Photocrosslinked liposome hydrogel microparticles and application thereof in cell co-culture and co-differentiation

Abstract

The application discloses a kind of photo-crosslinking liposome hydrogel microparticles and its application in cell co-culture co-differentiation, belong to cell culture technical field.The application is prepared by film dispersion method by soybean lecithin, DOPE, CHEMS and DSPE-PEG-Mal pH-sensitive liposome, and load rosiglitazone and insulin;Thiol-modified insulin is connected to the surface of liposome by thiol-alkene click reaction;5-norbornene-2,3-dicarboxylic anhydride is used to modify collagen, chitosan and hyaluronic acid respectively, under the action of LAP photo initiator and HS-PEG-SH crosslinking agent, hydrogel microparticle is prepared by microfluidic, and is cured by 405 nm light to form three-dimensional crosslinking network.The microparticle obtained has liposome drug-loading structure and hydrogel skeleton, can realize the wrapping of differentiation factor and pH-responsive release, has good structural stability and biocompatibility, and is suitable for muscle-fat tissue engineering, cell co-culture system construction and functional biomaterial development.

Description

Technical Field

[0001] This invention relates to a photocrosslinked liposome hydrogel microparticle and its application in cell co-culture and co-differentiation, belonging to the field of cell culture technology. Background Technology

[0002] With the rapid development of tissue engineering, biomanufacturing, and cell culture technologies, constructing three-dimensional tissue models with specific structural and functional characteristics in vitro using animal-derived stem cells has become an important research direction in the life sciences and biomedicine fields. Natural muscle tissue is typically composed of highly ordered muscle fibers and adipose tissue distributed within them. The spatial distribution, interaction, and maturation level between muscle cells and adipocytes have a significant impact on tissue function maintenance and microenvironment regulation. Therefore, how to achieve the co-culture of muscle cells and adipocytes in vitro and construct three-dimensional composite tissue structures with spatial heterogeneity and functional characteristics is a key technical problem that urgently needs to be solved in the fields of tissue engineering and biomanufacturing.

[0003] In recent years, modular tissue engineering has provided a new technical strategy for simulating the composition of natural tissues by encapsulating different types of cells in three-dimensional carriers with specific functions and then constructing complex tissue structures through modular assembly. Hydrogel microparticles, due to their good biocompatibility, high water content, adjustable mechanical properties, and excellent cell encapsulation capabilities, have been widely used in three-dimensional cell culture, tissue construction, and delivery of bioactive substances. By constructing hydrogel cell modules with different functions, microenvironments suitable for the growth and differentiation of muscle cells and adipocytes can be provided respectively, and further modular assembly can form composite tissue structures with multi-regional functional characteristics. However, traditional hydrogel materials are usually only used as cell support carriers, lacking the ability to continuously regulate cell behavior and differentiation processes. The inducing factors required during culture mainly rely on exogenous addition, which leads to problems such as insufficient local effective concentration due to diffusion and degradation, and unstable induction effects.

[0004] Liposomes, as biocompatible nanodelivery carriers, can encapsulate different types of functional molecules and achieve the protection and sustained release of active substances by regulating lipid composition. Combining liposomes with hydrogel systems to construct a composite delivery platform enables the local enrichment and time-sequential release of functional molecules, thereby providing a continuously stable microenvironment for cell differentiation. Rosiglitazone, as a peroxisome proliferator-activated receptor gamma (PPARγ) agonist, can promote the differentiation of mesenchymal stem cells into adipocytes, increasing lipid droplet formation and lipid accumulation. Insulin, as an important metabolic regulator, not only participates in adipocyte differentiation and lipid metabolism but also promotes muscle cell proliferation, differentiation, and protein synthesis by activating related signaling pathways such as PI3K / Akt. Therefore, achieving stable delivery of functional molecules related to adipose and muscle differentiation is of great significance for constructing muscle-adipocyte complex tissues.

[0005] Currently, relevant research mainly employs methods such as directly adding inducing factors to the culture system or delivering them via vectors. However, free inducing factors are easily affected by the culture environment, leading to diffusion, degradation, or inactivation, making it difficult to maintain a long-term stable stimulating environment around cells. Traditional liposome delivery systems may also experience initial burst release, resulting in fluctuations in local factor concentrations and affecting the efficiency of cell-directed differentiation. Therefore, developing novel composite material systems that combine stable loading of functional molecules, responsive release, and three-dimensional cell culture is of great significance for improving tissue construction efficiency. Summary of the Invention

[0006] Technical issues: To address the problems in existing cultured meat tissue engineering and muscle-adipose tissue co-culture systems, such as insufficient cell microenvironment regulation, low utilization of inducing factors, difficulty in maintaining stable release processes, and difficulty in modular construction of muscle and adipose tissues, this invention provides a method for preparing and applying photocrosslinked liposome hydrogel microparticles.

[0007] In existing technologies, muscle stem cells and adipose stem cells are typically cultured in two dimensions or directly mixed with three dimensions, making it difficult to simulate the spatial distribution of muscle and adipose tissue in natural marbled beef. Differentiation-inducing factors are mainly added through the culture medium, which suffers from problems such as rapid diffusion, poor stability, the need for frequent medium changes, and difficulty in maintaining local concentrations, resulting in low cell differentiation efficiency and tissue maturity. Furthermore, simply encapsulating bioactive factors within a carrier can easily lead to burst release, making it difficult to form a sustained and stable induction microenvironment, thus limiting its application in tissue engineering and cultured meat construction.

[0008] Therefore, there is an urgent need to develop a novel biomaterial that combines three-dimensional cell culture, continuous delivery of differentiation factors, and modular tissue construction functions to achieve efficient co-culture and directed differentiation of muscle stem cells and adipose stem cells, thereby improving the efficiency of cultured meat tissue construction.

[0009] Technical solution: This invention provides a method for co-culturing muscle cells and adipocytes based on photocrosslinked liposome hydrogel microparticles, comprising the following steps: (1) Soybean lecithin (SPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanol (DOPE), cholesterol hemisuccinate (CHEMS), maleimide polyethylene glycol distearate phosphatidylethanolamine (DSPE-PEG-Mal), rosiglitazone (Ros) and solvent were mixed, and then the solvent was removed to obtain Ros@Mal-SDCD lipid membrane; the Ros@Mal-SDCD lipid membrane was then hydrated by mixing it with an aqueous solution of PBS containing insulin, and then the hydrated mixture was repeatedly extruded using a polycarbonate membrane to obtain Ros / In membranes with uniform size and encapsulated rosiglitazone and insulin. Ros / Ins@Mal-SDCD liposomes were dispersed in PBS buffer, and thiol-modified insulin (Ins-SH) was added. The mixture was subjected to a light-protected reaction to obtain surface-coupled insulin liposomes (Ins-Lipo). Finally, borneol-modified chitosan, borneol-modified hyaluronic acid, borneol-modified collagen (Nor-Col), insulin-modified liposomes (Ins-Lipo), a cross-linking agent, a photoinitiator, and deionized water were mixed to form a precursor solution. This precursor solution was then used in a microfluidic manner to form droplets, which were then subjected to photocrosslinking and curing treatment to finally obtain liposome-composite hydrogel microparticles. (2) Adipose stem cells and muscle stem cells were seeded onto the surface of liposome composite hydrogel microparticles, then mixed and assembled. A three-dimensional culture microenvironment was gradually constructed through inter-microparticle interface binding and extracellular matrix secreted by the cells themselves for cell co-culture.

[0010] In one embodiment of the present invention, the specific process of inoculation in step (2) is as follows: After sterilization, liposome-based hydrogel microparticles were pre-immersed in complete culture medium and pre-equilibrated at 37 °C and 5% CO2 for 12–24 h. Subsequently, adipose-derived stem cells or muscle stem cells were suspended in complete culture medium, and the cell concentration was adjusted to 1 × 10⁻⁶ cells / mL. 6 Cells / mL were used to obtain a cell suspension; each 1 mL of cell suspension was mixed with 200-800 hydrogel microparticles with a diameter of 100-500 μm to control the cell seeding density at 1 × 10⁻⁶ cells / mL. 5Cells / cm² were placed on the surface of a hydrophilically treated culture medium; then the culture plate was placed in a 37 °C, 5% CO2 incubator and left to stand for about 2 h to allow the cells to adhere to the surface of the hydrogel particles.

[0011] In one embodiment of the present invention, in step (2), adipose stem cells are seeded on the surface of liposome composite hydrogel microparticles. When the liposome composite hydrogel microparticles are prepared, the mass ratio of borneol-modified chitosan to borneol-modified hyaluronic acid in the precursor solution is controlled to be 2:6.

[0012] In one embodiment of the present invention, in step (2), muscle stem cells are seeded onto the surface of liposome composite hydrogel microparticles. When the liposome composite hydrogel microparticles are prepared, the mass ratio of borneol-modified chitosan to borneol-modified hyaluronic acid in the precursor solution is controlled to be 4:4.

[0013] In one embodiment of the present invention, during step (2), the ratio of the number of hydrogel microparticle modules seeded with adipose stem cells to the number of hydrogel microparticle modules seeded with muscle stem cells is 1:2 to 2:1. Specifically, 1:2, 1:1, or 2:1 can be selected.

[0014] In one embodiment of the present invention, the method for preparing the liposome-composite hydrogel microparticles in step (1) combines pH-responsive dual-drug-carrying liposomes with thiol-norbornene photo-click hydrogels to achieve synergistic delivery of rosiglitazone (Ros) and insulin (Ins), and constructs a modular three-dimensional culture system suitable for cultured meat tissue engineering; specifically including the following steps: S1. Preparation of liposome membrane: Soybean lecithin (SPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanol (DOPE), cholesterol hemisuccinate (CHEMS), maleimide polyethylene glycol distearate phosphatidylethanolamine (DSPE-PEG-Mal), and rosiglitazone (Ros) were dissolved in chloroform solution in a certain proportion and mixed well. Then, the solvent was evaporated under vacuum at 40°C until dry to obtain Ros@Mal-SDCD lipid membrane. S2. Synthesis of liposomes: Add PBS aqueous solution containing insulin to the dried Ros@Mal-SDCD liposome membrane, hydrate and sonicate for 10 min, and then repeatedly extrude the above hydrate with polycarbonate membrane to obtain Ros / Ins@Mal-SDCD liposomes with uniform size and encapsulated rosiglitazone and insulin. S3. Thiol modification of insulin: Insulin was dissolved in PBS buffer (pH 7.4), and an appropriate amount of Traut's reagent (2-iminothione hydrochloride) was added. The mixture was reacted at 4°C in the dark for 60 min at a specific molar ratio to convert the amino group in the insulin molecule to a thiol group. After the reaction, unreacted reagent was removed by dialysis to obtain thiol-modified insulin (Ins-SH).

[0015] S4. Insulin conjugation on liposomes: Ros / Ins@Mal-SDCD liposomes were dispersed in PBS buffer, and thiol-modified insulin (Ins-SH) was added. The mixture was prepared at a predetermined molar ratio and reacted in the dark at room temperature or 4 °C for 2 h. During the reaction, the thiol groups on the Ins-SH surface reacted with the maleimide groups on the liposome surface via a thiol-maleimide click reaction, forming stable thioether bonds and achieving covalent conjugation of insulin to the liposome surface. After the reaction, unconjugated insulin was removed by ultrafiltration to obtain insulin-conjugated liposomes (Ins-Lipo).

[0016] S5. Norbornene Modification of Sodium Hyaluronate, Chitosan, and Collagen: Sodium hyaluronate was dissolved in 0.05 M acetic acid solution and stirred until completely dissolved at room temperature. Collagen was also dissolved in 0.05 M acetic acid solution and dissolved completely at 4 °C. Chitosan was dissolved in 0.05 M acetic acid solution, precipitated with 1 M NaOH, washed several times with water until neutral, freeze-dried, and ground into powder. Subsequently, 5-norbornene-2,3-dicarboxylic anhydride was dissolved in DMF and added dropwise to the polysaccharide solution under vigorous stirring. The reaction was carried out at 4 °C for 3 hours in the dark. After the reaction, sodium hyaluronate and chitosan were recovered by precipitation with ethanol / DMF (1:1, volume ratio), while collagen was directly dialyzed. The resulting product was then dialyzed in deionized water or decreasing concentration NaCl solution for 2-3 days, with the dialysate changed several times daily. The purified solutions were eventually lyophilized and stored at -20 °C (sodium hyaluronate and chitosan) or 4 °C (collagen), respectively.

[0017] S6. Synthesis of liposome hydrogel microparticles: Solutions of borneol-modified chitosan (Nor-CS) and borneol-modified hyaluronic acid (Nor-HA) with different final concentrations were prepared. Then, HS-PEG-SH as a crosslinking agent, borneol-modified collagen (Nor-Col), insulin-modified liposomes (Ins-Lipo), and photoinitiator LAP were added to each solution, and thoroughly mixed to obtain a homogeneous hydrogel precursor solution. Eight samples were prepared according to different Nor-CS to Nor-HA ratios, designated as Lipo-CS4, Lipo-CS2, Lipo-C4H2, Lipo-C4H4, Lipo-C2H6, Lipo-C2H4, Lipo-C2H2, and Lipo-HA6, with the concentrations of other components remaining consistent. Subsequently, each group of precursor liquids was injected into the microfluidic chip as a dispersed phase. Under the shearing action of the continuous phase, droplets of uniform size were formed. At the chip outlet, a 405 nm light-induced thiol-norbornene click reaction was carried out to achieve rapid in-situ cross-linking and curing, ultimately obtaining liposome composite hydrogel microparticles with a particle size of approximately 100-500 μm, uniform particle size distribution, and good sphericity.

[0018] In one embodiment of the present invention, in S1, the total concentration of the four fatty acids SPC:DOPE:CHEMS:DSPE-PEG-Mal relative to the solvent is 10-20 mg / mL.

[0019] In one embodiment of the present invention, in S1, the feed ratio (molar ratio) of fatty acids is SPC:DOPE:CHEMS:DSPE-PEG-Mal = 4:3:2:1. The total proportion of pH-sensitive fatty acids DOPE and CHEMS is 50%.

[0020] In one embodiment of the present invention, in S1, the mass ratio of rosiglitazone to the total fatty acids is 10-20:1. Specifically, 10:1, 15:1, and 20:1 can be selected.

[0021] In one embodiment of the present invention, in step S2, the mass ratio of insulin to total fatty acids is 20-40:1. Specifically, 20:1, 30:1, and 40:1 can be selected.

[0022] In one embodiment of the present invention, in the thiol modification of insulin described in S3, the molar ratio of insulin to Traut's reagent (2-iminothione hydrochloride) is 1:10.

[0023] In one embodiment of the present invention, the molar ratio of thioglycolic insulin to maleimide in S4 is 1:2.

[0024] In one embodiment of the present invention, in step S5, 5-norbornene-2,3-dicarboxylic anhydride is dissolved in DMF and added dropwise to an acetic acid solution of sodium hyaluronate, chitosan, or collagen under vigorous stirring, while maintaining the pH of the reaction solution at 9; the reaction is carried out at low temperature (4-10 °C) under light-protected conditions for 1-3 hours; after the reaction is completed, sodium hyaluronate and chitosan are recovered by ethanol / DMF precipitation, while collagen is directly dialyzed; subsequently, the obtained products are dialyzed in deionized water, with the dialysate changed multiple times, and the purified solution is finally lyophilized to obtain norbornene-modified sodium hyaluronate (Nor-HA), norbornene-modified chitosan (Nor-CS), and norbornene-modified collagen (Nor-Col), respectively.

[0025] In one embodiment of the present invention, the norbornene-modified collagen described in S5 is prepared by the following method: .

[0026] In one embodiment of the present invention, the norbornene modification of hyaluronic acid described in S5 is prepared by the following method: .

[0027] In one embodiment of the present invention, the chitosan modified with norbornene in step S5 is prepared by the following method: .

[0028] In one embodiment of the present invention, the molar ratio of the polysaccharide reactive group in the norbornene modification described in S5 to 5-norbornene-2,3-dicarboxylic anhydride is 1:2.

[0029] In one embodiment of the present invention, HS-PEG-SH is used as a crosslinking agent in the synthesis of the liposome hydrogel described in S6, and the concentration is 10 mM.

[0030] In one embodiment of the present invention, in S6, the amount of borneol-modified collagen (Nor-Col) is 0.5 wt.%.

[0031] In one embodiment of the present invention, in S6, the amount of insulin-modified liposomes (Ins-Lipo) is 1 wt.

[0032] In one embodiment of the present invention, in S6, the photoinitiator is LAP, and the amount used is 0.1 wt.%.

[0033] The present invention also provides a three-dimensional culture microenvironment for cell co-culture and co-differentiation obtained by the above method.

[0034] This invention constructs a liposome-hydrogel microparticle system, combining functional liposomes with a hydrogel network possessing tunable mechanical properties. This system enables liposomes to perform functions such as three-dimensional cell encapsulation, microenvironment regulation, and sustained release of bioactive molecules. During culture, the liposomes respond to changes in the microenvironment caused by cellular metabolism, undergoing structural regulation and releasing loaded functional molecules. Simultaneously, the hydrogel network gradually degrades and releases relevant regulatory factors, thereby providing continuous biochemical stimulation to different cell types. Furthermore, by adjusting the proportions of hydrogel components, cell culture modules with different mechanical characteristics can be constructed, and further, through modular assembly, spatial co-culture and directed differentiation of muscle cells and adipocytes can be achieved.

[0035] This invention provides a multi-cell co-culture and tissue construction system based on liposome-composite hydrogel microparticles, which can be used to construct three-dimensional tissue models with muscle-fat composite structures. This system can be applied not only to soft tissue engineering, muscle injury repair, adipose tissue regeneration, tissue model construction, and drug evaluation, but also to providing new technical solutions for the construction of cell-cultured meat and biomimetic food structures in the future.

[0036] This invention also provides the application of the above-mentioned three-dimensional culture microrings in the fields of muscle-fat tissue engineering and biomanufacturing.

[0037] This invention also provides the application of the above-mentioned three-dimensional culture microrings in the fields of regenerative medicine and the development of functional biomaterials.

[0038] The beneficial effects achieved by this invention are as follows: (1) The present invention uses liposomes and natural polymer hydrogels to construct photocrosslinked hydrogel microparticles, which have the good drug loading performance of liposomes and the three-dimensional support structure of hydrogels, which can provide a suitable three-dimensional culture microenvironment for cells and improve the stability of active factors.

[0039] (2) The present invention utilizes pH-responsive liposomes to load rosiglitazone and insulin, and combines them with a hydrogel network to form a composite delivery system, which can achieve local sustained release and continuous release of differentiation factors, reduce the rapid diffusion of active factors, improve induction efficiency, and help maintain the microenvironment required for cell differentiation.

[0040] (3) In this invention, thiol-modified insulin is covalently linked to the surface of liposomes via a thiol-maleimide click reaction, thereby stabilizing insulin on the liposome carrier, improving its local enrichment capacity and stability, which is beneficial to promoting the continuous differentiation of muscle stem cells and adipose stem cells.

[0041] (4) The present invention uses microfluidics combined with photocrosslinking technology to prepare hydrogel microparticles with uniform size. The preparation process is mild and controllable. The resulting microparticles have good morphological uniformity, structural stability and biocompatibility, which facilitates cell embedding and subsequent modular assembly.

[0042] (5) The present invention uses hydrogel microparticles to construct muscle stem cell modules and adipose stem cell modules respectively, realizing modular co-culture of muscle tissue and adipose tissue. This is beneficial to simulating the tissue structure of muscle and fat interspersed in natural marbled beef. It can be applied to fields such as cultured meat tissue engineering, muscle-fat tissue construction, cell co-culture system and development of functional biomaterials. Attached Figure Description

[0043] Figure 1 This is a flowchart illustrating the preparation process of the photocrosslinked liposome hydrogel microparticles of the present invention; Figure 2 MALDI-TOF characterization of thioglycolic insulin: Figure 3 Characterization of insulin-modified liposomes: electron micrographs of Mal-SDCD (A) and @Ins-SDCD (B), particle size distribution (C) and infrared spectral characterization (D); Figure 4 Characterization of liposome synthesis: MEL-SDCD (A), Ros / Ins@Ins-SDCD (B) electron microscopy images, particle size distribution (C), zeta potential distribution (D), UV spectrum (E) and IR spectrum (F); Figure 5 In vitro release diagram of Ros / Ins@Ins-SDCD liposomes: (A) Ros, (B) Ins; Figure 6 Infrared spectra (A) and 1H NMR spectra (B) of hyaluronic acid before and after norbornene modification. Figure 7 The infrared spectrum (A) and the 1H NMR spectrum (B) of collagen before and after modification with borneol. Figure 8 Infrared spectra (A) and 1H NMR spectra (B) of chitosan before and after norbornene modification. Figure 9 Microscopic observation of liposome hydrogel microparticles; where A, B, C, and D correspond to microscopic observation of Lipo-C4H2, Lipo-C4H4, Lipo-C2H6, and Lipo-C2H4 liposome hydrogel microparticles, respectively. Figure 10 Microscopic observations of C2C12 muscle cells induced by liposome Ros / Ins@Ins-SDCD on days 1 (A), 3 (B), and 5 (C); Figure 11 Microscopic observations of 3T3L1 adipocytes on days 1 (A), 3 (B), and 5 (C) after liposome Ros / Ins@Ins-SDCD induction; Figure 12Live and dead staining observation of muscle cells C2C12 (A) and adipocytes 3T3L1 (B) cultured in Ros / Ins@Ins-SDCD liposome hydrogel microparticles.

[0044] Figure 13 Optical micrographs of bovine skeletal muscle satellite cells (bMuSCs) cultured in Lipo-C4H4 (A) and adipose-derived stem cells (bMuCFs) cultured in Lipo-C2H6 hydrogel (B) 14 days after differentiation; Figure 14 BODIPY fluorescence confocal microscopy image (A) and laser confocal 3D reconstruction image (B) of adipose-derived stem cells (bMuCFs) cultured in Lipo-C2H6 hydrogel after 14 days of differentiation; macroscopic assembly image (C) and magnified image (D) of bovine skeletal muscle satellite cells (bMuSCs) cultured in Lipo-C4H4 and adipose-derived stem cells (bMuCFs) cultured in Lipo-C2H6 hydrogel after 14 days of differentiation. Detailed Implementation

[0045] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0046] Example 1: Preparation of Ros / Ins@Mal-SDCD liposomes Soybean lecithin (SPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), cholesterol hemisuccinate (CHEMS), and maleimide polyethylene glycol distearate phosphatidylethanolamine (DSPE-PEG2000-Mal) were weighed in a molar ratio of 4:3:2:1, dissolved thoroughly in chloroform, and mixed well (total concentration of the four fatty acids was 10 mg / mL). Rosiglitazone (Ros) was then thoroughly mixed at a mass ratio of 20:1 to the total amount of the four fatty acids. The resulting mixture was evaporated under reduced pressure at 40 °C using a rotary evaporator to remove the organic solvent, forming a homogeneous lipid film. This film was then further dried under vacuum to constant weight to obtain the Ros@Mal-SDCD lipid membrane. The obtained lipid membrane was hydrated by adding 1 mg / mL insulin 0.1×PBS buffer to achieve a lipid concentration of 10 mg / mL. The membrane was then sonicated for 3 min to promote thorough dispersion, forming a liposome suspension. Liposomes were extruded through a 0.22 µm polycarbonate membrane at least six times and subjected to multiple ultrafiltrations (MWCO 10 kDa) at room temperature to remove free Ros and Ins. The final Ros / Ins@Mal-SDCD liposomes were collected and stored at 4°C for later use.

[0047] In addition, the following reference standards were synthesized: Mal-SDCD blank liposomes: Soybean lecithin (SPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), cholesterol hemisuccinate (CHEMS), and maleimide polyethylene glycol distearate phosphatidylethanolamine (DSPE-PEG-Mal) were weighed in a molar ratio of 4:3:2:1, dissolved thoroughly in an appropriate amount of chloroform, and mixed well. The resulting mixture was then evaporated under reduced pressure at 40 °C using a rotary evaporator to remove the organic solvent, forming a uniform lipid film. This film was further dried under vacuum to constant weight to obtain the Mal-SDCD lipid film.

[0048] The obtained lipid membrane was then hydrated with preheated 0.1×PBS buffer (pH 7.4) to room temperature to achieve a lipid concentration of 10 mg / mL, and hydrated for 30 min under suitable conditions. Following this, sonication was performed for 3 min to promote thorough dispersion of the lipid membrane and the formation of a homogeneous liposome suspension. The resulting liposome suspension was continuously extruded through a 0.22 μm polycarbonate membrane at least six times to obtain Mal-SDCD blank liposomes with uniform particle size distribution. Ultrafiltration centrifugation (MWCO 10 kDa) was then used to remove any possible small molecule impurities, finally obtaining Mal-SDCD blank liposomes, which were stored at 4 °C for later use.

[0049] Example 2: Synthesis of Thio-Insulin Insulin was dissolved in 0.01 M HCl (concentration 1 mg / mL), and the pH was adjusted to 7.6 by adding 0.1 M NaOH to prepare an insulin solution. Traut's Reagent (2-iminothiolane) was added to the insulin solution at a molar ratio of insulin:Traut's Reagent = 1:10. The reaction was carried out at 4°C for 1-2 h under light-protected conditions to introduce thiol groups. After the reaction, excess Traut's reagent was removed by dialysis, and the thiolized insulin was obtained by lyophilization at -80°C.

[0050] MALDI-TOF mass spectrometry analysis showed that insulin successfully incorporated thiol groups after modification with Traut's reagent. Characteristic peaks corresponding to single-thiol modification (Ins-1SH) and double-thiol modification (Ins-2SH) were observed in the mass spectrum, indicating that Traut's reagent can effectively thiolize insulin. Based on the relative abundance of each mass spectrometry peak, the average number of thiol modifications in insulin was approximately 0.9 SH / insulin, indicating that most insulin molecules successfully incorporated about one thiol group, providing sufficient reaction sites for subsequent thiol-maleimide click coupling with maleimide-modified liposomes.

[0051] Example 3: Insulin Modification of Liposomes Because insulin is a hydrophilic protein, its encapsulation efficiency in liposomes is relatively low, and insulin is also expensive. Relying solely on liposome encapsulation often requires increasing the insulin dosage to achieve the desired induction effect. Therefore, this invention employs a dual delivery strategy combining in vivo insulin encapsulation with surface modification to achieve synergistic regulation of sustained release and local presentation. The insulin modification process in liposomes specifically includes: Thiol-modified insulin was slowly added to the maleimide-modified fully encapsulated liposomes Ros / Ins@Mal-SDCD and the blank liposome Mal-SDCD prepared in Example 1, respectively, controlling the molar ratio of insulin to maleimide structure in the liposomes to be 1:2. Then, 0.1% (w / v, g / mL) LAP was added, and the mixture was irradiated at 405 nm for 60 s. After the reaction was complete, free insulin was removed by gel filtration (Bio-Gel P-60), and the insulin-coupled liposomes were collected. After ultrafiltration at 4°C, surface-coupled insulin liposomes (Ros / Ins@Ins-SDCD (abbreviated as Ins-Lipo) and @Ins-SDCD) were finally obtained.

[0052] Transmission electron microscopy (TEM) results showed that both the blank liposome Mal-SDCD and insulin-coupled liposomes (@Ins-SDCD) exhibited regular spherical vesicle structures with good dispersibility. Compared with uncoupled liposomes, Ros / Ins@Ins-SDCD maintained a complete spherical vesicle structure, with a clearer lipid bilayer outline and a relatively uniform coating layer observed at the vesicle edge, indicating successful insulin modification of the liposome surface. Dynamic light scattering (DLS) results showed that the average particle size of the liposomes increased slightly from approximately 197 nm to 212 nm after insulin coupling, while the polydispersity index (PDI) remained below 0.20, indicating that the coupling process did not cause significant liposome aggregation and the system maintained good dispersion stability. Fourier transform infrared spectroscopy (FT-IR) results showed that, compared with INS, both Ins-SH and @Ins-SDCD retained the characteristic absorption peak of insulin, with the peak at 1655 cm⁻¹. - ¹ and 1544 cm - ¹ These correspond to the amide I band (C=O stretching vibration) and amide II band (N–H bending vibration) of proteins, respectively, indicating that the basic structure of insulin is maintained during modification and coupling. Furthermore, @InsSDC at 1737 cm⁻¹ - The characteristic peak of C=O stretching vibration appears at ¹, at 1083 cm⁻¹. - ¹ and 1051 cm - A COC / CS-related absorption peak appears at ¹, and at 654 cm⁻¹ - ¹The characteristic absorption of CS bonds can be observed near the surface, indicating that the thiol group and the maleimide group undergo a coupling reaction to form a thioether bond, further confirming that insulin has been successfully covalently modified on the carrier surface.

[0053] Example 4: Characterization of Ros / Ins@Ins-SDCD liposomes The average particle size, particle size distribution index (PDI), and zeta potential of the Mal-SDCD blank liposomes, @Ins-SDCD liposomes, and Ros / Ins@Ins-SDCD liposomes prepared in Example 1 were determined using dynamic light scattering (DLS). The synthesized liposomes were characterized using Fourier transform infrared spectroscopy (FTIR) to verify the successful encapsulation of rosiglitazone and insulin within the liposomes.

[0054] Results showed that: DLS particle size and PDI analysis revealed that all liposomes exhibited a unimodal distribution and relatively concentrated particle size distribution, indicating good uniformity of the prepared liposomes. Compared with the Mal-SDCD blank liposomes, the average particle size of @Ins-SDCD liposomes and Ros / Ins@Ins-SDCD liposomes gradually increased. This is because insulin and rosiglitazone were successfully encapsulated inside the liposomes or inserted into the lipid bilayer, increasing the hydrodynamic particle size of the liposomes. The PDI of all liposomes was at a low level (generally <0.20), indicating that the liposomes were uniformly dispersed, had a narrow particle size distribution, and exhibited good reproducibility and colloidal stability. Zeta potential analysis showed that all liposomes exhibited a negative potential. The Mal-SDCD blank liposomes had a certain negative charge, and the absolute value of the Zeta potential decreased after loading insulin, indicating that insulin was successfully adsorbed or encapsulated on the surface and inside the liposomes, altering the charge distribution on the liposome surface. After further encapsulation with rosiglitazone, the absolute value of the Zeta potential of the Ros / Ins@Ins-SDCD liposomes changed again, but the overall potential remained high (generally >20 mV), indicating that the system has good electrostatic repulsion, which can effectively reduce liposome aggregation and improve system stability. FTIR structural analysis: the blank liposomes mainly showed the characteristic absorption peaks of lipid materials. The insulin sample showed a typical amide I band (approximately 1650 cm⁻¹). - ¹) and amide II band (approximately 1540 cm) - ¹), rosiglitazone exhibits its characteristic functional group absorption peaks. When prepared as Ros / Ins@Ins-SDCD liposomes, characteristic peaks of insulin and rosiglitazone were observed in the liposome spectrum. Some peak positions showed slight shifts or decreased peak intensity, but no new characteristic peaks appeared. This indicates that the drugs are mainly bound to the liposomes through physical encapsulation and intermolecular interactions (such as hydrogen bonds and hydrophobic interactions), without any new chemical reactions. This proves that both insulin and rosiglitazone have been successfully encapsulated in the liposome system.

[0055] Example 5: Transmission electron microscopy sample preparation of Ros / Ins@Ins-SDCD liposomes The liposomes (1 mg / mL) from Example 1 were picked up with an inoculation loop and dropped onto a carbon membrane copper mesh (200 mesh). After standing for 10 min and allowing to dry, 5 μL of uranium phosphate staining agent (2%) was quickly added. After standing for 30-60 s, the staining agent was blotted dry with filter paper and dried at room temperature for 10 min.

[0056] The results showed that the Mal-SDCD blank liposomes prepared in Example 1 had a regular spherical or near-spherical structure with clear edges, intact membrane, and relatively uniform particle size distribution, and no obvious aggregation was observed. After loading insulin and rosiglitazone (Ros / Ins@Ins-SDCD liposomes), the liposomes still maintained an intact spherical vesicle structure, clear membrane, and no significant change in morphology, only a slight increase in particle size, indicating that drug loading did not destroy the liposome structure and that the liposomes had good morphological stability.

[0057] Example 6: Detection of Wrap-up and Loading Rate of Ros / Ins@Ins-SDCD Standard curves for rosiglitazone (Ros) and insulin (Ins) were established using high-performance liquid chromatography (HPLC). Standard solutions of different concentrations of Ros and Ins were prepared, and peak areas were measured under optimized chromatographic conditions. Standard curves were plotted with drug concentration on the x-axis and peak area on the y-axis. After liposome preparation, free drug and drug-loaded liposomes were separated by ultrafiltration centrifugation. The filtrate was collected, and the contents of unencapsulated Ros and Ins were determined. The encapsulation efficiency (EE) and loading efficiency (LE) of the differentiation factors were calculated using the standard curves. The calculation formulas are as follows: EE (%) = (Total input - Free input) / Total input × 100%; LE (%) = Encapsulation amount / Total mass of loaded liposomes × 100%.

[0058] The specific tests are shown in Table 1.

[0059] Table 1

[0060] The results showed that, compared with the single-encapsulation system, Ros@Mal-SDCD exhibited higher encapsulation and loading efficiencies for rosiglitazone (encapsulated in a hydrophobic bilayer), reaching 89.6 ± 1.4% and 4.25 ± 0.06%, respectively. For insulin (encapsulated within a hydrophilic core), Ins@Mal-SDCD showed an encapsulation efficiency of 56.6 ± 3.8% and a loading efficiency of 1.48 ± 0.32%. In contrast, rosiglitazone exhibited higher encapsulation and loading levels, which may be related to its physicochemical properties and its interaction with the liposome's internal / membrane structure.

[0061] After further constructing the rhodamine and insulin co-encapsulation system, the encapsulation efficiency and loading efficiency of rosiglitazone in Ros / Ins@Ins-SDCD were 85.4 ± 3.8% and 4.15 ± 0.35%, respectively, while the encapsulation efficiency and loading efficiency of insulin were 54.2 ± 2.7% and 1.31 ± 0.39%, respectively. Compared with the corresponding single encapsulation systems, the encapsulation efficiency and loading efficiency of both rosiglitazone and insulin decreased to some extent after co-encapsulation, but remained at a high level overall, indicating that the two active molecules can be effectively co-encapsulated in the Mal-SDCD liposome system.

[0062] Example 7: Releasing Ros and Ins from Ros / Ins@Ins-SDCD One mL of Ros / Ins@Ins-SDCD liposome suspension was placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa and then in 20 mL of PBS buffer (pH 6.8). In vitro release experiments were conducted under constant temperature shaking at 37 °C and 100 rpm. PBS buffer (pH 7.4) served as a control group. At predetermined time points (0, 2, 4, 8, 12, 24, 36, and 48 h), 1 mL of release medium was collected, and an equal volume of preheated fresh PBS buffer was immediately added to maintain a constant release volume. The concentrations of Ros and Ins in the release medium were determined by high-performance liquid chromatography (HPLC), and the cumulative release rate was calculated based on a standard curve. A 48-h in vitro release curve was plotted.

[0063] The results showed that Ros / Ins@Ins-SDCD liposomes exhibited significant sustained-release characteristics under pH 6.8 conditions. In the initial release phase (0-12 h), the release rates of Ros and Ins were relatively rapid, mainly attributed to drug adsorption on the liposome surface and rapid diffusion of the superficially loaded drug. Subsequently, the release rate gradually decreased, entering a sustained and stable release phase. Within 48 h, both Ros and Ins maintained a continuous release trend, with Ros's cumulative release rate at approximately 80% and Ins's at approximately 65%. Compared to the cumulative release rates of approximately 35% for Ros and 20% for Ins under neutral conditions (pH 7.4), the weakly acidic environment promoted the loosening and rearrangement of the liposome membrane structure, significantly improving the release efficiency of Ros and Ins, which is beneficial for the sustained release of drugs in the inflammatory microenvironment and the exertion of local therapeutic effects.

[0064] Example 8: Norbornene Modification with Sodium Hyaluronate, Chitosan, and Collagen Preparation of three second-component solutions: Sodium hyaluronate was dissolved in 0.05 M acetic acid solution and stirred until completely dissolved at room temperature to prepare a 5% (w / v, 50 mg / mL) sodium hyaluronate solution. Collagen (0.3 w / v%) was also dissolved in 0.05 M acetic acid solution and dissolved completely at 4 °C to obtain a collagen solution. Chitosan was dissolved in 0.05 M acetic acid solution, precipitated with 1 M NaOH, washed several times with water until neutral, freeze-dried, and ground into powder; subsequently dissolved in 0.05 M acetic acid solution and stirred until completely dissolved to prepare a 5% (w / v, 50 mg / mL) chitosan solution.

[0065] Adjust the pH of the sodium hyaluronate solution, collagen solution, and chitosan solution to 9.

[0066] 5-Norbornene-2,3-dicarboxylic anhydride was dissolved in DMF (10 mg / mL) and added dropwise to the second component solution prepared above under vigorous stirring, while maintaining the pH of the reaction solution at 9 with NaOH. The reaction was carried out at low temperature (4-10 °C) under light-protected conditions for 1-3 hours. After the reaction, sodium hyaluronate and chitosan were recovered by precipitation with ethanol / DMF (1:1, volume ratio), while collagen was directly dialyzed. Subsequently, the obtained products were dialyzed in deionized water for 3 days, with the dialysate changed several times a day. The purified solution was finally lyophilized to prepare norbornene-modified sodium hyaluronate (Nor-HA), norbornene-modified chitosan (Nor-CS), and norbornene-modified collagen (Nor-Col), respectively. They were stored at -20 °C (Nor-HA and Nor-CS) or 4 °C (Nor-Col), respectively.

[0067] Nor-HA modification results: FT-IR results show that, compared with HA, Nor-HA at 1774 cm⁻¹... -1 A new characteristic absorption peak for anhydride groups (-CO-O-CO-) appears nearby, along with a peak at 1600 cm⁻¹. -1 Nearby carboxylates (-COO) - The weakening of the characteristic absorption peak intensity indicates that the carboxyl group of HA is involved in the coupling reaction. Furthermore, at 880 cm⁻¹... -1 The presence of the CH out-of-plane bending vibration absorption peak characteristic of the bornene structure further proves that the bornene group has been successfully grafted onto the HA molecular chain. 11H NMR spectra further confirmed the successful synthesis of Nor-HA. The characteristic peak of the sugar ring protons in HA is located at δ 3.0-5.0 ppm. After modification, the chemical environment of the adjacent sugar ring protons is changed due to the formation of amide bonds, resulting in a slight signal shift accompanied by peak broadening. At the same time, the characteristic resonance peak of the borneol double bond protons appears at δ 6.0-6.5 ppm. This peak is not present in unmodified HA, providing direct evidence for the successful grafting of borneol.

[0068] Nor-CS modification results: FT-IR results show that, compared with unmodified CS, Nor-CS at 1760 cm⁻¹... -1 A new characteristic absorption peak for an anhydride group (-CO-O-CO-) appears nearby; at 1689 cm⁻¹ -1 The characteristic peak of amide I band (C=O stretching vibration) appears at 1543 cm⁻¹. -1 The significantly enhanced absorption of the amide II band (NH bending vibration) indicates that the carboxyl group of borneol undergoes an amidation reaction with the amino group of CS, successfully forming an amide bond and achieving covalent modification of CS by the borneol group. 1 1H NMR spectra further confirmed the successful synthesis of Nor-CS. The characteristic signal of the sugar ring protons in CS is located at δ 3.0-5.0 ppm. After modification, due to the formation of amide bonds, the chemical environment of the protons adjacent to the sugar ring changes, resulting in a slight signal shift accompanied by peak broadening. Simultaneously, a characteristic resonance peak of the bornene ring double bond protons appears at δ 6.0-7.0 ppm, a signal absent in unmodified CS, further demonstrating that the bornene group has been successfully grafted onto the CS molecular chain.

[0069] Nor-Col modification results: FT-IR results showed that, compared with unmodified collagen, Nor-Col showed a higher concentration at 1780 cm⁻¹. -1 A new characteristic absorption peak for an anhydride group (-CO-O-CO-) appears nearby; at 1080 cm⁻¹ -1 and 1046 cm -1 The absorption peaks of COC and CO stretching vibrations are significantly enhanced at 880 cm⁻¹. -1 The presence of the C–H out-of-plane bending vibration absorption peak characteristic of the borneol structure indicates that the borneol group has been successfully introduced into the collagen molecular chain. 1 The 1H NMR spectrum further confirmed the successful preparation of Nor-Col. Compared with unmodified collagen, Nor-Col exhibited a characteristic resonance peak of the bornene double bond proton at δ 6.0-6.5 ppm, a signal absent in unmodified collagen, providing direct evidence for the successful grafting of the bornene group.

[0070] Example 9: Synthesis of liposome hydrogels Mixed solutions containing different final concentrations of borneol-modified chitosan (Nor-CS) and borneol-modified hyaluronic acid (Nor-HA) were prepared. Then, 10 mM HS-PEG-SH as a crosslinking agent, 0.5 wt.% borneol-modified collagen (Nor-Col), 1 wt.% insulin-modified liposomes (Ros / Ins@Ins-SDCD), and 0.1 wt.% photoinitiator LAP were added to the mixed solutions, and the mixtures were thoroughly mixed to obtain a homogeneous hydrogel precursor solution. Based on the different ratios of Nor-CS to Nor-HA in the precursor solution (as shown in Table 1), eight groups of samples were prepared, designated as Lipo-CS4, Lipo-CS2, Lipo-C4H2, Lipo-C4H4, Lipo-C2H6, Lipo-C2H4, Lipo-C2H2, and Lipo-HA6, with the concentrations of the remaining components remaining consistent. Subsequently, the precursor solutions were injected into the microfluidic chip as the dispersed phase. Under the shearing action of the continuous phase (98% mineral oil and 2% Span 80), uniformly sized droplets were formed. At the chip outlet, a 405 nm light-induced thiol-norbornene click reaction was carried out to achieve rapid in-situ cross-linking and solidification, ultimately yielding liposome composite hydrogel microparticles with a particle size of approximately 100-500 μm, uniform particle size distribution, and good sphericity. Specific product information is shown in Table 2.

[0071] Table 2

[0072] Optical microscope ( Figure 9 The results showed that groups 3-6 all formed microspheres with regular morphology and good sphericity, uniform particle size distribution, and no obvious adhesion or aggregation between microspheres. Furthermore, the shear rate could be adjusted to form hydrogel microspheres of different diameters, indicating that microfluidics can effectively control the particle size of liposome-composite hydrogel microparticles, ultimately obtaining spherical microparticles with uniform size and diameters of approximately 100-500 μm.

[0073] The obtained hydrogel microparticles (100-500 μm) were added to an equal volume of 1% (v / v) Span 80 solution. The mixture was gently shaken to fully demulsify the oil phase. After centrifugation to collect the hydrogel microparticles, they were washed 2-3 times with 1×PBS to remove residual oil phase and surfactants. The microparticles were then resuspended in 1×PBS or complete culture medium to prepare hydrogel microparticle suspensions for later use. The dispersion concentration of the obtained hydrogel microparticle suspensions of different sizes was controlled to approximately 1000 microparticles / mL.

[0074] Example 10: Mechanical testing of liposome hydrogels Uniaxial compression tests were performed on the hydrogel using an Instron mechanical testing frame (model 5565A). Stress was determined based on the force curve. The calculated values, F and A0, represent the forces used to compress the sample and the initial area of ​​the sample. The modulus of the gel is obtained through G(t) = The results were calculated. Samples were tested at least three times. Before testing, carefully inspect the hydrogel for cracks or deformation. Align the gel centered on a stainless steel compression plate.

[0075] The results showed that the compressive modulus of hydrogels with different ratios differed significantly. As the Nor-CS content decreased and the Nor-HA content increased, the compressive modulus of the hydrogels gradually decreased, from approximately 17.6 kPa in the Lipo-CS4 group to approximately 2.7 kPa in the Lipo-HA6 group. This indicates that Nor-CS can form a denser cross-linked network, improving the overall stiffness of the hydrogel; while with an increase in the Nor-HA ratio, the network structure tends to be looser, resulting in a lower elastic modulus. The results demonstrate that by controlling the ratio of Nor-CS to Nor-HA, the mechanical properties of liposome-composite hydrogels can be continuously regulated within the range of approximately 2–18 kPa. Tissue engineering studies have shown that cells are highly sensitive to the mechanical properties of the matrix, and cells from different tissue sources tend to maintain their specific phenotypes and perform their corresponding biological functions in a microenvironment with a stiffness close to that of natural tissues. Skeletal muscle tissue is a relatively stiff soft tissue, with an elastic modulus typically around 10–20 kPa, while mature adipose tissue is a soft tissue, with an elastic modulus generally around 2–5 kPa. Therefore, this study selected Lipo-C4H4 hydrogel with a compressive modulus of approximately 15 kPa as the culture medium for muscle cells and Lipo-C2H6 hydrogel with a compressive modulus of approximately 3.5 kPa as the culture medium for adipocytes. The higher matrix stiffness of Lipo-C4H4 more closely resembles the mechanical properties of natural skeletal muscle tissue, promoting myogenic cell adhesion, spreading, and myotube formation, and facilitating the expression of myogenic genes (such as MyoD, Myogenin, and MyHC). Conversely, the lower elastic modulus of Lipo-C2H6 more closely resembles the microenvironment of natural adipose tissue, reducing cytoskeletal tension, promoting lipid droplet formation, and the expression of adipocyte differentiation markers such as PPARγ, C / EBPα, and FABP4, thereby promoting adipocyte maturation. Therefore, subsequent experiments used Lipo-C4H4 and Lipo-C2H6 as culture carriers for muscle cells and adipocytes, respectively, to construct in vitro culture systems that better conform to the natural tissue mechanical microenvironment.

[0076] Example 11: Induction of muscle cells C2C12 and adipocytes 3T3L1 by liposomes Mouse skeletal muscle myoblasts C2C12 and 3T3-L1 preadipocytes were seeded in DMEM high-glucose medium containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin, and cultured in a 37 ℃, 5% CO2 incubator. When the cell confluence reached approximately 80%, the medium was replaced with differentiation medium containing 2% horse serum, and 100 µg / mL of Ros / Ins@Ins-SDCD liposomes were added. The culture medium was changed every 2 days.

[0077] C2C12 induction results ( Figure 10 The results showed that on day 1, C2C12 cells successfully adhered to the culture substrate, exhibiting a spindle-shaped or short spindle shape, relatively uniform distribution, and low cell density, with some cells beginning to extend. By day 3, the cell number significantly increased, and the cell morphology further elongated, exhibiting a typical spindle-shaped arrangement. Contact between adjacent cells increased, and a parallel alignment trend emerged, suggesting that Ros / Ins@Ins-SDCD liposomes can promote the proliferation and spread of C2C12 cells. By day 5, the cells reached a high degree of confluence, with a large number of cells aligning in the same direction. Some cells fused together to form slender myotube-like structures, and the number of myotubes significantly increased compared to day 3, indicating that Ros / Ins@Ins-SDCD liposomes can continuously promote the myogenic differentiation and maturation of C2C12 cells.

[0078] 3T3-L1 induction results ( Figure 11 The results showed that on day 1 of culture, 3T3-L1 cells had successfully adhered and grown, exhibiting a typical spindle or fusiform shape with relatively uniform cell arrangement. Some cells began to transform from a slender shape to a polygonal shape. By day 3, the number of cells increased significantly, the cell volume gradually increased, and the cell morphology gradually changed from spindle-shaped to round or polygonal. The cytoplasm increased, and a small number of refractive lipid droplets could be observed in some cells, indicating that the cells had begun to enter the adipogenic differentiation stage. On day 5 of culture, the cells further hypertrophied, the number and size of lipid droplets in the cytoplasm increased significantly, and some lipid droplets fused together to form larger lipid droplets. The cells exhibited a typical mature adipocyte morphology, indicating that the Ros / Ins@Ins-SDCD liposome can continuously promote the differentiation of 3T3-L1 cells into mature adipocytes.

[0079] Example 12: Observation of the liveness and death of liposome hydrogel microparticles in the culture of muscle cells C2C12 and adipocytes 3T3L1 Cell seeding: 3T3-L1 cells were seeded onto Lipo-C2H6 hydrogel microparticle modules. The specific procedures were as follows: 1) After sterilization, the hydrogel microparticle modules were pre-soaked in complete culture medium and pre-equilibrated at 37 °C and 5% CO2 for 12–24 h; 2) ADSCs were trypsinized, collected by centrifugation, and resuspended in complete culture medium, adjusting the cell concentration to 1 × 10⁻⁶. 6 cells / mL; take 1 mL of cell suspension (approximately 1 × 10⁻⁶ cells / mL); 5 The number of microparticles (100-500 μm) is adjusted according to their actual particle size to ensure a theoretical total surface area of ​​approximately 1 cm², thereby controlling the cell seeding density at 1 × 10⁻⁶ cells. 5 cells / cm². For example, for hydrogel microparticles with an average diameter of approximately 200 μm, approximately 800 microparticles (approximately 0.8 mL of microparticle suspension) are inoculated each time. For hydrogel microparticles of different sizes, the number of microparticles is adjusted accordingly based on their actual average diameter (200-800 / 100-500 μm) to maintain a constant inoculation density of 1 × 10⁻⁶ cells / cm². 5 Cells / cm². The plates were placed on the surface of a hydrophilically treated culture medium; subsequently, the plates were incubated in a 37 °C, 5% CO2 incubator for three-dimensional culture. On day 3 of culture, cells were stained for live / dead cell activity using the Calcein-AM / PI live / dead cell staining kit.

[0080] C2C21 cells were seeded onto Lipo-C4H4 hydrogel microparticle modules, following the same procedure as above.

[0081] Calcein-AM can label the cytoplasm of live cells and emit green fluorescence, while PI can penetrate the membrane of dead cells and bind to nucleic acids, emitting red fluorescence. After staining, the survival of cells in liposome hydrogel microparticles was observed using a fluorescence microscope.

[0082] The results show that ( Figure 12 Both cell types were uniformly distributed on the surface of the liposome hydrogel microparticles, exhibiting predominantly green fluorescence with only a small amount of red fluorescence signal, indicating that the hydrogel microparticles possess good cell compatibility and low cytotoxicity. After 3 days of culture, C2C12 cells maintained high survival rates in Lipo-C4H4 hydrogel, and 3T3-L1 cells also maintained good activity in Lipo-C2H6 hydrogel, demonstrating their adaptability to the three-dimensional culture environment. These results indicate that the Ros / Ins@Ins-SDCD liposome composite hydrogel microparticles can provide a suitable three-dimensional growth microenvironment for muscle cells and adipocytes, which is beneficial to cell survival, proliferation, and subsequent tissue-specific differentiation, providing a good biomaterial basis for the construction of muscle-adipocyte composite tissue engineering.

[0083] Example 13: Co-differentiation of primary muscle cells and adipocytes and modular tissue construction by liposome-composite hydrogel microparticles To evaluate the application potential of liposome-composite hydrogel microparticles in multi-cell co-culture and tissue construction, a modular tissue engineering system with spatial partitioning and functional regulation capabilities was constructed.

[0084] (1) Cell seeding: Adipose-derived mesenchymal stem cells (ADSCs) and muscle-derived stem cells (MDSCs) were seeded into liposome-based hydrogel microparticles with different mechanical properties. Specifically, ADSCs were seeded onto Lipo-C2H6 hydrogel microparticle modules with a lower elastic modulus. The specific procedures were as follows: 1) After sterilization, the hydrogel microparticle modules were pre-soaked in complete culture medium and pre-equilibrated at 37 °C and 5% CO2 for 12–24 h; 2) After trypsin digestion, the ADSCs were collected by centrifugation and resuspended in complete culture medium, adjusting the cell concentration to 1 × 10⁻⁶. 6 cells / mL; mix 1 mL of cell suspension with approximately 800 hydrogel particles with a diameter of 200 μm (approximately 0.8 mL of hydrogel particle suspension) to control the cell seeding density at 1 × 10⁻⁶ cells / mL. 5 cells / cm² (the surface area of ​​hydrogel particles with a diameter of 200 nm is 1.26 × 10⁻⁶) 3 cm 2 ), and place them on the surface of the hydrophilically treated culture medium; then place the culture plate in a 37 °C, 5% CO2 incubator and let it stand for about 2 h to allow the cells to adhere to the surface of the hydrogel microparticles; 3) then slowly add complete culture medium along the well wall to completely immerse the hydrogel microparticles, and continue 3D culture; change the culture medium every 2–3 days during the culture process, and evaluate subsequent proliferation and adipogenic differentiation according to the experimental design. To simulate the extracellular matrix environment of adipose tissue and soft connective tissue regions; seed MDSCs on Lipo-C4H4 hydrogel microparticle modules with high mechanical strength, and operate in the same way as above, to simulate the mechanical microenvironment of muscle tissue regions.

[0085] (2) Co-culture: After cell seeding, the hydrogel microparticle modules of the two cell types were assembled into microspheres of the same cell type. Through interfacial bonding between microparticles and extracellular matrix secreted by the cells themselves, a stable three-dimensional composite tissue structure was gradually formed. During the culture process (the proliferation culture conditions were DMEM medium containing 10% fetal bovine serum with 5 ng / ml bFGF to maintain cell stemness, and the differentiation conditions were DMEM medium containing 2% horse serum), cells from different microparticles migrated, adhered, and interconnected, promoting tissue fusion between modules. Under the influence of local microenvironmental changes caused by cell metabolism, the liposome structure gradually underwent responsive changes and released the loaded functional molecules, realizing the continuous delivery of inducing factors; at the same time, the hydrogel network gradually degraded in the culture environment, further promoting the release of loaded factors and providing continuous and stable biochemical stimulation for cell differentiation. Through the synergistic effect of dynamic liposome delivery and hydrogel microenvironment regulation, the behavior of cell regions, tissue fusion, and directed differentiation process were continuously regulated, promoting the construction of muscle / fat composite tissue structures. After a certain period of cultivation, the modular composite tissues were frozen sections, and immunofluorescence staining, histological staining, and related protein expression analysis were used to evaluate the formation of functional regions and the level of tissue-specific differentiation.

[0086] Culture results as follows Figure 13 As shown, liposome-composite hydrogel microparticles can effectively support the long-term survival of ADSCs and MDSCs and maintain the spatial independence of different cell modules. During culture, both types of cell-loaded modules maintained good structural integrity, and the cells gradually proliferated and spread in their respective microenvironments, exhibiting a clear tissue-specific differentiation trend.

[0087] Frozen section and immunofluorescence analysis revealed that the Lipo-C4H4 muscle module showed significant expression of muscle-specific markers, with MDSCs gradually elongating and forming a directional cell arrangement. Increased expression levels of related muscle proteins indicated that a high-mechanical-strength microenvironment combined with sustained liposome delivery effectively promoted muscle cell maturation and myogenic differentiation. In the Lipo-C2H6 adipose module, ADSCs showed a gradual transformation in morphology from spindle-shaped to round and polygonal, with increased intracellular lipid accumulation and the appearance of distinct lipid droplet structures. Histological staining and adipose-related protein detection results demonstrated that a low-stiffness hydrogel microenvironment combined with a liposome delivery system promoted ADSC differentiation towards adipocytes. In summary, liposome-composite hydrogel microparticles, through a combination of mechanical microenvironment regulation, sustained delivery of functional factors, and modular assembly strategies, achieved the co-culture and regional differentiation of primary muscle cells and adipocytes. This provides an effective method for constructing three-dimensional composite tissue models with multi-tissue characteristics and has potential applications in tissue engineering, regenerative medicine, and biomimetic tissue construction.

[0088] Example 14: Construction of muscle-fat composite tissue after induction and modular assembly To further evaluate the application capability of liposome-composite hydrogel microparticles in modular tissue construction, mature muscle cell modules and adipocyte modules were constructed. Specifically, adipocytes (ADSCs) were seeded into Lipo-C2H6 hydrogel microparticle modules (operation method as in Example 13) and cultured under adipogenic conditions (culture conditions: DMEM medium containing 2% horse serum) to form adipocyte modules with lipid droplet accumulation characteristics. Simultaneously, MDSCs were seeded into Lipo-C4H4 hydrogel microparticle modules (operation method as in Example 13) and cultured under myogenic conditions (culture conditions: DMEM medium containing 2% horse serum) to form muscle cell modules with myotube structure and muscle-specific protein expression. After the two types of cell modules completed directed differentiation, they were modularly assembled according to preset ratios (number ratios of 2:1, 1:1, and 1:2). Through interfacial bonding between microparticles and cell-cell and cell-matrix interactions, a muscle-adipocyte composite tissue structure with spatial partitioning characteristics was formed. After a certain period of assembly and culture, the resulting composite tissue was frozen sectioned, and the tissue formation was evaluated through immunofluorescence staining, histological staining, and related structural analysis. Specifically, MHC immunofluorescence staining was used to observe muscle region formation, and BODIPY staining was used to observe lipid deposition in the adipose region. Furthermore, the interfacial continuity and tissue fusion effects between different functional modules were analyzed.

[0089] The results are as follows Figure 14 (Mixed ratio 1:1) The assembled composite tissue maintained distinct spatial regional characteristics. The muscle cell region exhibited high MHC expression and formed a continuous muscle-like structure; the adipocyte region showed significant BODIPY-positive lipid droplet signals, forming an adipose-like tissue region. Frozen section observation revealed good interfacial connections between different functional modules, allowing cell migration and matrix remodeling along module boundaries, promoting inter-module fusion while maintaining the spatial distribution characteristics of the muscle and adipose regions. These results demonstrate that the liposome-based hydrogel microparticles provided by this invention can not only achieve synergistic induction of differentiation from stem cells of different origins but also construct spatially heterogeneous muscle-adipose composite tissues through modular assembly, providing a novel technical strategy for the construction of complex tissue engineering.

[0090] Comparative Example 1 Referring to Example 1, all other reaction parameters remained unchanged, except that the molar ratios of soybean lecithin (SPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), cholesterol hemisuccinate (CHEMS), and maleimide polyethylene glycol distearate phosphatidylethanolamine (DSPE-PEG-Mal) were adjusted to 10:3:2:1 and 6:3:2:1, respectively.

[0091] The results showed that when the SPC ratio was increased to 10, the resulting liposomes had larger particle sizes, wider particle size distributions, and decreased system stability. The encapsulation efficiency and loading of differentiation factors were lower than in Example 1. Analysis suggests that an excessively high SPC ratio increased the rigidity of the lipid bilayer, affecting the uniform formation of the liposome membrane structure and hindering the stable encapsulation of rosiglitazone and insulin. When the SPC ratio was reduced to 6, the uniformity of the resulting liposome particle size distribution improved, but due to insufficient main components of the lipid bilayer, the stability of the liposome membrane decreased, making it prone to drug leakage during storage and release, resulting in reduced encapsulation efficiency and sustained-release performance. Comprehensive comparison revealed that the SPC, DOPE, CHEMS, and DSPE-PEG-Mal molar ratio used in Example 1 effectively balanced liposome particle size uniformity, structural stability, drug encapsulation efficiency, and sustained-release performance, thus representing the preferred formulation of this invention.

[0092] Comparative Example 2 Referring to Example 1, all other reaction parameters remained unchanged, except that the mass ratio of insulin to lipids was adjusted to 20:1 and 30:1, respectively.

[0093] The results showed that when the insulin-to-lipid mass ratio was reduced to 20:1 or 30:1, the encapsulation efficiency and drug loading rate of insulin in the resulting liposomes were significantly lower than those in Example 1. Analysis suggests that reducing the insulin ratio decreases the total amount of insulin that can be encapsulated in the system, reducing the drug concentration gradient within the liposomes and thus decreasing the liposomes' insulin encapsulation capacity. Simultaneously, the reduced drug content in the liposomes further decreases the drug loading rate, which is detrimental to the sustained release of the drug and the exertion of its biological functions. In contrast, Example 1, using an insulin-to-lipid mass ratio of 40:1, achieved a higher insulin encapsulation efficiency and drug loading rate, indicating that this ratio is more conducive to the stable encapsulation of insulin in liposomes. While ensuring the structural stability of the liposomes, it also improves drug utilization efficiency, thus representing a preferred embodiment of the present invention.

[0094] Comparative Example 3 Referring to Example 10, all other reaction parameters were exactly the same, except that the concentration of the photoinitiator LAP was changed. The LAP concentration was adjusted from 0.1 wt.% to 0.05 wt.% and 0.20 wt.%, respectively, while the other components remained unchanged.

[0095] The results showed that when the LAP concentration decreased to 0.05 wt.%, the rate of free radical generation in the system decreased, and the click reaction of thiol-norbornene was incompletely cross-linked, resulting in insufficient cross-linking density of the formed hydrogel, reduced mechanical strength, and deformation or rupture of some microspheres during cultivation and soaking. Simultaneously, liposomes easily escaped from the loose network, affecting the stability of the system and the sustained release of differentiation factors. When the LAP concentration was increased to 0.20 wt.%, although the hydrogel solidification speed was accelerated, the excessively rapid cross-linking reaction resulted in an overly dense network structure, leading to decreased hydrogel elasticity, hindered diffusion of differentiation factors, and reduced sustained drug release efficiency. Furthermore, excessively high concentrations of photoinitiators and their photolyzed free radicals may increase cytotoxicity, which is detrimental to long-term cell survival and tissue regeneration. Comprehensive comparison revealed that when the LAP concentration was 0.1 wt.%, the hydrogel could achieve rapid and uniform cross-linking, exhibiting good structural integrity, mechanical properties, biocompatibility, and sustained drug release performance. Therefore, 0.1 wt.% was determined to be the preferred photoinitiator concentration for this invention.

[0096] Comparative Example 4 Referring to Example 10, all other reaction parameters were exactly the same, except that the concentration of insulin-modified liposomes (Ins-Lipo) was changed. The Ins-Lipo concentration was adjusted from 1 wt.% to 0.25 wt.% and 0.50 wt.%, respectively, while the other components remained unchanged.

[0097] The results showed that when the Ins-Lipo concentration was reduced to 0.25 wt.% or 0.50 wt.%, the liposome content in the hydrogel decreased significantly, leading to a reduction in the total amount of rosiglitazone and insulin loaded in the system. In vitro release experiments showed that the cumulative release of the two differentiation factors was significantly lower than in Example 1, failing to maintain an effective local drug concentration over a longer culture period, thus weakening the sustained induction effect on muscle cell and adipocyte differentiation. Furthermore, due to the reduced number of liposomes, the drug reservoir formed inside the hydrogel was insufficient. Although it still had a certain sustained-release effect, the overall release dose was low and could not fully meet the demand for a continuous supply of differentiation factors during tissue repair. In contrast, Example 1, using 1 wt.% Ins-Lipo, achieved a higher loading of differentiation factors and sustained release capacity while ensuring the stability of the hydrogel structure, providing a stable and continuous induction signal for muscle cells and adipocytes. Therefore, 1 wt.% was determined to be the preferred liposome addition concentration of this invention.

[0098] Comparative Example 5 Referring to Example 1, except for the fatty acid composition, all other reaction parameters remained unchanged. 1,2-Dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE) was not added during liposome preparation, while the other components and their amounts remained constant, to prepare drug-loaded liposomes.

[0099] Comparative analysis of results: Compared with the drug-loaded liposomes obtained in Example 1, the liposomes prepared without DOPE exhibited higher structural stability. The results showed that after removing DOPE, the liposome particle size changed less, and the particle size distribution was more stable during storage, indicating improved liposome membrane structural integrity. Simultaneously, due to the more compact arrangement of the lipid bilayer, the liposomes provided better protection for the encapsulated drug. Analysis suggests that DOPE, with its unique cone-shaped molecular structure and strong membrane curvature tendency, can improve lipid bilayer fluidity and promote membrane fusion and structural rearrangement. While DOPE is beneficial for enhancing the interaction between liposomes and the cell membrane and promoting content release, its introduction may also reduce the compactness of the lipid bilayer. Removing DOPE resulted in more compact lipid molecular packing and increased membrane rigidity, thereby improving the physical stability of the liposomes and reducing drug leakage. The results indicate that DOPE in the liposome system of this invention mainly plays a role in regulating membrane fluidity, promoting drug release, and improving cell-cell interactions, rather than simply improving liposome stability. By controlling the DOPE content, a balance can be achieved between liposome stability and drug release efficiency.

[0100] Comparative Example 6 Referring to Example 2, except for the insulin modification method, the rest of the liposome preparation process, component ratios, and cell culture conditions remained unchanged. Specifically, insulin was dissolved in 0.01 M HCl solution, and the pH of the solution was adjusted to 7.0–7.4 using 0.1 M NaOH. Then, an appropriate amount of tris(2-carboxyethyl)phosphine (TCEP) was added as a reducing agent, and the reaction was carried out under light-protected and low-temperature conditions to break some of the disulfide bonds within the insulin molecule and expose free sulfhydryl groups. After the reaction, unreacted TCEP was removed by dialysis, and the reduced-modified insulin was obtained by lyophilization and analyzed by MALDI-TOF.

[0101] Comparative analysis of results: MALDI-TOF mass spectrometry was used to analyze insulin modified by TCEP reduction. The results showed that compared with insulin modified by Traut's Reagent, the TCEP-treated insulin exhibited less change in molecular weight and a significantly lower proportion of thiol modification. This indicates that this method mainly exposes existing thiol groups through partial disulfide bond reduction, making it difficult to achieve highly efficient thiol introduction. Further analysis suggests that the degree of disulfide bond breakage within insulin during TCEP reduction is greatly affected by reaction conditions. Excessive reduction may lead to changes in insulin's spatial conformation, while reducing the degree of reduction results in insufficient numbers of active thiol groups available for coupling. Therefore, the coupling efficiency between insulin obtained by the TCEP method and the maleimide groups on the surface of Mal-SDCD liposomes is low, leading to a decrease in insulin density on the liposome surface. In contrast, thiol modification using Traut's Reagent can effectively increase the proportion of active thiol groups introduced into insulin, maintaining the structural stability of insulin while improving its coupling efficiency with the Mal groups on the liposome surface, which is beneficial for constructing a stable insulin-functionalized liposome system.

[0102] Comparative Example 7 Referring to Example 12, the Ros / Ins@Ins-SDCD liposomes were replaced with liposomes that were only modified with the insulin surface but did not encapsulate insulin (Ros@Ins-SDCD), or with liposomes that only encapsulated insulin but did not have surface modification (Ros / Ins@Mal-SDCD). All other experimental steps, liposome composition, and dosage remained unchanged, and these were used for the induction culture of C2C12 cells and 3T3-L1 preadipocytes, respectively. The culture conditions, culture medium replacement frequency, and detection methods were consistent with Example 12.

[0103] The results showed that, compared with the Ros / Ins@Ins-SDCD liposomes using a dual insulin delivery strategy in Example 12, both the Ros@Ins-SDCD and Ros / Ins@Mal-SDCD liposomes exhibited reduced promoting effects on myogenic differentiation of C2C12 cells and adipogenic differentiation of 3T3-L1 cells. Specifically, the Ros@Ins-SDCD group, relying solely on surface insulin presentation, while enhancing the interaction between liposomes and cells, lacked internal insulin reserves, making it difficult to maintain a stable insulin-stimulated environment over a long period; its induction effect gradually weakened with prolonged culture time. Although the Ros / Ins@Mal-SDCD group achieved sustained insulin release through internal liposome encapsulation, the lack of surface insulin presentation resulted in reduced local insulin concentration around cells and decreased cellular response efficiency, thus its promoting effect on directed cell differentiation was weaker than that of Example 12. In contrast, Ros / Ins@Ins-SDCD liposomes, by combining internal loading and surface modification as delivery methods, achieved synergistic regulation of sustained insulin release and cell interface interactions, which can more effectively promote myotube formation in C2C12 cells and lipid droplet accumulation in 3T3-L1 cells. This indicates that the dual delivery strategy can significantly improve the regulatory ability of liposomes on the co-differentiation process of muscle and adipocytes.

[0104] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for co-culturing muscle cells and adipocytes based on photocrosslinked liposome hydrogel microparticles, characterized in that, Includes the following steps: (1) Soybean lecithin, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanol, cholesterol hemisuccinate, maleimide polyethylene glycol distearate phosphatidylethanolamine, rosiglitazone and solvent were mixed, and then the solvent was removed to obtain Ros@Mal-SDCD lipid membrane; the Ros@Mal-SDCD lipid membrane was then hydrated by mixing it with an aqueous solution of PBS containing insulin, and then the hydrated mixture was repeatedly extruded using a polycarbonate membrane to obtain Ros / Ins@M al-SDCD liposomes; Ros / Ins@Mal-SDCD liposomes were dispersed in PBS buffer, and thiol-modified insulin was added. The reaction was carried out in the dark to obtain liposomes with insulin-conjugated surfaces; finally, borneol-modified chitosan, borneol-modified hyaluronic acid, borneol-modified collagen, insulin-modified liposomes, crosslinking agents, photoinitiators, and deionized water were mixed to form a precursor solution; the precursor solution was formed into droplets through microfluidics and subjected to photocrosslinking and curing treatment to finally obtain liposome composite hydrogel microparticles; (2) Adipose stem cells and muscle stem cells were seeded onto the surface of liposome composite hydrogel microparticles, then mixed and assembled. A three-dimensional culture microenvironment was gradually constructed through interparticle interface binding and extracellular matrix secreted by the cells themselves for cell co-culture.

2. The method according to claim 1, characterized in that, In step (2), the specific process of vaccination is as follows: After sterilization, liposome-composite hydrogel microparticles were pre-immersed in complete culture medium and pre-equilibrated at 37 °C and 5% CO2 for 12–24 h. Subsequently, adipose-derived stem cells or muscle stem cells were suspended in complete culture medium, and the cell concentration was adjusted to 1 × 10⁶ cells / mL. 6 Cells / mL were used to obtain a cell suspension; each 1 mL of cell suspension was mixed with 200-800 hydrogel microparticles with a diameter of 100-500 μm to control the cell seeding density at 1 × 10⁻⁶ cells / mL. 5 Cells / cm² were placed on the surface of a hydrophilically treated culture medium; then the culture plate was placed in a 37 °C, 5% CO2 incubator and left to stand for about 2 h to allow the cells to adhere to the surface of the hydrogel particles.

3. The method according to claim 1, characterized in that, In step (2), adipose-derived stem cells are seeded onto the surface of liposome-modified hydrogel microparticles. During the preparation of the liposome-modified hydrogel microparticles, the mass ratio of borneol-modified chitosan to borneol-modified hyaluronic acid in the precursor solution is controlled to be 2:

6. Muscle stem cells are seeded onto the surface of the liposome-modified hydrogel microparticles. During the preparation of the liposome-modified hydrogel microparticles, the mass ratio of borneol-modified chitosan to borneol-modified hyaluronic acid in the precursor solution is controlled to be 4:

4.

4. The method according to claim 1, characterized in that, In step (2), during the mixed assembly, the ratio of the number of hydrogel microparticle modules seeded with adipose stem cells to the number of hydrogel microparticle modules seeded with muscle stem cells is 1:2 to 2:

1.

5. The method according to claim 1, characterized in that, In step (1), The total relative solvent concentration of the four fatty acids—soy lecithin, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanol, cholesterol hemisuccinate, and maleimide polyethylene glycol distearate phosphatidylethanolamine—was 10-20 mg / mL; the mass ratio of the four fatty acids was 4:3:2:

1. The feed ratio of rosiglitazone to the total amount of four fatty acids—soybean lecithin, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanol, cholesterol hemisuccinate, maleimide, and polyethylene glycol distearate phosphatidylethanolamine—is 10-20:

1. The mass ratio of insulin to the total amount of four fatty acids—soybean lecithin, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanol, cholesterol hemisuccinate, maleimide, and polyethylene glycol distearate phosphatidylethanolamine—is 20-40:

1.

6. The method according to claim 1, characterized in that, In step (1), the preparation methods for borneol-modified chitosan, borneol-modified hyaluronic acid, and borneol-modified collagen include: 5-Norbornene-2,3-dicarboxylic anhydride was dissolved in DMF and added dropwise to acetic acid solutions of sodium hyaluronate, chitosan, or collagen under vigorous stirring, while maintaining the pH of the reaction solution at 9. The reaction was carried out at low temperature (4-10 °C) under light-protected conditions for 1-3 hours. After the reaction was completed, sodium hyaluronate and chitosan were recovered by ethanol / DMF precipitation, while collagen was directly dialyzed. Subsequently, the obtained products were dialyzed in deionized water, with the dialysate changed multiple times. The purified solution was finally lyophilized to obtain norbornene-modified sodium hyaluronate, norbornene-modified chitosan, and norbornene-modified collagen, respectively.

7. The method according to any one of claims 1-6, characterized in that, In step (1), the precursor solution contains HS-PEG-SH as a crosslinking agent at a concentration of 10 mM; borneol-modified collagen at a concentration of 0.5 wt.%; insulin-modified liposomes at a concentration of 1 wt.%; and LAP as a photoinitiator at a concentration of 0.1 wt.%.

8. A three-dimensional culture microenvironment for cell co-culture and co-differentiation obtained by the method of any one of claims 1-7.

9. The application of the three-dimensional culture microrings according to claim 8 in the fields of muscle-fat tissue engineering and biomanufacturing.

10. The application of the three-dimensional culture microrings according to claim 8 in the fields of regenerative medicine and the development of functional biomaterials.

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