitrogen atmosphere. In some embodiments, selenium-containing water is injected into a three-necked reaction flask and maintained under nitrogen. Then 20 milliMohs of sodium borohydride (NaBH ₄ ) Slowly dripping into selenium-containing water in 8 ml of water, and stirring to colorless to completely dissolve selenium powder to obtain first mixed solution. Thereafter, an equal amount of 10 milli Mo Erxi powder was added to the first mixed solution and heated until reddish brown was exhibited to obtain a second mixed solution. In some embodiments, the heating temperature is between 80 ℃ and 130 ℃, such as 85 ℃, 90 ℃, 95 ℃, 100 ℃, 105 ℃, 110 ℃, 115 ℃, 120 ℃, 125 ℃, or any value in between these values. Subsequently, 20 mmole of 3-chloropropionic acid (3-chloropropionic acid) was added to the second mixed solution and stirred under nitrogen atmosphere at room temperature to obtain a third mixed solution. The third mixture was exposed to the atmosphere and stirred, followed by filtration to remove unreacted materials, to give a yellow supernatant. The yellow supernatant was adjusted to pH 3.5 with 1M hydrochloric acid (HCl) and extracted twice with anhydrous Ethyl Acetate (EA) to give an upper organic layer, which was then washed with water and extracted to remove water (e.g., absorbed in anhydrous magnesium sulfate powder) and ethyl acetate to give DSeDPA.

In one embodiment, 1.2 milliMoire DSeDPA is then dissolved in 5 milliliters anhydrous Tetrahydrofuran (THF), and added dropwise to a vessel under nitrogen, followed by 2.88 milliMoire N-hydroxysuccinimide (NHS) and stirred to obtain an initial solution. Then 2.88 milliMohr of diimine was dissolved in 5 ml of anhydrous tetrahydrofuran and added dropwise to the low temperature initial solution to control the reaction rate to avoid excessive speed. In some embodiments, the low temperature is between 0 ℃ and 20 ℃, such as 5 ℃, 10 ℃, 15 ℃, or any value in between these values. Then, the initial solution containing tetrahydrofuran was stirred at room temperature until the reaction was completed, and then impurities were filtered and tetrahydrofuran was removed to obtain DSeDPA-NHS. DSeDPA-NHS can also be collected and stored after 24 hours of vacuum oven drying.

In some embodiments, chemically preparing a soluble reduced temperature-sensitive microsphere (Gms-DTSP-Se-pnipam) or a soluble temperature-sensitive microsphere (Gms-Se-pnipam) comprises mixing a soluble reduced microcarrier, the reduced cross-linking agent, and the temperature-sensitive polymer to obtain the soluble reduced temperature-sensitive microcarrier; or mixing the soluble microcarrier, the reduced cross-linking agent and the temperature-sensitive polymer to obtain the soluble temperature-sensitive microcarrier. In one embodiment, after preparing the aqueous solution of the temperature-sensitive polymer, 0.18mM DSeDPA-NHS and soluble-reducible microspheres (Gms-DTSP) or soluble-type microspheres (Gms) are added, and stirred at a low temperature until the surface of the soluble-reducible microspheres is covered with the temperature-sensitive polymer and chemical bonding is completed. In some embodiments, the microspheres are stirred at a low temperature of 0 ℃ to 15 ℃, e.g., 1 ℃, 2 ℃, 3 ℃, 4 ℃, 5 ℃, 6 ℃, 10 ℃, 12 ℃, 14 ℃, or any value in between these values. After the bonding is completed, the microspheres are washed to remove residual temperature-sensitive polymers, and then dried to remove redundant water, so that the microspheres (Gms-DTSP-Se-pnipam) of soluble reduction temperature-sensitive type or the microspheres (Gms-Se-pnipam) of soluble temperature-sensitive type are obtained.

In some embodiments, the chemically prepared, soluble reduced temperature-sensitive microspheres (Gms-DTSP-Se-pnipam) correspond to pnipam, se, gms weight percent concentration (wt%) of: pnipam 3-10%, se 1-3%, gms-96%. pnipam, se, gms is divided into 3 to 10:1 to 3: 87-96, for example: pnipam is divided by weight into 3, 4, 5, 6, 7, 8, 9, 10 or any value in between these values; se weight is divided into 1, 2, 3 or any value between these values; gms is divided into 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 or any value in between these values.

Although the methods disclosed herein are illustrated below with a series of acts or steps, the order in which the acts or steps are performed should not be construed as a limitation of the present disclosure. For example, certain operations or steps may be performed in a different order and/or concurrently with other steps. Moreover, not all illustrated operations, steps, and/or features may be required to implement an embodiment of the present disclosure. Furthermore, each operation or step described herein may comprise several sub-steps or actions.

FIG. 1 is a schematic diagram of microcarrier preparation according to an embodiment of the present disclosure.

Preparation example 1 preparation of soluble reduced microspheres

Mixing the mineral oil with the surfactant sorbitol monooleate 80 to form a mixed solution. After gelatin and water were heated to prepare 5 mL of an aqueous gelatin solution of 0.25 g/mL (g/mL), the aqueous gelatin solution was slowly dropped into the mixture to obtain a water-in-oil (W/O) emulsion (oil: water v/v, 7:1). The water-in-oil emulsion is rapidly cooled to cool the microspheres in a set, and 0.1 to 1 g of the disulfide cross-linking agent DTSP is added and stirred to effect cross-linking until the microspheres solidify (where DTSP is about 0.25mM to 1.2 mM). And removing the oil phase after the crosslinking reaction is finished, washing, and drying the microspheres to obtain the soluble reduced microspheres (Gms-DTSP).

The weight ratio of gelatin to DTSP in the dried, reduced soluble microspheres (Gms-DTSP) was about 99:1 to 65:35 as measured by elemental analyzer.

Preparation example 2 preparation of soluble microspheres

Mixing the mineral oil with the surfactant sorbitol monooleate 80 to form a mixed solution. After gelatin and water were heated to prepare a gelatin aqueous solution of 0.25 g/mL (g/mL), the gelatin aqueous solution was slowly dropped into the mixed solution to obtain a water-in-oil (W/O) emulsion (oil: water v/v, 7:1). And rapidly cooling the water-in-oil emulsion to enable the microspheres to be shaped and cooled for a period of time, adding glutaraldehyde, stirring for a period of time, and performing a crosslinking reaction until the microspheres are solidified. Filtering to remove oil phase after the crosslinking reaction is finished, washing, and drying the microspheres to obtain the soluble microspheres (Gms).

Preparation example 3 preparation of temperature-sensitive Polymer

NIPAM, ALA, initiator (2, 2' -azolbis (2-methyl-precursor), AIBN, chain transfer agent (4-cyano-4- (phenylcarbothioyl) acid, CTA) were dissolved in 1,4-Dioxane (1, 4-Dioxane) by reversible addition fragmentation chain transfer polymerization (RAFT), and the mixture was obtained by dissolving with an ultrasonic vibrator. After the mixture was slowly added to the reaction flask and purged with nitrogen, the polymerization was performed with heating and continuous stirring. The condensing tube is arranged above the reaction bottle to maintain the reflux of the system, so that the loss of the reactant caused by the heated volatilization of the reactant with overhigh temperature is avoided. After the viscosity of the heated mixture of the reaction flask is no longer thickened, the reaction flask is placed in liquid nitrogen to terminate the reaction to obtain a polymerization solution, and the polymerization solution is reprecipitated in cold diethyl ether to ensure removal of unreacted monomers (e.g., NIPAM, ALA) and chain transfer agent. The precipitate was obtained as a pale yellow solid and dried to remove diethyl ether. Next, a purification step was performed using a dialysis membrane (molecular weight cut-off (molecular weight cut off, MWCO) =1000), the dried pale yellow solid was dialyzed and purified in water to obtain a semi-finished product, and the semi-finished product was dehydrated to obtain a dried thermosensitive polymer P (NIPAM-co-alliylamine), or thermosensitive block copolymer (yield: about 90%).

Preparation example 4 preparation of soluble reduction temperature-sensitive microsphere-physical method (Gms-DTSP-pnipam)

After preparing 3wt% of an aqueous solution of P (NIPAM-co-Allylamine) in ethanol, the soluble reduced type microspheres (Gms-DTSP) prepared in preparation example 1 were added and stirred at a low temperature of 0 to 5℃until the soluble type microspheres were covered with P (NIPAM-co-Allylamine). Washing the modified microspheres to remove residual P (NIPAM-co-Allylamine), and drying to remove water to obtain the soluble reduction temperature-sensitive microspheres (Gms-DTPS-pnipam).

Preparation example 5 preparation of soluble temperature-sensitive microspheres-physical method (Gms-pnipam)

After preparing 3 weight percent (wt%) of P (NIPAM-co-Allylamine) ethanol aqueous solution, the soluble microspheres (Gms) prepared in preparation example 2 were added and stirred at a low temperature of 0-5 ℃ until the soluble microspheres were covered with P (NIPAM-co-Allylamine) and physical bonding was completed. The modified microspheres were washed to remove residual P (NIPAM-co-Allylamine), and then dried to remove water, thereby obtaining soluble temperature-sensitive microspheres (Gms-pnipam).

Preparation example 6 preparation of a diselenide Cross-linker

10 mmoles (mmol) of selenium (Se) powder was placed in 3 ml of water and kept in a nitrogen atmosphere, followed by 20 mmoles of sodium borohydride (NaBH) ₄ ) Slowly dripping into selenium-containing water in 8 ml of water, and stirring to colorless to completely dissolve selenium powder to obtain first mixed solution. Then go downAn equal amount of 10 milliMoles of selenium powder was added to the first mixture and heated until a reddish brown color was obtained. 20 milliMole of 3-chloropropionic acid was added to the second mixed solution and stirred at room temperature under nitrogen atmosphere to obtain a third mixed solution. The third mixture was exposed to the atmosphere and stirred, followed by filtration to remove unreacted materials, to give a yellow supernatant. The yellow supernatant was adjusted to pH 3.5 with 1M hydrochloric acid and extracted with anhydrous ethyl acetate to give an upper organic layer, which was then extracted twice with water and ethyl acetate were removed to give DSeDPA (yield: 85%).

1.2 mMohr DSeDPA was dissolved in 5 ml anhydrous tetrahydrofuran, and then dropped into a vessel under nitrogen atmosphere, followed by adding 2.88 mMohr N-hydroxysuccinimide (NHS) and stirring to obtain an initial solution. Then 2.88 milliMohr of diimine is dissolved in 5 ml of anhydrous tetrahydrofuran and added dropwise to the low temperature initial solution, and the reaction speed is controlled to avoid excessive speed. Stirring an initial solution containing tetrahydrofuran at room temperature to completely react, filtering impurities and removing the tetrahydrofuran to obtain DSeDPA-NHS. DSeDPA-NHS can also be collected and stored after 24 hours of vacuum oven drying.

Preparation example 7 preparation of soluble reduction temperature-sensitive microsphere-chemical method (Gms-DTSP-Se-pnipam)

After preparing 3wt% P (NIPAM-co-Allylamine) ethanol aqueous solution, 1wt% DSeDPA-NHS and 96wt% soluble reduced microsphere (Gms-DTSP) of preparation example 1 were added, and stirred at low temperature until the soluble reduced microsphere was covered with P (NIPAM-co-Allylamine) and chemical bonding was completed. After the bonding is completed, the microspheres are washed to remove residual P (NIPAM-co-Allylamine), and then dried to remove excessive moisture, so as to obtain the soluble reduction temperature-sensitive microspheres (Gms-DTSP-Se-pnipam).

EXAMPLE 1 Synthesis of temperature-sensitive Polymer

The temperature-sensitive polymer is prepared from preparation example 3 by polymerizing ALA monomer and NIPAM, so that the structure has amine groups with positive electric property, which is beneficial to cell attachment application. Experimental Synthesis of proportions 1% ALA, 3% ALA, 5% ALA into three groups the following were formulated as shown in Table 1, and the synthesized polymers were reacted with each other via FT-IR ¹ H-NMR confirmed successful grafting and molecular weight was measured via gel permeation chromatography (gel permeation chromatography, GPC).

Table 1, P (NIPAM-co-Allylamine) synthetic formulation

1.1 ¹ H-NMR identification and constituent elements

The chemical structure of the P (NIPAM-co-Allylamine) polymer was first confirmed. During the reaction, initiator AIBN is added, and when the temperature reaches 70 ℃, free radicals are started to generate, so that the double bond (C=C) of the monomer is polymerized. Hydrogen spectrum using nuclear magnetic resonance spectrometer (NMR) ¹ H-NMR analysis identified the molecular structure (as shown in FIG. 2), corresponding to the formula delta=0.95-1.24 ppm (a, CH) ₃ From NIPAM), delta = 1.34-1.75ppm (b, CH) ₂ From NIPAM and Ala) (h, CH ₂ From CTA), δ=1.82-2.13 ppm (c, CH from NIPAM and Ala) (i, j, CH from CTA), δ=2.66 ppm (d, CH) ₂ From Ala), δ=3.82 ppm (e, CH from NIPAM), δ=7.32-7.79 ppm (f, CH from CTA), δ=8.38 ppm (g, NH from NIPAM). Will be ¹ The H-NMR signal was integrated to calculate the grafting ratio and composition (Table 2 below).

Table 2, composition ratio of P (NIPAM-co-Allylamine)

1-2 molecular weight measurement and identification

The molecular weight size and molecular weight distribution of the synthesized polymer were identified using gel permeation chromatography. Table 3 shows the molecular weight and molecular weight distribution of the P (NIPAM-co-Allylamine) temperature-sensitive polymer as measured by gel permeation chromatography. Table 3 shows that the polymers synthesized with molecular weight dispersity (polydispersity index, PDI) all have a narrow molecular weight distribution, with values mostly between 1.2 and 1.3, while the weight average molecular weights (Mw) of P (NIPAM-co-Allylamine) are 37666, 42803, 43056, respectively, demonstrating the successful polymerization of monomers into copolymers via RAFT.

TABLE 3 Property analysis of P (NIPAM-co-Allylamine)

Proportioning of	Mn*(g/mol)	Mw(g/mol)	PDI
				1％ALA	29739	37666	1.26
3％ALA	32341	42803	1.32
				5％ALA	33959	43056	1.26

* Mn: number average molecular weight

1-3 minimum critical solution temperature LCST measurement

After embedding monomeric ALA into NIPAM copolymer, PNIPAM hydrophobic force affects LCST due to increased electrostatic repulsive force. The LCST of the copolymer can be determined by uv/vis spectroscopy at a corresponding temperature at 50% transmission: when the temperature-rising copolymer is aggregated in a large amount, a non-flowing gel state is formed from the solution state, and the light penetration value is relatively lowered. FIG. 3 shows the transmittance of PNIPAM and 1%, 3% and 5% ALA at different temperatures, showing that the LCST of PNIPAM is 31.4 ℃; LCST of 1%, 3%, 5% ALA was 32.3 ℃, 32.5 ℃, 33.4 ℃, respectively, and thus LCST increased with ALA ratio. This demonstrates that the addition of hydrophobic monomers to the copolymer decreases the LCST, whereas hydrophilic monomers increase the LCST, mainly due to the hydrophilic-hydrophobic nature of the polymer tail groups.

1-4 Water contact Angle test

The water contact angle of the temperature sensitive polymer varies with temperature. The water generates circular water drops at the gas-liquid interface through self surface tension, and the water contact angle is calculated according to a Young formula by using an image graph, wherein γsv=γsl+γlvcos theta. Referring to FIG. 4, PNIPAM was used as a control group, and the PNIPAM had water contact angles of 44.83+ -0.47 DEG at room temperature, and water contact angles of 1% ALA, 3% ALA, 5% ALA after ALA was embedded were 37.13+ -0.55 DEG, 32.65+ -0.28 DEG, 29.4+ -0.95 deg. When the temperature is increased from room temperature to 40 ℃, the water contact angles of PNIPAM, 1% ALA, 3% ALA and 5% ALA are 63.23+/-1.85 degrees, 64.91 +/-0.89 degrees, 63.45+/-1.04 degrees and 53.83 +/-1.17 degrees respectively, the angle difference of the PNIPAM of the control group when the PNIPAM is converted into high temperature is 18.4 degrees, and the P (NIPAM-co-alliylamine) difference after ALA is embedded is more than 25 degrees, so that the copolymer has the temperature response characteristic. In addition, ALA is a hydrophilic monomer, and the contact angle of 5% ALA water at low temperature is smaller, and the ALA is most hydrophilic. As the ALA content of the copolymer increases, the contact angle gradually decreases. The hydrophobic contact angle of 1% ALA and 3% ALA is similar to that of PNIPAM of a control group at high temperature, the coincidence is slightly hydrophilic (the water contact angle is less than 90 ℃) and has better protein adsorption property, and the adsorption proliferation of cells is proved to be beneficial.

EXAMPLE 2 Synthesis of a diselenide Cross-linker

One of the purposes of the synthesis of diselenide linkages is to combine P (NIPAM-co-alliylamine) polymers with microspheres in chemical bonding to form thermally responsive (thermo-responsive) microspheres, and to compare them with thermally responsive polymers with physical coatings on the surface of the microspheres. The diselenide bond is a redox sensitive material, is easily broken by environmental change, and oxygen in carboxylic acid groups (-COOH) at two ends of DSeDPA is nucleophilic attacked by EDC to form high-activity intermediate (O-a)cyliosourea), followed by a second intermediate (O-acylisoourea) formed by reaction with NHS, is hydrolyzed to DSeDPA-NHS in water and is reacted with an amine group (-NH) ₂ ) The rapid reaction produces a stable amide linkage which is then crosslinked to a polymer by reaction with the tail amine group of P (NIPAM-co-Allylamine).

2.1 ¹ H-NMR identification and constituent elements

For structural correctness ¹ H-NMR identification. DSeDPA was found to correspond to structural formula δ=2.69-2.72 ppm (a) prior to activation (as shown in fig. 5); delta=3.04-3.06 ppm (b), and the structural formulas of the two points are CH ₂ The carboxylic acid group (-COOH) is arranged beside the point b, the signal which is subjected to the pulling displacement of larger electronegativity to the left and accords with the resonance frequency increase in nuclear magnetic resonance is positioned in a low field region, and after activation (shown in figure 6), DSeDPA-NHS structural formula delta=2.69-2.71 ppm (a, CH) ₂ From DSeDPA); delta = 3.03-3.06ppm (b, CH ₂ From DSeDPA); delta=2.59 ppm (c, CH from NHS), from which it can be demonstrated that there are four CH signal intensities on the NHS active group higher than the a and b points, the analysis of the above NMR results demonstrates that the diselenide crosslinking agent DSeDPA-NHS functional group is successfully synthesized.

2-2 Raman spectrometer analysis

Selenium belongs to the air-sensitive element. Fig. 7 is a raman spectrum of a symmetric structure of selenium. 290cm after analysis ^-1 、310cm ^-1 Se-Se double-selenium bond signal appears at the position of 276cm ^-1 The obvious Se-C functional group exists on the left and right sides, so that the activity and the reaction efficiency of carboxylic acid are improved through EDC activation, NHS is connected with carboxylic acid groups (-COOH), DSeDPA-NHS which is successfully synthesized is proved, and the existence of selenium element in the structure is confirmed by Raman.

2-3 Fourier transform infrared spectroscopy (FT-IR) analysis

The structure of the inorganic functional group can be more accurately observed by using FT-IR. From the profile analysis of FIG. 8, it was found that DSeDPA carboxylic acid groups bind to NHS before activation at 1688cm ^-1 Belonging to the group c=o functions; after activation, the NHS binding is pulled to 1776cm ^-1 A peak value. In addition, the spectra of the vibration of the N-O, C-N, C-O molecules of the other functional group signals are obviously different, which represents that the reaction is successful.

Example 3 identification of gelatin microspheres

Gelatin is soluble in hot water but insoluble in cold water, but human cell culture is carried out in an environment of 37 ℃, so that the network is required to be crosslinked firmly, and a crosslinking agent is added through an emulsification reaction to obtain the microsphere. Wherein, the microsphere prepared by the reducing crosslinking agent is a microsphere of a soluble reduction type (Gms-DTSP, as in preparation example 1), and the microsphere prepared by the non-reducing crosslinking agent is a microsphere of a soluble type (Gms, as in preparation example 2). Then, the temperature-sensitive polymer is combined on the surface of the microsphere by a physical coating or chemical bonding mode to respectively obtain a physical coating soluble reduction temperature-sensitive microsphere (Gms-DTPS-pnipam, as in preparation example 4) and a soluble temperature-sensitive microsphere (Gms-pnipam, as in preparation example 5) and a chemical bonding soluble reduction temperature-sensitive microsphere (Gms-DTSP-Se-pnipam, as in preparation example 7).

3.1 Raman Spectroscopy analysis

The microspheres of each group were analyzed using raman instrument and classified according to the use of cross-linking agents:

a first group: soluble microspheres (Gms, as in preparation 2), physical coated soluble temperature sensitive microspheres (Gms-pnipam, preparation 5);

second group: microspheres of the soluble reduction type (Gms-DTSP, as in preparation example 1), chemically bonded microspheres of the soluble reduction temperature-sensitive type (Gms-DTSP-Se-pnipam, preparation example 7), microspheres of the physical coating of the soluble reduction temperature-sensitive type (Gms-DTPS-pnipam, preparation example 4).

Since gelatin has no fixed structural formula, but a large number of hydroxyl groups and amine groups exist in the structure, raman peaks cannot indicate the structure after actual modification, but the difference of each microsphere can be observed through peak displacement. FIG. 9 shows the first group of 2000-2500cm ^-1 Two signals are present in the range, and after crosslinking of gelatin by glutaraldehyde (Gms), it was confirmed that both peaks were shifted left and right in the three reaction stages. FIG. 10 shows a second set of microspheres crosslinked with DTSP. Observed to approach 1400cm ^-1 In place, gelatin was crosslinked by DTSP and each group of microspheres gave more of this signal, suggesting that DTSP did indeed fix gelatin crosslinks to microspheres. In addition, 2000-2500cm ^-1 The two signals within the range slightly vary.

3.2 Fourier transform Infrared Spectrometry

Raman spectroscopy is complementary to FT-IR and is better suited for detecting symmetric bonds. The microsphere belongs to an asymmetric structure, and has relatively strong infrared absorption peak value by using FT-IR to analyze two groups of microspheres. FIG. 11 shows that amide I at 1629cm before gelatin is cross-linked ^-1 Is affected by C=O stretching vibration at 1634cm ^-1 The c=n stretching vibration of the Schiff base reaction (Schiff base) occurred, and the c=o stretching of the mixture of amide I and unreacted aldehyde groups after crosslinking resulted in confirmation of the Schiff base reaction formed between the amine group of gelatin and the carbonyl group of glutaraldehyde (c=o), confirming the success of crosslinking of gelatin with glutaraldehyde.

The main peak value of gelatin is 1600-1700cm ^-1 Amide I (stretching of c=o bond), 1500-1590cm ^-1 Amide II (NH bending vibration and CN stretching vibration) and 1200cm ^-1 The characteristic peaks were observed in fig. 12 and 13 for the amide III (NH bending vibration and CN stretching vibration) in the vicinity. In particular, the DTSP cross-linking agent is used at 3500-3000cm ^-1 Peaks of O-H and N-H vibrations were observed at 2940cm ^-1 where-CH is observed ₃ Symmetrical stretching vibration peak of group, DTSP has NHS active group and amino (-NH) group in gelatin network ₂ ) After the reaction, a stable amide bond is formed. At the same time NHS structure is released and at 1390cm ^-1 Characteristic peaks of s=o were observed.

3.3 Scanning Electron Microscope (SEM) analysis

The size of the dried crosslinked and cured gelatin microspheres was analyzed using SEM and the surface morphology was observed. FIG. 14a shows microspheres obtained without the use of a cross-linking agent; FIG. 14b shows the use of glutaraldehyde to crosslink the soluble microspheres (Gms) to form agglomerates, failing to screen, and non-uniform particle size range; FIG. 14c shows that after the temperature-sensitive-type microsphere (Gms-pnipam) is dissolved and coated on the surface, the microsphere has dispersibility, the particles are obviously dispersed to improve the agglomeration phenomenon, and the powder state on the surface is coated by the temperature-sensitive polymer; FIG. 14d shows the use of DTPS crosslinker 0.25mM (Gms-0.25 mM DTSP); FIG. 14e shows that DTPS crosslinker is 0.6mM (Gms-0.6 mM DTSP); FIG. 14f (50-fold magnification) and FIG. 14g (150-fold magnification) show that the DTPS crosslinker was 1.2mM (Gms-1.2 mM DTSP), and that the microsphere surface was progressively roughened from smooth as the crosslinker concentration increased. Then, microspheres with temperature sensitive polymers were observed: FIG. 14h shows that the microspheres were coated with a physical coating (Gms-DTSP-pnipam) to a smooth state after the surface of the microspheres was coated with a temperature sensitive polymer from the original roughness; another set of FIG. 14i shows that chemical bonding (Gms-DTSP-Se-pnipam) uses a selenium cross-linker to chemically bond P (NIPAM-co-Allylamine) to the gelatin surface with significantly varying shrinkage voids.

In addition to using SEM, elemental analysis composition was known using an energy-dispersive X-ray spectrometer (EDS) at the same time. The composition element of the microsphere crosslinked by glutaraldehyde is C, N, O element, C, N, O, S element is obtained by using a disulfide bond DTPS crosslinking agent, C, N, O, S, se element is obtained by chemically bonding P (NIPAM-co-Allylamine) to gelatin by using a diselenide bond crosslinking agent, and the crosslinking agent element is used for confirming the success of the crosslinking again. To understand the size of the microspheres in the dry state, the particle size was analyzed using Image J, and table 4 shows the average particle size of each set of gelatin microspheres.

TABLE 4 average particle size of gelatin microspheres of each group

Sample of	Average diameter (μm)
		(a)Gms	142.19±107.68
(b)Gms-pnipam	123.08±51.75
		(c)Gms-0.25mM DTSP	222.31±169.13
(d)Gms-0.6mM DTSP	226.37±69.25
		(e)Gms-1.2mM DTSP	251.13±81.14
(f)Gms-DTSP-pnipam	208.56±85.96
		(g)Gms-DTSP-Se-pnipam	230.17±71.36

3.4 swelling State particle size analysis

The microsphere particle size is defined as ranging from 100 to 300 microns. Gelatin microspheres smaller than 100 microns were first isolated using a screen and then immersed in a warm medium maintained in a 37 ℃ environment for 5 days to simulate a real cell culture environment. The expanded size and stability of the microspheres were then observed using an optical microscope. Table 5 shows the Gms and Gms-pnipam particle size distributions. The Gms particle size is agglomeration, cannot be screened for the first time, and the microspheres with small particles cannot be separated, so that the size dispersion is large; and after the surface of Gms-pnipam is coated with P (NIPAM-co-Allylamine), the agglomeration phenomenon can be improved, the agglomeration phenomenon can smoothly pass through a screen, and the size distribution interval of microspheres is smaller.

TABLE 5 particle diameters after swelling of gelatin microspheres (Gms), (Gms-pnipam)

Another group is microspheres crosslinked at 0.25mM, 0.6mM, 1.2mM DTSP. FIG. 15 and Table 6 show that 0.25mM DTSP microsphere was observed to swell to 900 microns in size and gradually rise at 0.5 hours, and the microsphere swelled and ruptured at 48 hours, and the group of crosslinking agents failed to stabilize the gelatin network, resulting in poor microsphere stability. The size of the 0.6mM DTSP was observed to be in a stable range within 48 hours, the decrease in size of the microspheres was observed at 72 hours, and the slow dissolution disappeared with time, while the 1.2mM DTSP crosslinked microspheres were stable in scale curve and about 300 microns in particle size range within 120 hours, confirming that the concentration of the crosslinking agent was increased, resulting in network-stabilized-size microspheres. The weight ratio of gelatin to 0.6-1.2mM DTSP is 1.25:0.4 to 1.25:1 (1:0.32-1:0.8).

TABLE 6 particle size after swelling of soluble reduced microspheres with different crosslinker concentrations

Table 7 shows Gms-DTSP-pnipam and Gms-DTSP-Se-pnipam microspheres formed by combining Gms-1.2mM DTSP with temperature sensitive polymer P (NIPAM-co-Allylamine). After 120 hours observation, the size is stable, and the cracking and swelling phenomena are avoided, so that the three groups are suitable for cell culture experiments, and the successful synthesis of the temperature-sensitive soluble gelatin microsphere is proved.

TABLE 7 particle size after swelling of soluble reduced and temperature sensitive microspheres

3.5 microsphere expansion test

10 mg dry weight microspheres (W1) were placed in a 15 ml centrifuge tube immersed in 37℃medium, the medium was removed at various time points, and excess liquid was removed using a wipe, leaving a wet sample weight (W3), and each group was repeated three times. The microsphere expansion ratio (sweeping ratio) is calculated as follows:

Swelling ratio＝(W3-W2)/W1

the water content (water content) calculation formula is:

Water content(％)＝[1-W1/(W3-W2)]×100

w1, drying the weight of the sample; w2, dry sample and centrifuge tube weight; w3 wet sample and centrifuge tube weight.

The comparative microsphere swelling degree can be used to evaluate the water absorption characteristics of the polymer microsphere at 37 ℃. Experiment after immersing the dried microspheres in the medium for 24 hours, the swelled weight was weighed. FIG. 16 shows the swelling properties of different gelatin microspheres, with increasing concentrations of 0.6mM and 1.2mM DTSP, and decreasing swelling, 0.25mM DTSP swelled to 18.05 in 24 hours, but the microspheres swelled and ruptured after 48 hours as shown in Table 6 above, and the set of cross-linking agents failed to stabilize the gelatin network. The swelling degree of the temperature-sensitive polymer of the surface physical coating is increased along with the swelling degree, and the water content is increased. The water absorption capacity is remarkably improved by incorporating P (NIPAM-co-Allylamine) into gelatin microspheres, and the most obvious is GMS-DTSP-Se-pnipam microspheres, the swelling rate is 12.62+/-0.07, and the water content is 93.32+/-0.97%.

TABLE 8 expansion and Water content after swelling of different microspheres

Sample of	Expansion ratio (w/w)	Moisture content (%)
			Gms	3.24±0.05	67.14±0.26
Gms-pnipam	4.59±0.43	80.82±1.27
			Gms-0.25mM DTSP	18.05±1.04	93.57±1.88
Gms-0.60mM DTSP	8.08±0.61	88.95±0.76
			Gms-1.20mM DTSP	6.94±0.17	89.13±2.71
Gms-DTSP-pnipam	7.29±0.31	90.02±0.57
			Gms-DTSP-Se-pnipam	12.62±0.07	93.32±0.97

3.6 dissolution microsphere collapse behavior

Disulfide bonds (-S-) are typically formed by natural cross-linking of two sulfur atoms between cysteine side groups, and alkylation of free cysteine using the chemical reducing agent DTT activity reduces disulfide bonds, breaking the cysteine into cysteine, while diselenide bonds have a similar redox mechanism as disulfide bonds. The oxidation response capability of the diselenide bond is larger than that of the disulfide bond, the diselenide has larger atomic radius and weaker electronegativity, the lower bond energy can be easily reduced, the diselenide bond is broken, and an intermediate of selenate is generated (RSe) ^- )。

The disclosure will conduct a disintegration experiment on oxidative dissolution type microspheres. To simulate the cell culture environment, gms-DTSP, gms-DTSP-pnipam, gms-DTSP-Se-pnipam were immersed in the medium and maintained at 37℃followed by the addition of 25mM reducing agent DTT to observe changes in the disintegration of the gelatin polymeric network. Table 9 shows that Gms-DTSP-Se-pnipam disappeared within 15 minutes, leaving only a clear solution, and the remaining two groups were completely collapsed within 30 minutes.

TABLE 9 disintegration behavior of reduced microspheres at 25mM DTT

Sample of	Collapse time (minutes)
		Gms-DTSP	30
Gms-DTSP-pnipam	30
		Gms-DTSP-Se-pnipam	15

Example 4 cell viability

4.1 sensitive Block copolymers

FIGS. 17, 18 and 19 show that the 1%, 3% ALA and 5% ALA temperature-sensitive polymers were co-cultured with MDCK cells for 24 hours, respectively, and the cell viability of the three copolymer concentrations was 97.4%, 96.5% and 96.3% at the highest 1000. Mu.g/mL, respectively, confirming that the temperature-sensitive polymers have good biocompatibility with MDCK cells (mammalian cell lines as standard in biomedical research). The cell apoptosis is caused by the change of cell types when the cells are stimulated by toxic substances, and the MTT test can act with living cell granulosa line body succinic dehydrogenase to generate purple crystals under the reduction reaction, and the qualitative living cell number is obtained through detection of absorbance by using a disk spectrospectrometer at 570 nm.

4.2 gelatin microspheres

The microsphere belongs to a carrier, can not be dissolved in a culture medium, and has non-uniform sample shape. The microspheres were immersed in 37℃medium at 0.1g/mL for 24 hours according to ISO-10993, and the medium after different percentages of immersion of the microspheres were extracted was co-cultured with MDCK for 24 hours. FIG. 20 shows that cell viability can reach 82% at a maximum concentration of 0.1g/mL using glutaraldehyde crosslinked soluble microspheres. The cell survival rate is improved by 96.3% under the highest concentration of Gms-pnipam. The surface physical coating of the temperature-sensitive polymer has good biocompatibility. FIG. 21 shows that another set of soluble reduced microspheres crosslinked using DTSP, three types of microspheres also give good biocompatibility, all capable of greater than 90%. The cell survival rate reaches 94% at the highest concentration, and each group of microspheres has no ability of killing cells, thus being beneficial to the subsequent attached culture experiment.

Example 5 test of cell attachment and detachment

5.1 temperature-sensitive Polymer cell De-attachment test

In the experiments of the adsorption and desorption characteristics of P (NIPAM-co-Allylamine), cells were grouped according to 1% ALA, 3% ALA, 5% ALA, and spin-coated on plastic coverslips, each 0.5X10 ⁶ cells/mL MDCK co-culture. Cells were grown in a tightly-packed cluster-like morphology at 37℃by plating on P (NIPAM-co-Allylamine) membranes, and exhibited good adhesion properties (not shown). When the temperature is reduced to 4 ℃, hydrophilic groups on the surface of the copolymer film interact with charged groups of cells, so that the cells float in a block shape and are suspended in PBS solution. In addition, a blank plastic coverslip (without temperature sensitive block copolymer coating) was used as a control, and when the temperature was adjusted to a low temperature, the morphology of the cells was not changed and desorption did not occur, and the cells were still adhered to the coverslip (not shown).

Cells detached via temperature, their activity was stained with live/dead cell imaging reagents. Living cells can easily penetrate into living cell membranes through Calcein-AM Calcein and remain after hydrolysisIn the cells, strong green fluorescence is emitted, while dead cells are subjected to BOBO ^TM -3Iodide released red fluorescence from nuclear staining across the broken cell membrane. FIG. 22 shows that cells desorbed from three groups of P (NIPAM-co-Allylamine) membranes were observed from fluorescence microscopy, with a significant proportion of green fluorescence in the overlaid images, demonstrating that temperature-induced desorption of cells remained well-activated.

The simple temperature control method is used for desorbing cells from the temperature-sensitive responsive surface P (NIPAM-co-Allylamine), so that the cells are not damaged, and the damage caused by desorption by the traditional enzymolysis method is avoided. In terms of engineering medicine, cells are cut off from cell-to-cell connection through a traditional enzymolysis method, the harvested cells are independent single cells, continuous cell sheets cannot be regenerated, cell sheets with complete cell-to-cell connection can be obtained through a temperature sensitive gel layer, the cells belong to non-invasive desorption, the cells after confluent culture need to be successfully harvested into cell sheets with tissue structures by reducing the temperature, and the purpose of regeneration and repair of biomedical tissues is achieved.

5.2 temperature-induced Desorption behavior of temperature-sensitive microspheres

For temperature-sensitive polymer P (NIPAM-co-Allylamine), ALA concentration increases amine (-NH) in the structure ₂ ) So that the positive charge and the hydrophilicity are increased. The cell culture was performed in the same manner as described in the above-mentioned 5.1. For the 5% ala copolymer, the cells had a sticky culture surface when attached. Cell desorption at low temperature, 5% ALA desorption time is longer than the other two groups, showing that more ALA content is helpful for cell adhesion, but too much ALA monomer is embedded, so that the copolymer is too hydrophilic to lose temperature sensing property. Therefore, when the temperature-sensitive gelatin microsphere is prepared, 5% ALA copolymer is finally selected for temperature-sensitive modification of the surface of the gelatin microsphere through the adsorption and desorption evaluation of the cells.

To test the temperature-induced cell desorption capacity of P (NIPAM-co-Allylamine) on microspheres, the desorption behavior of Gms-pnipam, gms-DTSP-pnipam was first tested. The gelatin microsphere is subjected to cell desorption by temperature induction, and after desorption is carried out at a low temperature of 4 ℃ for 30 and 60 minutes, cells can be seen to slightly drop (not shown), and the gelatin microsphere has poor effect of cell desorption by using temperature induction and needs longer time. Therefore, the traditional enzymolysis and disintegration method is needed to improve the desorption efficiency of cells and carriers.

5.3 De-attachment test of soluble microsphere cells

The cells were observed by fluorescence microscopy, and the presence or absence of adhesion of the cells to the microsphere surface was observed by Hoechst 33342 for growth. Hoechst 33342 can penetrate the cell membrane to emit blue fluorescence, and after observation by a fluorescence microscope, the blue cell is confirmed to be attached to five microspheres (including Gms, gms-pnipam, gms-DTSP-pnipam, gms-DTSP-Se-pnipam, not shown) so as to evaluate the capability of the five microspheres to adsorb cells.

To evaluate the microsphere cell adsorption and desorption test, after swelling Gms, gms-pnipam, each group was incubated at 0.5X10 ⁶ MDCK cells were cultured in cells/mL, and the adhesion of MDCK to the microspheres was observed with an optical microscope at 1, 3, 5, 7, and 24 hours, and the cell adhesion rate was calculated. The results showed that cells were observed to begin to adhere to the edges of the microspheres after 1 hour, but Gms was less likely to observe the presence or absence of adherent cells due to the aggregation of microspheres, non-uniform size, and small size microspheres. The Gms-pnipam is uniform in size, and it can be seen that cells are significantly attached, so that the cell attachment effect of Gms-pnipam is better than that before modification, and pnipam is beneficial to cell adhesion (not shown). In addition, the results also show that the trypsin method completely performed cell desorption experiments. After the microspheres are soaked in trypsin for 5 minutes at 37 ℃, cells drop from the surface of Gms, the trypsin can break peptide bonds formed by lysine or arginine with the increase of time, and the cell desorption effect is increased with the digestion of trypsin. After 10 minutes, the microspheres were washed with PBS to recover the cells, and the surface of the washed microspheres remained with a lot of cells, and the desorption effect was poor. Another group Gms-pnipam was subjected to trypsin for 5 minutes, and cells and the temperature-sensitive polymer were simultaneously cut off, and after 10 minutes, cells were almost completely dropped into the suspension, and after the cells were recovered by washing the microspheres with PBS, the surfaces of the microspheres were brought to a smooth and flat state, confirming that the temperature-sensitive polymer contributes to cell desorption (not shown).

In addition, cell-attached Gms, gms-pnipam were added with a live/dead stain to identify the degree of cell death on the surface of gelatin microspheres. Calcein (Calcein-AM) excites green fluorescence in live cells, while BOBO-3Iodide, which dyes dead cells, excites red fluorescence. The results show that cells attached to the microspheres have a large number of green fluorescent cells and few dead cells, and that cells can be damaged by damaging membrane proteins when the cells are detached with trypsin for too long. In addition, cells desorbed by Gms-pnipam showed green fluorescence, red cells were few, while Gms showed that cells were not completely desorbed after 10 minutes of desorption, and many healthy cells were still attached to the microspheres. Therefore, gms has the capability of adsorbing cells, but after the temperature-sensitive polymer is physically coated on the surface of the microsphere, the desorption capability of the cells released by the carrier can be improved.

5.4 cell attachment Rate and Desorption behavior of soluble microspheres

FIG. 23 shows the ability of microspheres Gms, gms-pnipam to adsorb cells. After 1 hour of incubation Gms-pnipam reached about 40% cell attachment, gms was 23% cell attachment, and after 24 hours Gms, gms-pnipam were 74%, 85% respectively. After the surface temperature-sensing polymer coating, the attachment rate is improved by 11 percent. Next, the desorption characteristics of the gelatin microspheres Gms and Gms-pnipam were evaluated, and section 5.3 above mentions that Gms and Gms-pnipam were digested by trypsin for the same time, and the Gms desorption efficiency was less than Gms-pnipam. After the collected and desorbed cells are subjected to washing and centrifugal calculation, the number of cells harvested by Gms and Gms-pnipam is 192500cell/mL and 355750cell/mL respectively, and the desorption proportion is improved by 45.8% after the cells are subjected to surface temperature-sensitive polymer coating.

5.5 De-attachment test of soluble reduced microspheres

The present disclosure synthesizes Gms-DTSP, gms-DTSP-pnipam and Gms-DTSPSe-pnipam by using a disulfide bond structure crosslinking agent DTSP with oxidation-reduction characteristics when preparing microspheres, and observes the adhesion of MDCK cells on the microspheres by an optical microscope after culturing for two days.

Gms-DTSP-pnipam reduced gelatin microspheres were evaluated for disruption using three GSH, L-cysteine (L-cysteine), and DTT reducing agent (25 mM concentration), and cell desorption was observed. As can be seen from fig. 24, the microspheres were not broken or dissolved after 60 minutes of use of GSH reducing agent; as can be seen from fig. 25, the microspheres were swollen and the cells were dropped in a lump after 30 minutes using the L-cysteine reducing agent, and the microspheres were completely dissolved and disappeared at 60 minutes; as can be seen from FIG. 26, the rapid disintegration of the swelling and deformation of the microspheres was observed 5 minutes with DTT reducing agent, leaving only the globoid cells at 30 minutes. Of the three tested reducing agents, DTT-containing dithiols showed the best effect in cleaving disulfide bonds, with higher redox potentials than GSH and L-cysteine, which were monothiols that required other thiol-containing molecular catalysis to accelerate the disulfide bond cleavage reduction.

Gms-DTSP and Gms-DTSP-pnipam take 30 minutes to degrade and disappear the microspheres by using a DTT reducer, and particularly Gms-DTSP dissolution type belongs to the DTT bursting type, and a cross-linked network is cut off by the reducer to lead the broken cells of the spheres to be in a single particle type finally. The Gms-DTSP-pnipam microsphere of the surface coating breaks down in a manner that the cells are separated in a bulk manner by DTT, and it is inferred that the outer temperature-sensitive polymer coating protects the coating and the cells from the outside before the sphere itself can be seen to slowly break down. The final cells are in the form of clusters, whereupon the agglomerated cells are detached together with the temperature sensitive polymer coating (not shown).

In addition, the temperature-sensitive polymer coating is hydrophilic at low temperature, and Gms-DTSP-pnipam is gradually disintegrated at low temperature (e.g. 4 ℃) for 5 minutes, and cells become dispersed at 10 minutes. While Gms-DTSP-Se-pnipam has the shortest collapse time (not shown).

Cells were attached to Gms-DTSP, gms-DTSP-pnipam, gms-DTSP-Se-pnipam and a live/dead stain was added to identify the degree of cell death on the surface of gelatin microspheres. The results show that the cells are attached to the microspheres and are coated by a large number of green fluorescent cells, and the desorbed cells are excited to green fluorescence after disintegration (not shown), which proves that the reduced gelatin microspheres have the capability of adsorbing cells and the capability of surviving the cells when the cells are disintegrated.

5.6 cell attachment Rate and Desorption behavior of reduced gelatin microspheres

FIG. 27 shows the capacities of the soluble reduced type and temperature sensitive microspheres Gms-DTSP, gms-DTSP-pnipam, gms-DTSP-Se-pnipam to adsorb cells. After 1 hour of culture, the cell attachment rates of Gms-DTSP, gms-DTSP-pnipam and Gms-DTSP-Se-pnipam are 40%, 39% and 29.5% respectively; gms-DTSP reaches 88% first after 8 hours; after 24 hours, the adsorption rates of Gms-DTSP, gms-DTSP-pnipam and Gms-DTSP-Se-pnipam were 95%, 90% and 47%, respectively. From SEM observation of example 3.3, the microsphere surface had micro concavo-convex holes to increase cell attachment, wherein Gms-DTSP-pnipam had better ability to adsorb cells than Gms-DTSP-Se-pnipam.

5.7 cell harvesting backculture test

To demonstrate the viability of cells harvested using gelatin microspheres and the ability for subsequent medical use, cells were desorbed from Gms, gms-pnipam, gms-DTSP-pnipam, gms-DTSP-Se-pnipam, and harvested cells were washed, centrifuged and placed in culture dishes for back culture. The results showed that cells cultured by Gms, gms-DTSP-Se-pnipam were observed after one day of culture, and the cells which were withdrawn originally were less likely to result in slower growth, while the remaining Gms-pnipam, gms-DTSP-pnipam were all in an octant state, and the cells were grown in a tightly packed cluster-like morphology, exhibiting good cell activity (not shown). Thus, the cells desorbed from the microspheres of the present disclosure have the ability to be repeatedly cultured.

The soluble and reducible microcarrier disclosed by the disclosure is beneficial to cell attachment through a reducing cross-linking agent, and the cell desorption can be easily realized through the reducing agent. In some embodiments, cells are detached within 30 minutes using a reducing agent, and the cells are viable after detachment, which proves to be non-toxic.

The soluble temperature-sensitive microcarrier disclosed by the invention is coated by the temperature-sensitive polymer, so that the attachment of cells is facilitated when the culture temperature is higher than LCST; the desorption of cells is facilitated by the passage temperature below the LCST. In some embodiments, the spherical shape becomes complete after soaking in the culture medium, the particle size is stably controlled between 280 and 350 microns, and the surface of the microsphere is protected by the temperature-sensitive polymer, so that the stability of the microsphere at 37 ℃ is facilitated. In some embodiments, the temperature-sensitive polymer is used for modifying, so that the cell attachment rate is improved by 11%, and the cell desorption rate is improved by 45.8% by using an enzymolysis method, so that the attachment and desorption capacity of the microcarrier is effectively improved.

While the present disclosure has been described with reference to the exemplary embodiments, it should be understood that the invention is not limited thereto, but may be variously modified and modified by those skilled in the art without departing from the spirit and scope of the present disclosure, and thus the scope of the present disclosure is defined by the appended claims.

Claims

1. A dissolvable microcarrier comprising:

a soluble polymer which bonds a plurality of soluble monomers to each other with a reducing crosslinking agent.

2. The microcarrier of claim 1, wherein the reduced cross-linking agent comprises a hydroxyl, amine, thiol, or carboxylic acid group linkage to the dissolved polymer.

3. The microcarrier of claim 1, wherein the reduced cross-linking agent comprises a disulfide cross-linking agent, or a diselenide cross-linking agent.

4. A microcarrier according to claim 3, wherein the disulfide cross-linking agent comprises bis (N-hydroxysuccinimide ester) 3,3' -dithiodipropionic acid, bis (sulfosuccinimidyl propionate), cystine or dimercaptodisuccinimidyl propionic acid.

5. The microcarrier of claim 3, wherein the diselenide cross-linking agent comprises bis (N-hydroxysuccinimide ester) of 3,3' -diselenodipropionic acid, 2' -diselenodiethylamine, 2' -diselenodiethanol, or a combination thereof.

6. The microcarrier of claim 1, wherein the dissolved polymer comprises cellulose, collagen, gelatin, sodium alginate, chitosan, hyaluronic acid, fruit acid, or a combination thereof.

7. Microcarrier according to claim 1, characterized in that the weight ratio of the dissolved polymer to the reduced cross-linking agent is 1:0.08 to 1:0.8.

8. the microcarrier of claim 1, further comprising a temperature-sensitive polymer coating the dissolved polymer.

9. The microcarrier of claim 8, wherein the temperature-sensitive polymer comprises poly (N-isopropylacrylamide), poly (N, N-diethylacrylamide), poly (N-vinylcaprolactam, poly (2-isopropyl-2-oxazoline), poloxamer, or a combination thereof.

10. The microcarrier of claim 9, wherein the temperature-sensitive polymer further comprises acrylic acid, acrylamide, [2- (methacryloyloxy) ethyl ] dimethyl- (3-sulfopropyl) ammonium hydroxide, diethylaminoethyl methacrylate, hydroxyethyl methacrylate, or a combination thereof.

11. The microcarrier of claim 10, wherein the temperature-sensitive polymer is poly (N-isopropylacrylamide-co-acrylic acid) or a combination thereof.

12. The microcarrier of claim 11, wherein the acrylic acid comprises 1% to 15% by weight of the poly (N-isopropylacrylamide-co-acrylic acid).

13. The microcarrier of claim 8, wherein the temperature-sensitive polymer is bound outside the dissolved polymer with the reduced cross-linking agent.

14. The microcarrier of claim 8, wherein the temperature-sensitive polymer is physically bound to the outside of the degradable polymer.

15. A method of preparing a dissolvable microcarrier comprising the steps of:

providing a dissolved polymer; and

the soluble polymer and a reducing cross-linking agent are subjected to a mixing process, and when the soluble polymer and the reducing cross-linking agent are contacted, the soluble microcarrier is obtained.

16. The method of claim 15, wherein the step of providing the dissolved polymer comprises:

heating the plurality of dissolved monomers to a liquid state;

mixing an oil with a surfactant to obtain a mixed solution;

mixing the mixed solution with the plurality of dissolved monomers to obtain a water-in-oil emulsion; and

cooling the water-in-oil emulsion to a fixed shape to obtain the dissolved polymer.

17. The method as recited in claim 15, further comprising:

Providing a temperature-sensitive polymer; and

mixing the soluble microcarrier with the temperature-sensitive polymer to obtain a soluble temperature-sensitive microcarrier.

18. The method of claim 15, wherein the mixing process comprises micro-fluidic channels, titration, electrospinning, emulsion polymerization, thin film emulsification, or a combination thereof.

19. A method of using the dissolvable microcarrier of claim 1, wherein the dissolvable reduced microcarrier disintegrates when the dissolvable microcarrier contacts a reducing agent.

20. A method of using the dissolvable microcarrier of claim 8, wherein the dissolvable microcarrier disintegrates when the dissolvable microcarrier is contacted with a reducing agent, contacted with a lower critical solution temperature, contacted with the reducing agent before the lower critical solution temperature, or contacted with the lower critical solution temperature before the reducing agent.