TWI856287B - Soluble and thermosensitive microcarrier, method for manufacturing and method of use thereof - Google Patents

Abstract

The present disclosure provides a soluble and thermosensitive microcarrier, including soluble polymer and a thermosensitive polymer covering the soluble polymer, wherein the soluble polymer is formed by a plurality of soluble monomers binding to each other with a crosslinking agent. The soluble and reducible microcarrier of present disclosure facilitates the attachment of cells, and the use of reducing agents can facilitate the detachment of cells.

Description

Soluble temperature-sensitive microcarrier, preparation method thereof and use method thereof

The present disclosure relates to a microcarrier, a preparation method thereof and a method of using the microcarrier, in particular to a soluble thermosensitive microcarrier, a preparation method thereof and a method of using the microcarrier.

Microcarriers are considered the best technology to achieve high-density stem cell expansion for regenerative medicine. Microcarriers are typically 100 micrometers (μm) to 400 μm in diameter, providing a high surface area for cell expansion. Traditional microcarrier designs target high cell attachment and proliferation rates, however, low cell recovery rates make the cell manufacturing process difficult. As can be seen, when culturing cells on microcarriers, the cell collection step is technically challenging.

Recently, Corning ^® has developed a soluble microcarrier that is crosslinked with calcium ions and coated with Synthemax ^® II matrix. Cell harvesting from the soluble microcarrier can be achieved by adding ethylenediaminetetraacetic acid (EDTA), pectinase, and trypsin.

In addition, the use of non-enzymatic methods can avoid cell damage and changes in immune morphology. Among them, temperature-induced detachment is a non-invasive behavior, which grafts the surface of temperature-sensitive materials on the culture dish or modifies them on the surface of microcarriers. When the temperature is higher than the lower critical solution temperature (LCST), it is hydrophobic and can adsorb cells; when the temperature is adjusted below the LCST, it will change from hydrophobic to hydrophilic and irregular curling, causing cell detachment. Thermal induction has been used in two-dimensional cell culture, using sensitive materials including pluronic, methylcellulose (MC) and poly (N-isopropylacrylamide) (PNIPAM). However, this detachment method is time-consuming or less efficient than the enzyme detachment method.

Therefore, based on the above shortcomings, the existing technology needs to be improved.

One embodiment of the present disclosure provides a soluble thermosensitive microcarrier, comprising a soluble polymer, wherein the soluble polymer uses a crosslinking agent to bond multiple soluble monomers to each other; and the thermosensitive polymer encapsulates the soluble polymer.

In some embodiments, the crosslinker comprises a zero-length crosslinker and a non-zero-length crosslinker.

In some embodiments, the zero-length crosslinker comprises carbodiimide (EDC), N, N'- dicyclohexylcarbodiimide (DCC), or a combination thereof.

In some embodiments, the non-zero length crosslinking agent comprises formaldehyde, glutaraldehyde, acrylamide, isocyanate, genipin, DTSP (3,3'-dithiodipropionic acid di(N-hydroxysuccinimide ester)), DSeDPA-NHS (3,3'-diselanediyldi-propanoic acid-N-hydroxysuccinimide), BSSS (bis(sulfosuccinimidyl) suberate, BS3), DSG (disuccinimidyl glutarate), sulfo-EGS (ethylene glicolbis(sulfosuccinimidilsucci-nato), DSS (disuccinimidyl suberateS), EGS (ethylene glicolbis(succinimidylsuccinate)), BS2G(bis(sulfosuccinimidyl)glutarate), DTSSP(3,3’-dithiobis(sulfosuccinimidylpropionate), DST(disuccinimidyl tartrate), BSOCOES(bis(2-(succinimidooxycarbonyloxy]ethyl)sulfone), DPDPB(1,4-di-(3’-(2’pi ridilditio)-propionamido)butane), sulfo DST (sulfodisuccinimidil tartrate), or DSP (dithiobis (succinimidyl propionate)).

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

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

In some embodiments, the temperature-sensitive polymer further comprises acrylic acid (AAC), allylamine (ALA), acrylamide (AAm), [2-(Methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (DMAPS), 2-(Diethylamino)ethyl methacrylate (DEAEMA), 2-Hydroxyethyl methacrylate (HEMA), or a combination thereof.

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

In some embodiments, the weight percentage of acrylamine in the poly(N-isopropylacrylamide-co-acrylamine) is 1% to 15%.

In some embodiments, the thermosensitive polymer is bonded to the outside of the soluble polymer by the crosslinking agent.

In some embodiments, the temperature-sensitive polymer is physically bonded to the soluble polymer.

One embodiment of the present disclosure further provides a method for preparing a soluble thermosensitive microcarrier, comprising the following steps: providing a soluble polymer; performing a mixing process on the soluble polymer and a crosslinking agent, wherein the soluble polymer and the crosslinking agent are crosslinked when in contact to obtain a soluble microcarrier; providing a thermosensitive polymer; and mixing the soluble microcarrier and the thermosensitive polymer to obtain a soluble thermosensitive microcarrier.

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

In some embodiments, the mixing process includes microfluidics, titration, electrostatic spinning, emulsion crosslinking, thin film emulsification, or a combination thereof.

In some embodiments, the step of providing the thermosensitive polymer includes: polymerizing the temperature-sensitive polymer and the hydrophilic monomer by free radical polymerization to obtain the thermosensitive polymer.

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

In some embodiments, the hydrophilic monomer comprises acrylic acid (AAC), allylamine (ALA), acrylamide (AAm), [2-(Methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (DMAPS), 2-(Diethylamino)ethyl methacrylate (DEAEMA), 2-Hydroxyethyl methacrylate (HEMA), or a combination thereof.

In some embodiments, the method further comprises: providing a reducing crosslinking agent; and mixing a soluble microcarrier, a reducing crosslinking agent and a temperature-sensitive polymer to obtain a soluble temperature-sensitive microcarrier, wherein the temperature-sensitive polymer is bonded to the soluble polymer by the reducing crosslinking agent.

One embodiment of the present disclosure further provides a method for using the aforementioned soluble thermosensitive microcarrier, wherein the soluble thermosensitive microcarrier is disintegrated when it contacts a reducing agent, contacts a lower critical solution temperature (LCST), contacts a reducing agent first and then contacts a lower critical solution temperature, or contacts a lower critical solution temperature first and then contacts a reducing agent.

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

The various aspects of the present disclosure will be most easily understood when the following detailed description is read in conjunction with the accompanying figures. It should be noted that, in accordance with industry standard operating procedures, the various feature structures may not be drawn to scale. In fact, for the clarity of the discussion, the size of the various feature structures may be arbitrarily increased or decreased. In order to make the above and other purposes, features, advantages and embodiments of the present disclosure more obvious and easy to understand, the attached drawings are described as follows: Figure 1 shows a schematic diagram of the preparation of a microcarrier in one embodiment of the present disclosure.

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

Figure 3 is a line graph showing the minimum critical values of thermosensitive polymers with different ALA ratios at different temperatures according to one embodiment of the present disclosure.

Figure 4 is a bar graph showing the water contact angles of thermosensitive polymers with different ALA ratios at different temperatures according to one embodiment of the present disclosure.

FIG. 5 shows a ¹ H-NMR spectrum of a DSeDPA di-selenide bond crosslinker according to an embodiment of the present disclosure.

FIG. 6 shows a ¹ H-NMR spectrum of DSeDPA-NHS di-selenide bond crosslinking agent according to an embodiment of the present disclosure.

Figure 7 shows the Raman spectrum of the di-selenide bond crosslinking agent of one embodiment of the present disclosure.

Figure 8 shows the Fourier-transform infrared spectroscopy (FT-IR) spectrum of the di-selenide bond crosslinking agent of one embodiment of the present disclosure.

Figure 9 shows Raman spectra of various soluble microspheres according to one embodiment of the present disclosure.

Figure 10 shows the Raman spectra of various soluble and reducible microspheres of one embodiment of the present disclosure.

Figure 11 shows the Fourier transform infrared spectrum of the soluble microspheres (Gms) before and after cross-linking according to one embodiment of the present disclosure.

Figure 12 shows the Fourier transform infrared spectra of various soluble microspheres of one embodiment of the present disclosure.

Figure 13 shows the Fourier transform infrared spectra of various soluble and reducible microspheres of one embodiment of the present disclosure.

Figures 14a to 14i show scanning electron microscope (SEM) images of various microspheres of one embodiment of the present disclosure; the scale bars are all 100 micrometers (μm), Figures 14a, b, c, d, h, and i are SEI 15.0kV, 150 times, WD 11.4~12.2, and Figures 14d, e, and f are SEI 5.0kV 50 times, WD 10.6~11.7.

Figure 15 shows a line graph of the particle size of soluble reducible microspheres (Gms-DTSP) after swelling at different crosslinking agent concentrations according to one embodiment of the present disclosure.

Figure 16 shows a line graph of the expansion rate of different microspheres after swelling according to one embodiment of the present disclosure.

Figure 17 shows a bar graph of cell survival rate of 1% ALA-containing thermosensitive polymer and canine renal epithelial cells (madin-darby canine kidney cells, MDCK cells) cultured in one embodiment of the present disclosure; n=8.

Figure 18 shows a bar graph of cell survival rate of canine renal epithelial cells cultured with a thermosensitive polymer containing 3% ALA according to one embodiment of the present disclosure; n=8.

Figure 19 shows a bar graph of cell survival rate of canine renal epithelial cells cultured with a thermosensitive polymer containing 5% ALA according to one embodiment of the present disclosure; n=8.

Figure 20 shows a bar graph of cell survival rates of soluble and thermosensitive microspheres according to one embodiment of the present disclosure.

Figure 21 shows a bar graph of cell survival rates of soluble reduced and thermosensitive microspheres according to one embodiment of the present disclosure.

Figure 22 shows a fluorescence staining image of a thermosensitive polymer containing different ratios of ALA and canine renal epithelial cells cultured according to one embodiment of the present disclosure.

Figure 23 shows a line graph of the attachment rate of MDCK cells to soluble and thermosensitive microspheres according to one embodiment of the present disclosure.

Figure 24 shows an image of the soluble reduced and temperature-sensitive microspheres of one embodiment of the present disclosure when they are decomposed by GSH; the scale bar is 100 micrometers.

Figure 25 shows an image of the soluble reduced and temperature-sensitive microspheres of one embodiment of the present disclosure when they are disintegrated by L-cysteine; the scale bar is 100 micrometers.

Figure 26 shows an image of the soluble reduced type and temperature sensitive microspheres of one embodiment of the present disclosure when they are disintegrated by DTT; the scale bar is 100 microns.

Figure 27 shows a line graph of the attachment rate of MDCK cells to soluble reducing microspheres and thermosensitive microspheres according to one embodiment of the present disclosure.

In order to make the description of the present disclosure more detailed and complete, the following is an illustrative description of the implementation and specific embodiments of the present disclosure, but this is not the only form of implementing or using the specific embodiments of the present disclosure. The embodiments disclosed below can be combined or replaced with each other in beneficial situations, and other embodiments can be added to one embodiment without further recording or explanation. In the following description, many specific details will be described in detail so that the reader can fully understand the following embodiments. However, the embodiments of the present disclosure can also be implemented without such specific details.

In addition, spatially relative terms, such as "lower", "upper", etc., are used to conveniently describe the relative relationship between an element or feature and other elements or features in the drawings. These spatially relative terms are intended to include different orientations of the device when in use or operation in addition to the orientation shown in the drawings. The device can be positioned differently (for example, rotated 90 degrees or other orientations), and the spatially relative descriptions used in this article can also be interpreted accordingly.

In this article, unless the context specifically limits the article, "one" and "the" may refer to one or more. It will be further understood that "include", "include", "have" and similar words used in this article indicate the characteristics, regions, integers, steps, operations, elements and/or components recorded therein, but do not exclude the described or additional one or more other characteristics, regions, integers, steps, operations, elements, components, and/or groups thereof.

In some embodiments, the preparation of the soluble reduced microcarrier or the soluble microcarrier includes a mixing process of a soluble polymer and a crosslinking agent, and when the soluble polymer and the crosslinking agent come into contact, crosslinking is performed to obtain the soluble reduced microcarrier or the soluble microcarrier. In one embodiment, the microcarrier may be in the form of microspheres including, but not limited to, approximately spherical microspheres. In some embodiments, the particle size of the microspheres in the dry state is between 100-300 microns (μm). The microspheres are suitable for the growth of adherent cells, for example, 110 μm, 120 μm, 140 μm, 200 μm, 220 μm, 250 μm, 280 μm, or any value between these values. In some embodiments, the microcarriers are immersed in the culture medium and swell to become complete spheres, and the particle size is between 150-400 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 between these values.

The charge and hydrophilicity of the microcarrier surface affect cell attachment behavior. Positively charged chemical groups, such as amines (-NH ₂ ) have better cell attachment than negatively charged carboxyl groups (-COOH). In addition, slightly hydrophilic surfaces have better protein adsorption properties than hydrophobic (water contact angle > 90°) and superhydrophobic (water contact angle > 150°).

In one embodiment, the soluble polymer comprises cellulose, collagen, gelatin, sodium alginate, chitosan, hyaluronic acid, fruit acid or a combination thereof. In one embodiment, gelatin is composed of 85% to 92% protein, mineral salts and water. It is a water-soluble mixture extracted from the extracellular matrix collagen in animal skin, bones and connective tissue. It is a highly biocompatible and biodegradable non-toxic macromolecule composed of 300-4000 amino acid heterogeneous single-chain and multi-chain polypeptides. The preparation method is type A gelatin (pH 3.8-6.0; isoelectric point 6-8) obtained by acid hydrolysis of pork skin; type B gelatin (pH 5.0-7.4; isoelectric point 4.7-5.3) obtained by alkaline hydrolysis of animal bones and skin. Gelatin has a unique amino acid sequence, consisting of three parallel left-handed α chains, each chain consisting of a repeated amino acid sequence Gly-Xaa-Yaa (Gly: glycine, Xaa: proline, Yaa: hydroxyproline), and is easily soluble in high-temperature aqueous solvents and forms a gel after cooling. When the temperature is at the highest critical solution temperature (UCST) of 30-35°C, a sol-gel phase transition will occur, which is a reversible gelation phenomenon. In one embodiment, collagen is the main structural protein in the extracellular matrix of many tissues, rich in arginine-glycine-aspartic acid (RGD) sequences, and promotes cell adhesion and proliferation and growth.

In one embodiment, the oil and the surfactant are mixed to form a mixed solution. In one embodiment, the oil includes mineral oil, stearic acid, cottonseed oil, oleyl alcohol, white wax oil or a combination thereof. In one embodiment, the surfactant includes sorbitan monooleate 80, hydroxylated lanolin, polyoxyethylene sorbitol beeswax derivative, propylene glycol fatty acid ester, anhydrous sorbitan monooleate, propylene glycol monolaurate, diethylene glycol monooleate, polyoxyethylene oleyl alcohol ether, polyoxyethylene sorbitol beeswax derivative, diethylene glycol fatty acid ester, diethylene glycol fatty acid ester or a combination thereof. In one embodiment, a hydrophilic-lipophilic balance (HLB) of 0 indicates a completely lipophilic molecule, and a larger value indicates a more hydrophilic molecule. In some embodiments, the gelatin oil-in-water emulsion system selects an appropriate surfactant according to the HLB, which is between 3 and 5, such as 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, or any value in between; wherein, the HLB of sorbitan monooleate 80 is 4.3, which is more lipophilic, suitable for dispersion in the oil phase and avoiding the merging of droplets in the water phase, thereby improving the stability of the emulsion.

In one embodiment, the soluble polymer and water are then heated to form a soluble polymer aqueous solution, and then the soluble polymer aqueous solution is slowly dripped into the mixed solution to obtain an oil-in-water (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 between these values). The amount of oil here should not be too low to avoid poor shaping effect. In one embodiment, the soluble polymer and water are heated to make multiple soluble polymers or their monomers present in liquid form, and the heating temperature includes, but is not limited to, 30°C to 90°C, such as 40°C, 50°C, 60°C, 70°C, 80°C, or any value between these values.

In one embodiment, the water-in-oil emulsion is then cooled to allow the microspheres to set. After cooling, a crosslinking agent is added and stirred to perform a crosslinking reaction until the microspheres solidify. In this article, "setting" refers to stabilizing the form of water in oil. In this article, "solidification" means that the form of the water droplets no longer changes; the emulsified material undergoes a slight surface and property modification, and the material is reshaped and no longer changes according to the modified properties. In one embodiment, the temperature of cooling the water-in-oil emulsion described herein is less than the aforementioned heating temperature. In one embodiment, in the preparation of a soluble, reduced microcarrier, the crosslinking agent is a reducing crosslinking agent, and the reducing crosslinking agent bonds to the hydroxyl, amine, thiol, or carboxylic acid group of the soluble polymer. In some embodiments, the reducing crosslinking agent includes, but is not limited to, a disulfide bond crosslinking agent or a diselenide bond crosslinking agent. The disulfide bond crosslinking agent includes, but is not limited to, 3,3'-dithiodipropionate (N-hydroxysuccinimidyl) (DTSP), 3,3'-dithiobis (sulfonic acid succinimidyl propionate) (DTSSP), cystine, or dibutyl disuccinimidyl propionate (DSP). The di-selenide bond crosslinking agent includes, but is not limited to, DSeDPA-NHS (3,3'-Dithiodipropionic acid di(N-hydroxysuccinimide ester), 3,3'-diselanediyldipropionic acid acid), 2,2'-diselanediylbis(ethan-1-amine), 2,2'-diselanediylbis(ethan-1-ol) or a combination thereof. In another embodiment, in preparing the soluble microcarrier, the crosslinking agent comprises a zero-length crosslinking agent and a non-zero-length crosslinking agent. The so-called zero-length crosslinking agent will be removed after the catalytic degradation polymer crosslinking is completed; the zero-length crosslinking agent includes, but is not limited to, carbodiimide (EDC), N,N'-dicyclohexylcarbodiimide (DCC), or a combination thereof. Non-zero length crosslinkers are eventually incorporated into the polymer network, react with the degradable polymer, and form covalent bonds between the degradable polymers. Non-zero length crosslinkers include, but are not limited to, formaldehyde, glutaraldehyde, acrylamide, isocyanate, dextrin, DTSP, DSeDPA-NHS, BSSS, DSG, sulfo-EGS, DSS, EGS, BS2G, DTSSP, DST, BSOCOES, DPDPB, sulfo DST, or DSP.

In one embodiment, the microspheres are then solidified and cross-linked, and the oil phase is filtered out and washed. The microspheres are dried to obtain soluble reduced microspheres (Gms-DTSP) or soluble microspheres (Gms). In some embodiments, the oil phase is removed by a vacuum filter after the cross-linking reaction, and then washed several times with an acetone/water solution (v/v, 5:1~1:1, such as 4:1, 3:1 or 2:1), and finally the microspheres are freeze-dried. In some embodiments, the ratio of acetone/water solution here can be fixed or sequentially decreased. The oil can be quickly removed under high concentration acetone/water solution, but it cannot be washed with all acetone, which will destroy the surface or morphology of the microspheres.

In one embodiment, the weight ratio of the soluble polymer to the reducing crosslinking agent is 1:0.08 to 1:0.8.

In one embodiment, the weight ratio of the soluble polymer to the reducing crosslinking agent 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 99:1-1.86:1, such as about 90:1-2:1, 80:1-2:1, 70:1-2:1, 60:1-2:1, 50:1-2:1, 40:1-2:1, 30:1-2:1, 20:1-2:1, 10:1-2:1, 7:1-2:1, 5.67:1-1.86:1, 5:1-1.86:1, 4:1-1.86:1, 3:1-1.86:1, or any value between any two of these values, as measured by an elemental analyzer.

In some embodiments, preparing the thermosensitive polymer includes: polymerizing the temperature-sensitive polymer and the hydrophilic monomer by a free radical polymerization method to obtain the thermosensitive polymer. In one embodiment, the temperature sensitive polymer includes, but is not limited to, poly(N-isopropylacrylamide), poly(N,N-diethylacrylamide) (poly(PDEAAM), poly(N-vinylcaprolactam (PVCL), poly(2-isopropyl-2-oxazoline) (PIOZ), poloxamer, or a combination thereof. In one embodiment, the hydrophilic monomer includes, but is not limited to, acrylic acid (AAC), acrylamine (ALA), acrylamide (AAm), [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonic acid propyl) ammonium hydroxide (DMAPS), diethylaminoethyl methacrylate (DEAEMA), hydroxyethyl methacrylate (HEMA) or a combination thereof. In one embodiment, the free Radical polymerization methods include, but are not limited to, stable free radical polymerization (SFRP), atom transfer radical polymerization (ATRP), or reversible addition-fragmentation chain transfer polymerization (RAFT). In one embodiment, the hydrophilic monomer includes, but is not limited to, acrylic acid, acrylamine, acrylamide, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonatepropyl)ammonium hydroxide (DMAPS), diethylaminoethyl methacrylate (DEAEMA), hydroxyethyl methacrylate (HEMA), or a combination thereof. In some embodiments, the thermosensitive polymer comprises N-isopropylacrylamide and acrylamine, wherein the acrylamine accounts for 1% to 5% by weight of the thermosensitive polymer, such as 2%, 3%, 4%, or any value in between.

In one embodiment, a temperature-sensitive polymer, a hydrophilic monomer, an initiator, and a chain transfer agent are dissolved in an organic solvent by a reversible addition-fragmentation chain transfer polymerization method, and an ultrasonic oscillator is placed to dissolve the mixture. The mixture is added to a reaction bottle and purged with nitrogen, and then heated and continuously stirred to perform a polymerization reaction. A condenser is installed above the reaction bottle to maintain reflux in the system to prevent the reactants from volatilizing due to heat due to excessive temperature. When the viscosity of the mixture after heating the reaction bottle no longer thickens, the reaction bottle is placed in liquid nitrogen to terminate the reaction and obtain a polymerization solution. In some embodiments, the heating temperature may be between 50°C and 90°C, such as 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, or any value therebetween. In some embodiments, the reaction time is between 4 hours and 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 therebetween. In some embodiments, conventional chain transfer agents include mercaptans, dodecyl mercaptan DDM, or halogenated alkanes, such as carbon tetrachloride. Chain transfer agents are also known as modifiers and control agents.

In one embodiment, the polymerization solution is precipitated in cold ether to ensure the removal of unreacted monomers and chain transfer agents. After precipitation, a solid is obtained and dried to remove the ether. Then, a purification step is performed using a dialysis membrane, and the dried solid is dialyzed in water to obtain a semi-finished product, and the semi-finished product is dehydrated to obtain a dry thermosensitive polymer, or a thermosensitive block copolymer.

In some embodiments, the soluble reductive temperature-sensitive microspheres (Gms-DTPS-pnipam) or soluble temperature-sensitive microspheres (Gms-pnipam) are prepared physically, including: mixing the soluble reductive microcarrier with the temperature-sensitive polymer to obtain the soluble reductive 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 temperature-sensitive polymer aqueous solution, the soluble reductive microspheres (Gms-DTSP) or soluble microspheres (Gms) are added and stirred at low temperature until the surface of the soluble microspheres or soluble reductive microspheres is covered with the temperature-sensitive polymer. The modified microspheres are washed to remove the residual thermosensitive polymer, and then dried to remove moisture to obtain soluble reduced thermosensitive microspheres (Gms-DTPS-pnipam) or soluble thermosensitive microspheres (Gms-pnipam). In some embodiments, the microspheres are stirred at a low temperature of 0°C to 15°C, such as 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 10°C, 12°C, 14°C, or any value in between. In some embodiments, the microspheres are freeze-dried to remove moisture. In one embodiment, the thermosensitive polymer is physically coated on the surface of the microcarrier through intermolecular forces, such as Van der Waals force, secondary bonds including but not limited to hydrogen bonds, etc.

In some embodiments, the preparation of the di-selenium bond crosslinking agent includes: disposing 10 mmol of selenium (Se) powder in 3 ml of water and maintaining it in a nitrogen environment. In some embodiments, the selenium-containing water is injected into a three-neck reaction bottle and maintained in nitrogen. Then, 20 mmol of sodium borohydride (NaBH ₄ ) is slowly dripped into the selenium-containing water in 8 ml of water, and stirred until it becomes colorless to completely dissolve the selenium powder, thereby obtaining a first mixed solution. Then, an equal amount of 10 mmol of selenium powder is added to the first mixed solution and heated until it becomes reddish brown to obtain a second mixed solution. In some embodiments, the heating temperature is between 80°C and 130°C, such as 85°C, 90°C, 95°C, 100°C, 105°C, 110°C, 115°C, 120°C, 125°C, or any value therebetween. Then, 20 mmol of 3-chloropropionic acid is added to the second mixed solution and stirred under a nitrogen environment at room temperature to obtain a third mixed solution. The third mixed solution is exposed to the atmosphere and stirred, and then filtered to remove unreacted substances to obtain a yellow supernatant. The yellow supernatant is adjusted to pH 3.5 with 1 M hydrochloric acid (HCl), and extracted twice with anhydrous ethyl acetate (EA) to obtain an upper organic layer, which is then washed with water, extracted and water is removed (for example, water is absorbed with anhydrous magnesium sulfate powder) and then ethyl acetate to obtain DSeDPA.

In one embodiment, 1.2 mmol of DSeDPA is then dissolved in 5 ml of anhydrous tetrahydrofuran (THF), and then dripped into a container under a nitrogen environment, and then 2.88 mmol of N-hydroxysuccinimide (NHS) is added and stirred to obtain an initial solution. Then 2.88 mmol of diimine is dissolved in 5 ml of anhydrous tetrahydrofuran and dripped into the low-temperature initial solution to control the reaction speed to avoid too fast. In some embodiments, the low temperature is between 0°C and 20°C, such as 5°C, 10°C, 15°C, or any value between these values. Next, the initial solution containing tetrahydrofuran is stirred at room temperature until the reaction is complete, and then DSeDPA-NHS is obtained after filtering out impurities and removing tetrahydrofuran. DSeDPA-NHS can also be collected and stored after drying in a vacuum oven for 24 hours.

In some embodiments, the soluble reductive thermosensitive microspheres (Gms-DTSP-Se-pnipam) or soluble thermosensitive microspheres (Gms-Se-pnipam) are prepared by chemical methods, including mixing the soluble reductive microcarrier, the reductive crosslinking agent and the thermosensitive polymer to obtain the soluble reductive thermosensitive microcarrier; or, mixing the soluble microcarrier, the reductive crosslinking agent and the thermosensitive polymer to obtain the soluble thermosensitive microcarrier. In one embodiment, after preparing the thermosensitive polymer aqueous solution, 0.18 mM DSeDPA-NHS and the soluble reductive microspheres (Gms-DTSP) or the soluble microspheres (Gms) are added, and stirred at low temperature until the surface of the soluble reductive microspheres is covered with the thermosensitive polymer and the chemical bonding is completed. In some embodiments, the microspheres are stirred at a low temperature of 0°C to 15°C, such as 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 10°C, 12°C, 14°C, or any value in between. After the bonding is completed, the microspheres are washed to remove the residual thermosensitive polymer, and then dried to remove excess water to obtain soluble reduced thermosensitive microspheres (Gms-DTSP-Se-pnipam) or soluble thermosensitive microspheres (Gms-Se-pnipam).

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

Although a series of operations or steps are used below to illustrate the methods disclosed herein, the order in which these operations or steps are shown should not be interpreted as a limitation of the present disclosure. For example, certain operations or steps may be performed in a different order and/or simultaneously with other steps. In addition, not all operations, steps, and/or features shown must be performed to implement the present disclosure. In addition, each operation or step described herein may include a number of sub-steps or actions.

Figure 1 shows a schematic diagram of microcarrier preparation in one embodiment of the present disclosure.

Preparation Example 1 Preparation of soluble and reducible microspheres

Mix mineral oil and surfactant sorbitan monooleate 80 to form a mixture. Heat gelatin and water to prepare 5 ml of 0.25 g/ml (g/mL) gelatin aqueous solution, then slowly drip the gelatin aqueous solution into the mixture to obtain an oil-in-water (W/O) emulsion (oil: water v/v, 7:1). Rapidly cool the oil-in-water emulsion to make the microspheres solidify and cool, add 0.1 g to 1 g of disulfide bond crosslinking agent DTSP and stir to perform crosslinking reaction until the microspheres solidify (DTSP is about 0.25 mM to 1.2 mM here). After the crosslinking reaction is completed, remove the oil phase and wash, and dry the microspheres to obtain soluble reduced microspheres (Gms-DTSP).

According to the measurement by elemental analyzer, the weight ratio of gelatin to DTSP in the dried soluble reduced microspheres (Gms-DTSP) is about 99:1~65:35.

Preparation Example 2 Preparation of soluble microspheres

Mix mineral oil and surfactant sorbitan monooleate 80 to form a mixture. Heat gelatin and water to prepare a 0.25 g/ml (g/mL) gelatin aqueous solution, then slowly drip the gelatin aqueous solution into the mixture to obtain an oil-in-water (W/O) emulsion (oil: water v/v, 7:1). Rapidly cool the oil-in-water emulsion to make the microspheres solidify. After cooling for a period of time, add glutaraldehyde and stir for a period of time to perform a crosslinking reaction until the microspheres solidify. After the crosslinking reaction is completed, filter to remove the oil phase and wash, and dry the microspheres to obtain soluble microspheres (Gms).

Preparation Example 3 Preparation of temperature-sensitive polymer

NIPAM, ALA, initiator (2,2’-azobis(2-methyl-propionitrile), AIBN), and chain transfer agent (4-cyano-4-(phenylcarbonothioylthio)acid, CTA) were dissolved in 1,4-dioxane by reversible addition fragmentation chain transfer polymerization (RAFT), and then placed on an ultrasonic oscillator to dissolve the mixture. The mixture was slowly added to a reaction bottle and flushed with nitrogen, then heated and continuously stirred to carry out the polymerization reaction. A condenser was installed above the reaction bottle to keep the system refluxed to prevent the reactants from volatilizing due to excessive temperature. When the viscosity of the mixture after heating the reaction bottle no longer thickens, the reaction bottle is placed in liquid nitrogen to terminate the reaction to obtain a polymerization solution, and the polymerization solution is precipitated in cold ether to ensure the removal of unreacted monomers (such as NIPAM, ALA) and chain transfer agents. After precipitation, a light yellow solid is obtained and dried to remove the ether. Then, a dialysis membrane (molecular weight cut off, MWCO) = 1000) is used for purification. The dried light yellow solid is dialyzed in water to obtain a semi-finished product. The semi-finished product is dehydrated to obtain a dry thermosensitive polymer P (NIPAM-co-Allylamine), or a thermosensitive block copolymer (yield: about 90%).

Preparation Example 4 Preparation of soluble and reductive thermosensitive microspheres - physical method (Gms-DTSP-pnipam)

After preparing a 3wt% P(NIPAM-co-Allylamine) ethanol aqueous solution, add the soluble reduced microspheres (Gms-DTSP) prepared in Preparation Example 1 and stir at a low temperature of 0~5℃ until the soluble microspheres are covered with P(NIPAM-co-Allylamine). Wash the modified microspheres to remove the residual P(NIPAM-co-Allylamine), and then dry them to remove moisture to obtain soluble reduced temperature-sensitive microspheres (Gms-DTPS-pnipam).

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

After preparing a 3% (wt%) P(NIPAM-co-Allylamine) ethanol aqueous solution, add the soluble microspheres (Gms) prepared in Preparation Example 2 and stir at a low temperature of 0~5℃ until the soluble microspheres are covered with P(NIPAM-co-Allylamine) and the physical bonding is completed. The modified microspheres are washed to remove the residual P(NIPAM-co-Allylamine), and then dried to remove the water to obtain soluble temperature-sensitive microspheres (Gms-pnipam).

Preparation Example 6 Preparation of diselenide bond crosslinking agent

Prepare 10 mmol of selenium (Se) powder in 3 ml of water and keep it in a nitrogen environment. Then slowly drip 20 mmol of sodium borohydride (NaBH ₄ ) in 8 ml of water into the selenium-containing water and stir until it becomes colorless and the selenium powder is completely dissolved to obtain a first mixed solution. Then add an equal amount of 10 mmol of selenium powder to the first mixed solution and heat it until it becomes reddish brown to obtain a second mixed solution. Add 20 mmol of 3-chloropropionic acid to the second mixed solution and stir it in a nitrogen environment at room temperature to obtain a third mixed solution. The third mixed solution is exposed to the atmosphere and stirred, and then filtered to remove unreacted substances to obtain a yellow supernatant. The pH value of the yellow supernatant was adjusted to 3.5 with 1 M hydrochloric acid and extracted with anhydrous ethyl acetate to obtain the upper organic layer, which was then washed and extracted twice with water and the water and ethyl acetate were removed to obtain DSeDPA (yield: 85%).

Dissolve 1.2 mmol of DSeDPA in 5 ml of anhydrous tetrahydrofuran, then drip into a container under nitrogen atmosphere, then add 2.88 mmol of N-hydroxysuccinimide (NHS) and stir to obtain the initial solution. Then, dissolve 2.88 mmol of diimine in 5 ml of anhydrous tetrahydrofuran and drip into the low-temperature initial solution to control the reaction speed to avoid too fast. Stir the initial solution containing tetrahydrofuran at room temperature until the reaction is complete, filter out impurities and remove tetrahydrofuran to obtain DSeDPA-NHS. DSeDPA-NHS can also be collected and stored after drying in a vacuum oven for 24 hours.

Preparation Example 7 Preparation of soluble reducing thermosensitive microspheres - chemical method (Gms-DTSP-Se-pnipam)

After preparing 3wt% P(NIPAM-co-Allylamine) ethanol aqueous solution, add 1wt% DSeDPA-NHS and 96wt% soluble reduced microspheres (Gms-DTSP) of Preparation Example 1, and stir at low temperature until the soluble reduced microspheres are covered with P(NIPAM-co-Allylamine) and chemical bonding is completed. After bonding is completed, the microspheres are washed to remove residual P(NIPAM-co-Allylamine), and then dried to remove excess water, and soluble reduced temperature-sensitive microspheres (Gms-DTSP-Se-pnipam) are obtained.

Example 1 Synthesis of temperature-sensitive polymer

The thermosensitive polymer was obtained from the polymerization of ALA monomer and NIPAM in Preparation Example 3, so that the structure has positively charged amine groups, which is beneficial for cell attachment applications. The following three groups of 1% ALA, 3% ALA, and 5% ALA experimental synthesis ratio formulas are shown in Table 1. The synthesized polymer was confirmed to be successfully grafted by FT-IR and ¹ H-NMR, and the molecular weight was measured by gel permeation chromatography (GPC).

1.1 ¹ H-NMR Identification and Composition Elements

First, the chemical structure of the P(NIPAM-co-Allylamine) polymer was confirmed. When the initiator AIBN was added during the reaction, free radicals began to be generated when the temperature reached 70°C, which promoted the monomer double bond (C=C) polymerization reaction. The molecular structure was identified by hydrogen spectroscopy ( ¹ H-NMR) analysis using a nuclear magnetic resonance spectrometer (NMR) (as shown in Figure 2), corresponding to the structural formula δ=0.95-1.24ppm (a, CH ₃ comes from NIPAM), δ=1.34-1.75ppm (b, CH ₂ comes from NIPAM and Ala)(h, CH ₂ comes from CTA), δ=1.82-2.13ppm (c, CH comes from NIPAM and Ala)(i,j, CH comes from CTA), δ=2.66ppm (d, CH ₂ comes from Ala), δ=3.82ppm (e, CH comes from NIPAM), δ=7.32-7.79ppm (f, CH comes from CTA), δ=8.38ppm (g, NH comes from NIPAM). The grafting rate and composition were calculated by integrating the ¹ H-NMR signal (see Table 2 below).

1-2 Molecular weight measurement and identification

The molecular weight and molecular weight distribution of the synthesized polymers were identified by gel permeation chromatography. Table 3 shows the molecular weight and molecular weight distribution of the P(NIPAM-co-Allylamine) thermosensitive polymer measured by gel permeation chromatography. Table 3 shows that the molecular weight dispersity (polydispersity index, PDI) of the synthesized polymers has a narrow molecular weight distribution, and its value is mostly between 1.2-1.3, and the weight average molecular weight (Mw) of P(NIPAM-co-Allylamine) is 37666, 42803, and 43056, respectively, proving that the monomers are successfully polymerized into copolymers through RAFT.

1-3 Minimum critical solution temperature LCST measurement

After the monomer ALA is embedded in the NIPAM copolymer, the electrostatic repulsion increases, and the hydrophobic force of PNIPAM affects the LCST. The LCST of the copolymer can be measured by UV/Vis spectrometer, which is the temperature corresponding to 50% transmittance: when the temperature rises, the copolymer aggregates in large quantities, and forms a non-flowing gel state from a solution state, and the light transmittance value decreases relatively. Figure 3 shows the transmittance of PNIPAM and 1% ALA, 3% ALA, and 5% ALA at different temperatures. It is known that the LCST of PNIPAM is 31.4℃; the LCST of 1% ALA, 3% ALA, and 5% ALA are 32.3℃, 32.5℃, and 33.4℃, respectively. Therefore, LCST increases with the proportion of ALA. This proves that the addition of hydrophobic monomers to copolymers will reduce LCST, whereas hydrophilic monomers will increase LCST. The main reason depends on the hydrophilicity and hydrophobicity of the polymer tail end groups.

1-4 Water contact angle test

The water contact angle of thermosensitive polymers changes with temperature. Water forms a round droplet at the air-liquid interface due to its own surface tension. The water contact angle is calculated based on the image according to Young's formula: γsv=γsl+γlvcosθ. Please refer to Figure 4. Using commercially available PNIPAM as a control group, the water contact angle of PNIPAM at room temperature is 44.83±0.47°. After embedding ALA, the water contact angles of 1% ALA, 3% ALA, and 5% ALA are 37.13±0.55°, 32.65±0.28°, and 29.4±0.95°, respectively. When the temperature rises from room temperature to 40°C, the water contact angles of PNIPAM, 1% ALA, 3% ALA, and 5% ALA are 63.23±1.85°, 64.91±0.89°, 63.45±1.04°, and 53.83±1.17°, respectively. It is known that the angle of the control group PNIPAM changes from low temperature to high temperature by 18.4°, and the difference of P(NIPAM-co-Allylamine) after embedding ALA is more than 25°, which proves that this copolymer has temperature-responsive characteristics. In addition, ALA is a hydrophilic monomer, and the water contact angle of 5% ALA is smaller at low temperature, making it the most hydrophilic. As the ALA content of the copolymer increases, the contact angle gradually decreases. 1% ALA and 3% ALA are closer to the hydrophobic contact angle of the control group PNIPAM at high temperature, slightly hydrophilic (water contact angle <90°), and have better protein adsorption properties, which is beneficial to cell adsorption and proliferation.

Example 2 Synthesis of di-selenide bond crosslinking agent

One of the purposes of synthesizing the diselenide bond is to chemically bond the P(NIPAM-co-Allylamine) polymer to the microspheres to form thermos-sensitive microspheres, and to compare the results with physically coating the microspheres with thermos-sensitive polymers. Diselenide bonds are redox-sensitive materials and are easily broken by environmental changes. The oxygen in the carboxyl groups (-COOH) at both ends of DSeDPA is attacked by EDC nucleophilically to form a highly active intermediate (O-acylisourea), which then reacts with NHS to form a second intermediate (O-acylisourea), which is hydrolyzed in water to DSeDPA-NHS and reacts rapidly with the amine group (-NH ₂ ) to produce a stable amide bond, which then reacts with the tail amine group of P(NIPAM-co-Allylamine) to crosslink into a polymer.

2.1 ¹ H-NMR Identification and Composition Elements

The correctness of the structure was confirmed by ¹ H-NMR. Before activation of DSeDPA (as shown in Figure 5), the corresponding structure was found to be δ=2.69~2.72 ppm (a); δ=3.04~3.06ppm (b). The structure of both points is CH _2. Next to point b is the carboxylic acid group (-COOH), which is pulled to the left by the larger electronegativity, which is consistent with the resonance frequency increase signal in the nuclear magnetic resonance located in the low field region. After activation (as shown in Figure 6), the structure of DSeDPA-NHS is δ=2.69-2.71ppm (a, CH ₂ comes from DSeDPA); δ=3.03-3.06ppm (b, CH ₂ comes from DSeDPA); δ=2.59ppm (c, CH comes from NHS). Point c shows that there are four CH signals on the NHS active group with higher intensities than those at points a and b. The above NMR results show that the double selenide bond crosslinker DSeDPA-NHS functional group was successfully synthesized.

2-2 Raman spectrometer analysis

Selenium is an air-sensitive element. Figure 7 is a Raman spectrum of the symmetrical structure of selenium. After analysis, Se-Se double selenium bond signals appeared at 290cm ^-1 and 310cm ^-1 , and there was an obvious Se-C functional group around 276cm ^-1 , which proved that the activity and reaction efficiency of carboxylic acid were improved through EDC activation, so that NHS was connected to the carboxylic acid group (-COOH), and DSeDPA-NHS was successfully synthesized. Raman confirmed the presence of selenium in the structure.

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

Using FT-IR can more accurately observe the structure of inorganic functional groups. From the spectrum analysis of Figure 8, we know that before activation, the carboxylic acid group of DSeDPA is bound to NHS, and the peak at 1688cm ^-1 belongs to the C=O functional group; after activation, it is pulled by NHS binding and changes to the peak at 1776cm ^-1 . In addition, the spectra of other functional group signals NO, CN, and CO molecular vibrations of the two are significantly different, indicating that the reaction is successful.

Example 3 Gelatin microsphere identification

Gelatin is soluble in hot water but not in cold water, but human cell culture needs to be in an environment of 37°C, so the network cross-linking needs to be stabilized, and the microspheres are obtained by adding a cross-linking agent through an emulsification reaction. Among them, the microspheres prepared with a reducing cross-linking agent are soluble reduced microspheres (Gms-DTSP, as shown in Preparation Example 1), and the microspheres prepared with a non-reducing cross-linking agent are soluble microspheres (Gms, as shown in Preparation Example 2). Then, the thermosensitive polymer is bonded to the surface of the microspheres by physical coating or chemical bonding, and physically coated soluble reductive thermosensitive microspheres (Gms-DTPS-pnipam, as in Preparation Example 4), soluble thermosensitive microspheres (Gms-pnipam, as in Preparation Example 5), and chemically bonded soluble reductive thermosensitive microspheres (Gms-DTSP-Se-pnipam, as in Preparation Example 7) are obtained.

3.1 Raman spectrometer analysis

The microspheres were analyzed by Raman instrument and divided into the following groups according to the crosslinking agent used: Group 1: soluble microspheres (Gms, as in Preparation Example 2), physically coated soluble thermosensitive microspheres (Gms-pnipam, Preparation Example 5); Group 2: soluble reductive microspheres (Gms-DTSP, as in Preparation Example 1), chemically bonded soluble reductive thermosensitive microspheres (Gms-DTSP-Se-pnipam, Preparation Example 7), physically coated soluble reductive thermosensitive microspheres (Gms-DTPS-pnipam, Preparation Example 4).

Since gelatin has no fixed structure, but has a large number of hydroxyl and amine groups in its structure, the Raman peak cannot indicate the actual structure after modification, but the difference between microspheres can still be observed through peak shift. As shown in Figure 9, there are two signals in the range of 2000-2500cm ^-1 for the first group. After gelatin is crosslinked with glutaraldehyde (Gms), it can be confirmed that the two peaks in the three reaction stages are shifted left and right. Figure 10 shows the second group of microspheres crosslinked with DTSP. It is observed that the position close to 1400cm ^-1 , after gelatin is crosslinked with DTSP, each group of microspheres has this signal, and it is inferred that DTSP does crosslink gelatin to stabilize it into microspheres. In addition, there are slight changes in the two signals in the range of 2000-2500cm ^-1 .

3.2 Fourier transform infrared spectrum analysis

Raman spectrometer and FT-IR are complementary and more suitable for detecting symmetric bonds. The microspheres have an asymmetric structure. The two groups of microspheres were analyzed using FT-IR and had relatively strong infrared absorption peaks. Figure 11 shows that amide I of gelatin before cross-linking is affected by C=O stretching vibration at 1629cm ^-1 , and Schiff base C=N stretching vibration occurs at 1634cm ^-1. After cross-linking, it is caused by the C=O stretching of amide I and the unreacted aldehyde mixture, confirming the Schiff base reaction formed between the amino group of gelatin and the carbonyl group (C=O) of glutaraldehyde, confirming the successful cross-linking of gelatin and glutaraldehyde.

The main peaks in gelatin are amide I (C=O bond stretching) at 1600-1700cm ^-1 , amide II (NH bending vibration and CN stretching vibration) at 1500-1590cm ^-1 , and amide III (NH bending vibration and CN stretching vibration) at around 1200cm ^-1 , all of which are observed as characteristic peaks in Figures 12 and 13. In particular, the peaks of OH and NH vibrations were observed at 3500-3000cm ^-1 using DTSP crosslinking agent, and the symmetric stretching vibration peak of -CH ₃ group was observed at 2940cm ^-1 . DTSP has NHS active group, which reacts with amine group (-NH ₂ ) in gelatin network to form stable amide bond. At the same time, the NHS structure was released and a characteristic peak of S=O was observed at 1390 cm ^-1 .

3.3 Scanning electron microscope (SEM) analysis

SEM was used to analyze the size of the dried cross-linked and cured gelatin microspheres and observe the surface morphology. Figure 14a shows the microspheres obtained without the use of a crosslinking agent; Figure 14b shows that the soluble microspheres (Gms) cross-linked with glutaraldehyde formed agglomerates that could not be sieved and had an uneven particle size range; Figure 14c shows that after the surface of the soluble thermosensitive microspheres (Gms-pnipam) was physically coated, the microspheres were dispersed, the particles were significantly dispersed and the agglomeration phenomenon was improved, and at the same time, it was observed that the surface was in a powder state covered by the thermosensitive polymer; Figure 14d shows the use of 0.25mM DTPS crosslinking agent (Gms-0.25mM DTSP); Figure 14e shows the use of 0.6mM DTPS crosslinking agent (Gms-0.6mM DTSP); Figure 14f (50 times magnification) and Figure 14g (150 times magnification) show DTPS crosslinker 1.2mM (Gms-1.2mM DTSP), and it is known that as the concentration of the crosslinker increases, the surface of the microspheres gradually becomes rough from smooth. Then observe the microspheres with thermosensitive polymers: Figure 14h shows the use of physical coating (Gms-DTSP-pnipam), and it is found that the surface of the microspheres using physical coating changes from the original roughness to a smooth state after being coated with thermosensitive polymers; another group of Figures 14i shows chemical bonding (Gms-DTSP-Se-pnipam) using selenium crosslinker to chemically bond P (NIPAM-co-Allylamine) to the gelatin surface, and there are obvious changes in shrinkage holes.

In addition to using SEM, energy-dispersive X-ray spectroscopy (EDS) was also used to obtain the elemental analysis composition. The microspheres crosslinked with glutaraldehyde contain C, N, and O elements. The disulfide bond DTPS crosslinker obtains C, N, O, and S elements. The diselenide bond crosslinker chemically bonds P (NIPAM-co-Allylamine) to gelatin to obtain C, N, O, S, and Se elements. The presence of crosslinker elements once again confirms that the crosslinking is successful. In order to understand the size of the microspheres in the dry state, Image J was used to analyze the particle size. Table 4 shows the average particle size of each group of gelatin microspheres.

3.4 Particle size analysis in swollen state

The particle size of microspheres is defined as 100-300 μm. First, gelatin microspheres smaller than 100 μm were separated using a sieve and then immersed in a warm culture medium at 37°C for 5 days to simulate the real cell culture environment. The expanded size and stability of the microspheres were then observed using an optical microscope. Table 5 shows the particle size distribution of Gms and Gms-pnipam. When Gms is dried, the particle size is agglomerated and cannot be screened for the first time. Some small microspheres cannot be separated and the size dispersion is large. However, after the surface of Gms-pnipam is coated with P (NIPAM-co-Allylamine), the agglomeration phenomenon is improved and it can pass through the screen smoothly. The size distribution range of the microspheres is smaller.

The other group is microspheres crosslinked with 0.25mM, 0.6mM, and 1.2mM DTSP. Figure 15 and Table 6 show that the 0.25mM DTSP microspheres were observed to expand to 900 microns in size at 0.5 hours and gradually increased. The microspheres expanded and broke at 48 hours. This group of crosslinkers cannot stabilize the gelatin network, resulting in poor stability of the microspheres. The 0.6mM DTSP was observed to be in a stable range within 48 hours, and the microsphere size decreased at 72 hours, slowly dissolving and disappearing with time. The 1.2mM DTSP crosslinked microspheres had a stable size curve within 120 hours, and the particle size range was about 300 microns, confirming that the increase in crosslinker concentration can obtain microspheres with a stable network size. The weight ratio of gelatin to 0.6-1.2mM DTSP is 1.25:0.4~1.25:1 (1:0.32~1:0.8).

Table 7 shows Gms-DTSP-pnipam and Gms-DTSP-Se-pnipam microspheres formed by combining Gms-1.2mM DTSP with the thermosensitive polymer P (NIPAM-co-Allylamine). After 120 hours of observation, the size changes steadily without rupture or swelling. Therefore, these three groups are suitable for cell culture experiments, proving the successful synthesis of reduced thermosensitive soluble gelatin microspheres.

3.5 Microsphere expansion test

Place 10 mg dry weight microspheres (W1) in a 15 ml centrifuge tube and immerse in 37°C culture medium. Remove the culture medium at different time points and use lens tissue to remove excess liquid, leaving the wet sample to weigh (W3). Repeat three times for each group. The microsphere swelling ratio is calculated as follows: Swelling ratio = (W3-W2)/W1. The water content is calculated as follows: Water content (%) = [1-W1/(W3-W2)] × 100. W1: dry sample weight; W2: dry sample and centrifuge tube weight; W3: wet sample and centrifuge tube weight.

Comparison of microsphere swelling can be used to evaluate the water absorption characteristics of polymer microspheres at 37°C. In the experiment, the dried microspheres were immersed in the culture medium for 24 hours and the weight after swelling was measured. Figure 16 shows the swelling properties of different gelatin microspheres. It is observed that the swelling decreases with the increase of 0.6mM and 1.2mM DTSP concentrations. The swelling rate of 0.25mM DTSP reaches 18.05 within 24 hours. However, as shown in Table 6 above, the microspheres swell and rupture after 48 hours. This set of crosslinkers cannot stabilize the gelatin network. The increase in the swelling of the temperature-sensitive polymer of the surface physical coating is accompanied by an increase in water content. Adding P(NIPAM-co-Allylamine) into gelatin microspheres significantly improves the water absorption capacity, and the most obvious one is GMS-DTSP-Se-pnipam microspheres, with a solubility of 12.62±0.07 and a water content of 93.32±0.97%.

Table 8. Expansion rate and moisture content of different microspheres after swelling

3.6 Disintegration behavior of dissolved microspheres

Disulfide bonds (-SS-) are usually formed by the natural cross-linking of two sulfur atoms between the side groups of cysteine. The chemical reducing agent DTT is used to reduce the disulfide bond by alkylation of active free cysteine, so that dicysteine is broken into cysteine. The diselenide bond has a similar redox mechanism to the disulfide bond. The oxidation response of the diselenide bond is greater than that of the disulfide bond. Selenium has a larger atomic radius and weaker electronegativity. Its lower bond energy can be easily reduced, causing the diselenide bond to break and generate a selenate intermediate ( ^RSe- ).

This disclosure will conduct a disintegration experiment on oxidative dissolution microspheres. In order to simulate the cell culture environment, Gms-DTSP, Gms-DTSP-pnipam, and Gms-DTSP-Se-pnipam were immersed in the culture medium and maintained at 37°C. Then 25mM reducing agent DTT was added to observe the changes in the disintegration of the gelatin polymer network. Table 9 shows that Gms-DTSP-Se-pnipam disappeared within 15 minutes, leaving only a clear solution, and the other two groups were completely disintegrated within 30 minutes.

Table 9. Disintegration behavior of reduced microspheres in 25 mM DTT

Example 4 Cell survival rate

4.1 Sensitive block copolymers

Figures 17, 18, and 19 show that 1% ALA, 3% ALA, and 5% ALA thermosensitive polymers were co-cultured with MDCK cells for 24 hours. The cell viability at the highest concentration of 1000 μg/mL for the three copolymers was 97.4%, 96.5%, and 96.3%, respectively, confirming that the thermosensitive polymer has good biocompatibility with MDCK cells (a standard mammalian cell line in biomedical research). When cells are stimulated by toxic substances, they will change their cell morphology and cause cell apoptosis. Through the MTT test, they will react with the mitochondrial succinate dehydrogenase of living cells, and purple crystals will be produced under the reduction reaction. The number of living cells can be qualitatively determined by the absorbance detection using a disk spectrometer at 570nm.

4.2 Gelatin microspheres

Microspheres are carriers and cannot be dissolved in culture medium. The sample shapes are not uniform. According to ISO-10993, the microspheres were immersed in 37℃ culture medium at 0.1g/mL for 24 hours. The culture medium after microspheres were immersed in different percentages was extracted and co-cultured with MDCK for 24 hours. Figure 20 shows that the cell survival rate of soluble microspheres cross-linked with glutaraldehyde can reach 82% at the highest concentration of 0.1g/mL. The cell survival rate of Gms-pnipam increased to 96.3% at the highest concentration. Due to the physical coating of the thermosensitive polymer on the surface, the thermosensitive polymer has good biocompatibility. Figure 21 shows another set of soluble reduced microspheres cross-linked with DTSP. The three types of microspheres also have good biocompatibility, all of which are greater than 90%. At the highest concentration, the cell survival rate reached 94%, proving that each group of microspheres had no ability to kill cells, which was beneficial for subsequent adhesion culture experiments.

Example 5 Cell attachment and detachment test

5.1 Thermosensitive polymer cell detachment test

In the adsorption and desorption experiment of P(NIPAM-co-Allylamine), cells were divided into 1% ALA, 3% ALA, and 5% ALA groups, and then spun onto plastic cover slips and co-cultured with 0.5×10 ⁶ cells/mL MDCK. Under an optical microscope, cells were observed to be attached to the P(NIPAM-co-Allylamine) membrane at 37°C, growing in a densely flattened cluster, fully demonstrating good adhesion properties (not shown). When the temperature dropped to 4°C, the hydrophilic groups on the copolymer membrane surface interacted with the charged groups of the cells, causing the cells to float in blocks and suspend in the PBS solution. In addition, a blank plastic cover slip (not coated with the temperature-sensitive block copolymer) was used as a control group. When the temperature was adjusted to a low temperature, the cell morphology did not change and no detachment occurred. The cells remained adhered to the cover slip (not shown).

The activity of the cells detached by temperature was stained with live/dead cell imaging reagent. Live cells can easily penetrate into the live cell membrane through Calcein-AM, and after hydrolysis, Calcein remains in the cell and emits strong green fluorescence, while dead cells are stained by BOBO ^TM -3 Iodide through the damaged cell membrane to stain the cell nucleus and release red fluorescence. Figure 22 shows the cells detached from three sets of P (NIPAM-co-Allylamine) membranes observed under a fluorescence microscope. In the superimposed images, the proportion of green fluorescence is the majority, proving that the cells detached by temperature still maintain good activity.

Using a simple temperature-controlled method, cells are detached from the thermoresponsive surface P (NIPAM-co-Allylamine) without causing damage to the cells. This also avoids the concerns about damage caused by traditional enzymatic detachment. From an engineering medicine perspective, traditional enzymatic detachment cuts off the cell-to-cell connections, and the harvested cells are independent single cells that cannot regenerate continuous cell sheets. However, through the temperature-sensitive gel layer, cell sheets with complete cell-to-cell connections can be obtained, which is a non-invasive detachment. After confluent culture, the cells need to lower the temperature to successfully harvest the cell sheets of the tissue structure, thereby achieving the purpose of biomedical tissue regeneration and repair.

5.2 Temperature-induced desorption behavior of temperature-sensitive microspheres

For the thermosensitive polymer P (NIPAM-co-Allylamine), the ALA concentration will increase the amine groups (-NH ₂ ) in the structure, which will increase the positive charge and hydrophilicity. The cell culture method is the same as described in 5.1 above. For the copolymer with 5% ALA, the cells have a more adherent culture surface when attached. When the cells detach at low temperature, the detachment time of 5% ALA is longer than that of the other two groups, indicating that a higher ALA content is conducive to cell adhesion, but embedding too many ALA monomers will make the copolymer too hydrophilic and lose its thermosensitive properties. Therefore, when preparing thermosensitive gelatin microspheres, after the above cell adsorption and detachment evaluation, the 5% ALA copolymer was finally selected for thermosensitive modification of the gelatin microsphere surface.

In order to detect the temperature-induced cell detachment ability of P(NIPAM-co-Allylamine) on microspheres, the detachment behavior of Gms-pnipam and Gms-DTSP-pnipam was tested first. The gelatin microspheres induced cell detachment by temperature. After 30 and 60 minutes of desorption at a low temperature of 4°C, it can be seen that the cells only slightly fell off (not shown), proving that the gelatin microspheres have a poor effect of cell detachment using temperature induction and require a long time. Therefore, it is necessary to use traditional enzymatic hydrolysis and disintegration methods to improve the efficiency of cell detachment from the carrier.

5.3 Deattachment test of soluble microsphere cells

Fluorescence microscopy was used to observe whether cells adhered to the surface of the microspheres and grew using Hoechst 33342. Hoechst 33342 can penetrate the cell membrane and emit blue fluorescence. After observation under a fluorescent microscope, it was confirmed that blue cells adhered to five types of microspheres (including Gms, Gms-pnipam, Gms-DTSP, Gms-DTSP-pnipam, Gms-DTSP-Se-pnipam, not shown in the figure), so as to evaluate the ability of the five types of microspheres to adsorb cells.

In order to evaluate the microsphere cell adsorption and detachment test, Gms and Gms-pnipam were swollen, and 0.5×10 ⁶ cells/mL MDCK cells were cultured in each group. The attachment of MDCK to the microspheres was observed under an optical microscope at 1, 3, 5, 7, and 24 hours, and the cell attachment rate was calculated. The results showed that cells could be observed to begin to attach to the edge of the microspheres in 1 hour, but Gms microspheres were agglomerated and uneven in size, and it was difficult to observe whether cells were attached to small-sized microspheres. Gms-pnipam was more uniform in size, and it could be seen that cells were clearly attached. Therefore, the cell attachment effect of Gms-pnipam was better than that before modification, so pnipam was conducive to cell adhesion (not shown). In addition, the results also showed that the trypsinization method was completely used for the cell detachment experiment. After the microspheres were soaked in trypsin and kept at 37°C for 5 minutes, cells fell off the surface of Gms. As time went on, trypsin could break the peptide bonds formed by lysine or arginine, and the cell detachment effect increased with the trypsin digestion. After 10 minutes, the microspheres were washed with PBS to recover the cells. After washing, there were still many residual cells on the surface of the microspheres, and the detachment effect was not good. In another group of Gms-pnipam, after 5 minutes of trypsinization, the cells and the thermosensitive polymer were cut off at the same time. After 10 minutes, the cells were almost completely dropped into the suspension. After the microspheres were washed with PBS and the cells were recovered, the surface of the microspheres tended to be smooth and flat, proving that this thermosensitive polymer helps the cell detachment effect (not shown).

In addition, Gms and Gms-pnipam attached to cells were added with live/dead dyes to identify the degree of cell death on the surface of the gel microspheres. Calcein-AM, which dyes live cells, will stimulate green fluorescence, while BOBO-3 Iodide, which dyes dead cells, will stimulate red fluorescence. The results showed that there were a large number of green fluorescent cells and a small number of dead cells on the microspheres, and the cells would be damaged by damaging the membrane protein if the trypsin detachment time was too long. In addition, the cells detached from Gms-pnipam showed green fluorescence, with a small number of red cells, while the cells of Gms were not completely detached after 10 minutes of detachment, and many healthy cells were still attached to the microspheres. This proves that Gms has the ability to adsorb cells, but after physically coating the surface of the microspheres with a thermosensitive polymer, the detachment ability of the carrier to release cells can be enhanced.

5.4 Cell attachment rate and detachment behavior of soluble microspheres

Figure 23 shows the ability of Gms and Gms-pnipam microspheres to adsorb cells. After 1 hour of incubation, Gms-pnipam reached a cell attachment rate of about 40% and Gms reached a cell attachment rate of 23%. After 24 hours, Gms and Gms-pnipam reached 74% and 85%, respectively. After surface coating with thermosensitive polymer, the attachment rate increased by 11%. Next, the desorption characteristics of gelatin microspheres Gms and Gms-pnipam were evaluated. As mentioned in Section 5.3 above, when Gms and Gms-pnipam were digested with trypsin for the same time, the desorption efficiency of Gms was lower than that of Gms-pnipam. After collecting the detached cells and washing and centrifuging them, the number of cells collected by Gms and Gms-pnipam was 192,500 cells/mL and 355,750 cells/mL respectively. After coating the surface with a thermosensitive polymer, the detachment ratio increased by 45.8%.

5.5 De-attachment test of soluble and reducible microspheres

This disclosure discloses that DTSP, a crosslinking agent with a disulfide bond structure and redox properties, is used to synthesize Gms-DTSP, Gms-DTSP-pnipam, and Gms-DTSPSe-pnipam. After two days of culture, the attachment of MDCK cells to the microspheres is observed under an optical microscope.

Gms-DTSP-pnipam reduced gelatin microspheres were evaluated for disintegration behavior using three GSH, L-cysteine, and DTT reducing agents (all at 25mM concentration) to observe cell detachment. As shown in Figure 24, after 60 minutes of using the GSH reducing agent, the microspheres did not swell or dissolve; as shown in Figure 25, after 30 minutes of using the L-cysteine reducing agent, the microspheres swelled and the cells fell off in clumps, and the microspheres completely dissolved and disappeared after 60 minutes; as shown in Figure 26, after 5 minutes of using the DTT reducing agent, the microspheres swelled and deformed and quickly disintegrated, and only clumps of cells remained after 30 minutes. Among the three reducing agents tested, DTT containing dithiol showed the best effect in cleaving disulfide bonds. It has a higher redox potential than GSH and L-cysteine. GSH and L-cysteine are monothiols and require other thiol-containing molecules to catalyze the acceleration of disulfide bond cleavage reduction.

It takes 30 minutes for Gms-DTSP and Gms-DTSP-pnipam to degrade and disappear the microspheres using the DTT reducing agent. In particular, the dissolution pattern of Gms-DTSP is that DTT swells the microspheres, and the cross-linked network is cut by the reducing agent, causing the spheres to rupture and the cells to eventually become single cells. The disintegration pattern of the surface-coated Gms-DTSP-pnipam microspheres is that DTT removes the cells in a cluster. It is inferred that the outer layer of the thermosensitive polymer coating protects the coating and cells, causing them to fall off first, and then the spheres themselves can be seen to slowly disintegrate. The final cells are in a clustered form, so the clustered cells and the thermosensitive polymer coating fall off together (not shown).

In addition, the thermosensitive polymer coating is hydrophilic at low temperatures. Gms-DTSP-pnipam gradually disintegrates in 5 minutes when placed at low temperatures (such as 4°C), and the cells become dispersed in 10 minutes. Gms-DTSP-Se-pnipam has the shortest disintegration time (not shown).

Live/dead dye was added to Gms-DTSP, Gms-DTSP-pnipam, and Gms-DTSP-Se-pnipam attached to cells to identify the degree of cell death on the surface of the gel microspheres. The results showed that cells attached to the microspheres were surrounded by a large number of green fluorescent cells, and the detached cells were stimulated to emit green fluorescence after disintegration (not shown), proving that the reduced gel microspheres have the ability to adsorb cells and the cells are still alive when they disintegrate.

5.6 Cell attachment rate and detachment behavior of reduced gelatin microspheres

Figure 27 shows the ability of soluble reduced and temperature-sensitive microspheres Gms-DTSP, Gms-DTSP-pnipam, and 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 were 40%, 39%, and 29.5%, respectively; after 8 hours, Gms-DTSP was the first to reach 88%; after 24 hours, the cell attachment rates of Gms-DTSP, Gms-DTSP-pnipam, and Gms-DTSP-Se-pnipam were 95%, 90%, and 47%, respectively. From the SEM observation of Example 3.3, the microsphere surface has tiny concave and convex holes to increase cell adhesion, among which Gms-DTSP-pnipam has better ability to adsorb cells than Gms-DTSP-Se-pnipam.

5.7 Harvest cell regeneration test

In order to prove that the cells harvested using gelatin microspheres are active and capable of subsequent medical applications, the cells were detached from Gms, Gms-pnipam, Gms-DTSP, Gms-DTSP-pnipam, and Gms-DTSP-Se-pnipam, washed, centrifuged, and placed in a culture dish for culturing. The results showed that after one day of culture, the cells cultured with Gms and Gms-DTSP-Se-pnipam had fewer cells initially recovered, resulting in slower growth, while the rest of the cells cultured with Gms-pnipam, Gms-DTSP, and Gms-DTSP-pnipam were all 80% full, with cells growing in a dense, flat cluster shape, showing good cell activity (not shown). Therefore, the cells detached from the microspheres disclosed in the present invention have the ability to be cultured repeatedly.

The soluble reduced microcarrier disclosed herein facilitates cell attachment through a reduced crosslinking agent, and the use of a reducing agent can facilitate cell detachment. In some embodiments, cells can be detached within 30 minutes using a reducing agent, and all cells are living cells after detachment, proving that this method is non-toxic.

The soluble thermosensitive microcarrier disclosed herein is coated with a thermosensitive polymer, which helps cells to attach when the culture temperature is higher than LCST; it helps cells to detach when the temperature is lower than LCST. In some embodiments, the microspheres become complete after being soaked in the culture medium and swelled, and the particle size is stably controlled between 280-350 microns. At the same time, the surface of the microspheres is protected by the thermosensitive polymer, which helps the stability of the microspheres at 37°C. In some embodiments, the cell attachment rate is increased by 11% after being modified by the thermosensitive polymer, and the cell detachment rate is increased by 45.8% using the enzymatic method, effectively improving the attachment and detachment capabilities of the microcarriers.

Although the present disclosure has been disclosed in the form of implementation as above, it is not intended to limit the present disclosure. Anyone familiar with this art can make various changes and modifications without departing from the spirit and scope of the present disclosure. Therefore, the protection scope of the present disclosure shall be subject to the scope defined in the attached patent application.

Claims (15)

A soluble temperature-sensitive microcarrier comprises: a soluble polymer, wherein a plurality of soluble monomers are bonded to each other by a crosslinker, wherein the crosslinker comprises a zero-length crosslinker and a non-zero-length crosslinker; and a temperature-sensitive polymer, which covers the soluble polymer, wherein the temperature-sensitive polymer comprises a temperature-sensitive polymer and a hydrophilic monomer, wherein the temperature-sensitive polymer comprises poly(N-isopropylacrylamide) (PNIPAM, PNIPA, PNIPAAm, NIPA, or PNIPAA), poly(N,N-diethylacrylamide) (PDEAAM), poly(N,N ... (poly(N-vinylcaprolactam, PVCL), poly(2-isopropyl-2-oxazoline, PIOZ), poloxamer, or a combination thereof; wherein the hydrophilic monomer comprises allylamine (ALA), acrylamide (AAm), [2-(Methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (DMAPS), diethylaminoethyl methacrylate (2-(Diethylamino)ethyl methacrylate, DEAEMA), hydroxyethyl methacrylate (2-Hydroxyethyl methacrylate, HEMA) or a combination thereof. A microcarrier as described in claim 1, wherein the zero-length crosslinker comprises carbodiimide (EDC), N,N' - dicyclohexylcarbodiimide (DCC), or a combination thereof. The microcarrier as claimed in claim 1, wherein the non-zero length crosslinking agent comprises formaldehyde, glutaraldehyde, acrylamide, isocyanate, genipin, DTSP (3,3'-dithiodipropionic acid di(N-hydroxysuccinimide ester)), DSeDPA-NHS (3,3'-diselanediyldi-propanoic acid-N-hydroxysuccinimide), BSSS (bis(sulfosuccinimidyl) suberate, BS3), DSG (disuccinimidyl glutarate), sulfo-EGS (ethylene glicolbis(sulfosuccinimidilsucci-nato), DSS (disuccinimidyl suberateS), EGS (ethylene glicolbis(succinimidylsuccinate)), BS2G(bis(sulfosuccinimidyl)glutarate), DTSSP(3,3’-dithiobis(sulfosuccinimidylpropionate), DST(disuccinimidyl tartrate), BSOCOES(bis(2-(succinimidooxycarbonyloxy]ethyl) sulfone), DDPPB(1,4-di-(3’-(2’ piridilditio)-propionamido)butane), sulfo DST (sulfodisuccinimidil tartrate), or DSP (dithiobis (succinimidyl propionate)). The microcarrier as described in claim 1, wherein the soluble polymer comprises cellulose, collagen, gelatin, sodium alginate, chitosan, hyaluronic acid, fruit acid or a combination thereof. The microcarrier as described in claim 1, wherein the temperature-sensitive polymer is poly(N-isopropylacrylamide-co-allylamine) (P(NIPAM-co-ALA)) or a combination thereof. The microcarrier as described in claim 5, wherein the weight percentage of the acrylamine in the poly(N-isopropylacrylamide-co-acrylamine) is 1% to 15%. A microcarrier as described in claim 1, wherein the temperature-sensitive polymer is bonded to the outside of the soluble polymer by the crosslinking agent. A microcarrier as described in claim 1, wherein the temperature-sensitive polymer is physically bound to the dissolving polymer. A method for preparing a soluble temperature-sensitive microcarrier comprises the following steps: providing a soluble polymer; performing a mixing process on the soluble polymer and a crosslinking agent, wherein the soluble polymer and the crosslinking agent are crosslinked when in contact with each other to obtain a soluble microcarrier, wherein the crosslinking agent comprises a zero-length crosslinking agent and a non-zero-length crosslinking agent; providing a temperature-sensitive polymer; and mixing the soluble microcarrier and the temperature-sensitive polymer to obtain the soluble temperature-sensitive microcarrier, wherein the temperature-sensitive polymer comprises a temperature-sensitive The temperature sensitive polymer is formed by polymerizing a hydrophilic monomer; 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; wherein the hydrophilic monomer comprises acrylamine, acrylamide, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonic acid propyl)ammonium hydroxide, diethylaminoethyl methacrylate, hydroxyethyl methacrylate, or a combination thereof. The method as described in claim 9, wherein the soluble polymer comprises cellulose, collagen, gelatin, sodium alginate, chitosan, hyaluronic acid, fruit acid or a combination thereof. The method as described in claim 9, wherein the mixing process comprises microfluidics, titration, electrostatic spinning, emulsification crosslinking, thin film emulsification or a combination thereof. The method as described in claim 9, wherein the step of providing the temperature-sensitive polymer comprises: polymerizing the temperature-sensitive polymer and the hydrophilic monomer by a free radical polymerization method to obtain the temperature-sensitive polymer. The method as described in claim 12 further comprises: providing a reducing crosslinking agent; and mixing the soluble microcarrier, the reducing crosslinking agent and the temperature-sensitive polymer to obtain the soluble temperature-sensitive microcarrier, wherein the temperature-sensitive polymer is bonded to the outside of the soluble polymer by the reducing crosslinking agent. A method for using a soluble thermosensitive microcarrier as described in claim 1, wherein the soluble thermosensitive microcarrier disintegrates when the soluble thermosensitive microcarrier contacts a reducing agent, contacts a lower critical solution temperature (LCST), contacts the reducing agent first and then contacts the lower critical solution temperature, or contacts the lower critical solution temperature first and then contacts the reducing agent. The method of use as described in claim 14, wherein the reducing agent comprises dithiothreitol (DTT), β-mercaptoethanol, glutathione (GSH), cysteine, 2-mercaptoethanol β-mercaptoethanol (β-ME), Tris (2-carboxyethyl) phosphine (TCEP) or a combination thereof.