A biomimetic extracellular matrix porous hydrogel for dendritic cell three-dimensional culture, delivery and a preparation method thereof

Abstract

The application belongs to the technical field of three-dimensional culture and delivery of cells, and discloses a gelatin / hyaluronic acid porous hydrogel composed of a biomimetic extracellular matrix for three-dimensional culture and delivery of dendritic cells and a preparation method thereof. The method uses a semi-interpenetrating polymer network composed of methacrylic anhydride modified gelatin (GelMA) and oxidized hyaluronic acid (OHA) as a matrix, introduces temperature-responsive gelatin microspheres and dendritic cells at the same time, and then constructs a three-dimensional supramolecular hydrogel cell in-vitro culture model with decoupled pore size and stiffness after the gelatin microspheres are melted by increasing the temperature. The supramolecular hydrogel has the advantages of convenient use, simple operation, biological safety and the like, and can be used in the fields of three-dimensional culture, delivery and tumor immunotherapy of dendritic cells.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of three-dimensional culture and delivery of cells based on hydrogel, and particularly relates to a biomimetic extracellular matrix porous hydrogel for three-dimensional culture and delivery of dendritic cells and a preparation method thereof. BACKGROUND

[0002] Dendritic cells (DCs) are currently the strongest antigen-presenting cells found, and the surface molecules of DCs can process and present tumor antigens, secrete various cytokines to initiate T cell recognition and kill tumor cells in an antigen-specific manner. At the same time, it can also stimulate immune memory protection and is the initiator of the body's anti-tumor immune response. However, DC vaccines have problems such as low cell activity in vivo, low homing efficiency, and weak immune activation ability. First, although as many as 10 6 DCs are transferred back to tumor-bearing hosts, but most of the DCs die or flow to non-disease sites after injection, and only about 1% of the DCs can migrate to the draining lymph nodes. Second, mature DCs have a short lifespan of only a few days, and if the host's own DCs cannot be recruited to continue the antigen uptake and presentation process, it will further limit the tumor-specific immune response. It is now known that the stiffness, dimension, and pore size of the tumor microenvironment are important factors affecting tumor progression and the efficacy of immunotherapy, and changes in the tumor microenvironment during tumor occurrence and development also directly affect the anti-tumor function of DCs. However, due to the lack of methods to decouple the complex interaction between stiffness and pore size, little is known about the behavior and function of DCs in the tumor microenvironment.

[0003] Hydrogels are three-dimensional water-swellable polymer networks constructed based on covalent bonds or physical interactions, which have similar structure and properties to extracellular matrix, such as high hydration, high permeability, adjustable biodegradability and mechanical properties. In addition, the minimally invasive delivery of hydrogel scaffolds can be achieved by the design of hydrogel materials, which can effectively avoid complex surgical operations and postoperative complications. In situ forming hydrogels are usually injected in a flowable state to the lesion site, and form gels in situ under the action of chemical or physical crosslinking, and then realize the in situ fixation of cells and active molecules. Therefore, hydrogels can be used as co-delivery carriers of DCs and tumor antigens, which can effectively improve the activity of exogenous DCs and prolong their residence time at the injection site, enrich the cytokines secreted by DCs, and construct the in situ inflammatory microenvironment. Moreover, through the co-delivery of tumor antigens, adjuvants and chemotherapy drugs, the recruitment and activation of DCs in the host body can be further achieved, thereby expanding the T cell immune response and inhibiting tumor growth. However, in situ injectable hydrogels need appropriate gelation time. Too fast gelation time can easily lead to delivery blockage, and too slow gelation time can cause the precursor liquid to flow to the surrounding tissues or be diluted by body fluids, thereby affecting the final performance of the gel. Although the dynamic hydrogels constructed based on physical interactions or dynamic covalent bonds have self-repairing ability, which can effectively improve the performance of in situ hydrogels. However, the formed nano-sized pore structure is not conducive to the interaction between cells and the diffusion of bioactive molecules.

[0004] Compared with in situ nanopore injectable hydrogel, the effective pore size of porous hydrogel is greater than 10 μm, and the scaffold material with different pore structures can be prepared by adjusting the preparation parameters. The porous structure can provide sufficient space for cells to migrate and proliferate to its interior, and is also more conducive to cell-cell communication, which can effectively reduce the inflammatory response in vivo. However, the existing manufacturing technology still faces many limitations in realizing the pore structure. For example, bioprinting technology (such as nozzle printing and stereolithography) can generate large-scale structures, but it is difficult to manufacture fine features smaller than the nozzle or voxel; the photolithography technology is limited to small volume manufacturing due to heat accumulation and time cost. In addition, although many emerging printing technologies (such as two-photon lithography) can print complex shapes, they are difficult to adapt to natural extracellular matrix, and there are problems such as material limitations, low efficiency and small build volume. On the other hand, hydrogels can realize the dimension, stiffness and pore structure of the biomimetic tumor extracellular matrix due to their high permeability and adjustable biophysical properties. Subsequently, the DCs are inoculated in the biomimetic extracellular matrix hydrogel to explore the biological functions of the DCs under various biophysical properties. However, as a cell culture model in vitro, how to decouple the physical properties (stiffness, dimension, pore size) of the extracellular matrix and independently explore the influence of the single physical property of the extracellular matrix on the DCs is a difficulty. Based on this, it is urgent to develop a hydrogel that can better mimic the components of the extracellular matrix while decoupling the structure and stiffness. SUMMARY

[0005] To solve the shortcomings and deficiencies of the prior art, the purpose of the present application is to provide a biomimetic extracellular matrix porous hydrogel for three-dimensional culture and delivery of dendritic cells and a preparation method thereof. The present application takes a semi-interpenetrating polymer network composed of methacrylic anhydride modified gelatin (GelMA) and oxidized hyaluronic acid (OHA) as the matrix, and constructs a dendritic cell in vitro three-dimensional culture model with decoupled pore size and stiffness by introducing sacrificial gelatin microsphere templates. The in vitro culture model of the hydrogel has a stable double network structure, in which the aldehyde group on the OHA reacts with the amino group of the GelMA to form a first Schiff base network, and the Schiff base reaction has the advantages of fast reaction speed, no need for additional crosslinking agent, good adhesion ability, etc. Subsequently, the chemical crosslinking of GelMA after light induction forms a second network. The double network structure improves the mechanical strength of the hydrogel, and the network structure is similar to the three-dimensional network structure of the extracellular matrix. In addition, the pore structure inside the supramolecular porous hydrogel is beneficial to the diffusion and exchange of oxygen, nutrients and metabolites, and can provide a suitable three-dimensional growth microenvironment for cells embedded therein.

[0006] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0007] A kind of biomimetic extracellular matrix porous hydrogel for dendritic cell three-dimensional culture, delivery and preparation method thereof, comprising the following steps:

[0008] (1) gelatin is added to deionized water and stirred until completely dissolved to obtain a gelatin solution.

[0009] The gelatin is derived from pigskin;

[0010] The stirring condition is 300-500 rpm;

[0011] The stirring temperature is 40-60℃, and the reaction time is 0.5-1h;

[0012] The mass percentage of the gelatin solution is 3%-5%;

[0013] (2) add anhydrous ethanol, poloxamer and gum arabic to the product and stir overnight, then treat to obtain gelatin microgel.

[0014] The volume ratio of anhydrous ethanol to deionized water is 3:5-1:1;

[0015] The volume ratio of gum arabic to gelatin is 0.05-0.1;

[0016] The stirring temperature is 20-25℃;

[0017] The mass ratio of poloxamer to gelatin is 0.125-0.25;

[0018] The subsequent treatment is 300g centrifugation for 5min, removing the emulsion phase, resuspending in PBS buffer (pH=7.4, 0.1M), 1000g centrifugation for 5min, discarding the supernatant, and repeating three times.

[0019] (3) synthesis of oxidized sodium hyaluronate polymer: dissolve sodium hyaluronate in deionized water, add oxidizing agent, stir and react, add oxidizing agent terminator, and then treat to obtain oxidized hyaluronic acid polymer.

[0020] The molecular weight of the hyaluronic acid is 90-120 million;

[0021] The oxidizing agent is sodium periodate;

[0022] The oxidizing agent terminator is ethylene glycol;

[0023] The mass percentage of sodium hyaluronate is 0.1-2%;

[0024] The mass percentage of sodium periodate is 0.1-0.5%;

[0025] The mass percentage of ethylene glycol is 0.5-2%;

[0026] The temperature of the stirring reaction is 0℃;

[0027] The time of the stirring reaction is 1-3h;

[0028] The subsequent treatment is dialysis, freeze-drying.

[0029] (4) Preparation of methacrylic anhydride modified gelatin: after the gelatin is completely dissolved by heating in phosphate buffer solution, methacrylic anhydride is added dropwise, and the subsequent treatment is performed to obtain methacrylic anhydride modified gelatin.

[0030] The reaction temperature is 50-60℃, and the reaction time is 3-5h;

[0031] The pH of the phosphate buffer solution is 7.4;

[0032] The mass concentration of the gelatin aqueous solution is 5-10%;

[0033] The volume percentage of the methacrylic anhydride is 1-20%;

[0034] The subsequent treatment is to centrifuge the reaction solution, dialyze it in deionized water, and freeze-dry to obtain methacrylic anhydride modified gelatin.

[0035] (5) The methacrylic anhydride modified gelatin of step (4) and the oxidized hyaluronic acid of step (3) are added to the basic medium, and stirred for 3-5h to obtain a matrix gel solution.

[0036] The mass percentage of the methacrylic anhydride modified gelatin is 3%-10%;

[0037] The mass percentage of the hyaluronic acid is 0.4%-0.8%;

[0038] The stirring temperature is 50-60℃.

[0039] (6) A photoinitiator (2959) is added to the matrix gel solution of step (5) to obtain a hydrogel precursor solution;

[0040] The mass percentage of the photoinitiator is 0.2%-0.5%.

[0041] (7) The temperature-responsive gelatin microspheres prepared in step (2) are added to the hydrogel precursor solution of step (6) in a certain proportion, and mixed uniformly by blowing to obtain a porous hydrogel precursor solution.

[0042] The ratio of the gelatin microspheres to the matrix gel is 1:1-3:1.

[0043] (8) Gelatin / hyaluronic acid porous hydrogel for three-dimensional culture of DCs: mix the dendritic cells with the precursor liquid in step (7), solidify under ultraviolet light for 1 min, and realize three-dimensional uniform encapsulation of the DCs through subsequent treatment.

[0044] The dendritic cells are dendritic cells induced for 6 days by mouse mesenchymal stem cells through interleukin 4 (IL-4) and granulocyte macrophage colony-stimulating factor (GM-CSF);

[0045] The cell density is 5 x 10 6 ~ 1 x 10 7 cells / mL sol;

[0046] The subsequent treatment is to add complete culture medium and then place in a culture box for 24 h.

[0047] (9) After the culture model is placed in a culture box for 24 h, the number of DCs in the culture medium supernatant is measured at fixed points every 24 h.

[0048] Compared with the prior art, the present application has the following advantages:

[0049] The present application introduces different sizes of temperature-responsive gelatin microsphere templates into a methyl methacrylic anhydride modified gelatin (GelMA) and oxidized hyaluronic acid (OHA) matrix, and after the matrix is photocured, the gelatin microspheres are melted, and a three-dimensional hydrogel culture model with the same stiffness and different pore sizes is constructed. Among them, the effective pore size of the hydrogel is precisely controlled by regulating the size of the gelatin microspheres; the stiffness of the hydrogel matrix is independently controlled by adjusting the ratio of GelMA and OHA.

[0050] Compared with traditional hydrogels, the biomimetic extracellular matrix supramolecular hydrogel decoupled in pore size and stiffness is more conducive to independently exploring the influence of the biophysical properties (pore size, stiffness) of the extracellular matrix on the immunological function of DCs. In addition, the interconnected space inside the supramolecular porous hydrogel provides a three-dimensional microenvironment for DCs, which can effectively improve the cell activity and maturity of DCs, and the porous structure of the supramolecular hydrogel is beneficial to the sustained release of DCs encapsulated therein. Based on this, the present application can be used as a co-delivery carrier for DCs and tumor antigens, effectively improving the problems of low cell activity, low homing efficiency, and weak immune activation ability of the current DC vaccine. And the supramolecular porous hydrogel has the advantages of convenient use, simple operation, biological safety, etc., and can be used in the fields of cell culture, tissue engineering, drug delivery, and tumor immunotherapy. BRIEF DESCRIPTION OF DRAWINGS

[0051] To more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below.

[0052] Figure 1 The temperature-responsive gelatin microgel morphology and particle size distribution diagram provided for this invention

[0053] Figure 2 and Figure 3 The internal microstructure of the supramolecular hydrogel provided by the present invention

[0054] Figure 4 Stiffness characterization of the supramolecular hydrogel provided by the present invention

[0055] Figure 5 Three-dimensional culture of DCs provided by the present invention

[0056] Figure 6 The in vitro release of DCs after three-dimensional culture provided by the present invention Detailed Implementation

[0057] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0058] The porous supramolecular hydrogel of this invention uses methacrylic anhydride-modified gelatin and oxidized hyaluronic acid as a matrix, and introduces temperature-sensitive gelatin microspheres as a pore-forming agent. It is obtained through subsequent processing.

[0059] The specific preparation steps of the methacrylic anhydride modified gelatin in the example are as follows: Weigh 5g of gelatin into a 250mL two-necked round-bottom flask, add 50mL of phosphate buffer (pH=7.4) using a graduated cylinder, heat and stir at 60℃ until completely dissolved, then add 4mL of methacrylic anhydride dropwise using a syringe, maintain the reaction temperature at 50℃ and stir for 3h; after centrifuging the reaction solution at 10000rpm for 20h, place it in an 8000-14000 dialysis bag for dialysis for 5 days, changing the water 3 times a day; after dialysis, freeze-dry and store in a -20℃ refrigerator for later use.

[0060] The preparation steps of the oxidized hyaluronic acid polymer in the example are as follows: Take a clean 250mL round-bottom flask, weigh 1g of sodium hyaluronate, add it to 100mL of deionized water, and stir in an ice-water bath for 12 hours to completely dissolve the sodium hyaluronate. Slowly add 5mL of 0.5M sodium periodate dropwise, and stir at 24℃ in the dark for 3 hours. Add 1mL of ethylene glycol and continue stirring for 10 minutes to terminate the reaction. Collect the product and place it in an 8000 mL container. Dialysis was performed for three days using a 12,000-cell dialysis bag, with the water changed three times a day. The product was then freeze-dried to obtain a white, spongy substance, which was stored at 4°C for later use.

[0061] Example 1

[0062] A kind of biomimetic extracellular matrix porous hydrogel for dendritic cell three-dimensional culture, delivery and preparation method thereof, characterized by comprising the following steps:

[0063] (1) 200 mg of GelMA and 24 mg of OHA were weighed into a glass bottle, 4 mL of basal medium was added, and 300 rpm magnetic stirring was carried out at 60°C for 4 h

[0064] (2) 10 mg of photosensitizer 2959 was added, and stirring was continued for 1 h to obtain a hydrogel precursor solution;

[0065] (3) The hydrogel precursor solution of step (2) was sterile filtered to obtain reaction system 1;

[0066] (4) DCs were mixed with reaction system 1 of step (3) to achieve three-dimensional encapsulation of DCs, and composite 1 was obtained;

[0067] (5) Composite 1 was placed in a cell culture device, placed 5 cm directly below a 365 nm laser, and irradiated for 1 min to solidify, obtaining a dense hydrogel three-dimensional culture model.

[0068] (6) After the culture model was placed in an incubator for 24 h, the number of DCs in the supernatant of the culture medium was measured at fixed points every 24 h.

[0069] Example 2

[0070] (1) 0.4 g of gelatin was added to 10 mL of deionized water, the temperature of the constant temperature water tank was controlled at 50°C, and 400 rpm magnetic stirring was carried out until complete dissolution;

[0071] (2) 6 mL of anhydrous ethanol was added to the reaction solution of step (1), and the reaction system changed from transparent to milky white after the addition of anhydrous ethanol;

[0072] (3) 0.05 g of poloxamer and 0.02 g of alginic acid were weighed into step (2), and after complete dissolution, the temperature was lowered to 24°C and the stirring reaction was continued for 8 h;

[0073] (4) The solution of step (3) was centrifuged at 300 g for 5 min to remove the emulsion, and gelatin microgel was obtained;

[0074] (5) The product obtained in step (4) was resuspended with PBS, centrifuged at 1000 g for 5 min, repeated three times to remove residual emulsion, and the gelatin microgel precipitate was resuspended with PBS and stored at 4°C for standby;

[0075] (6) 400 mg of GelMA and 32 mg of OHA were weighed into a glass bottle, 4 mL of basal medium was added, 300 rpm magnetic stirring was carried out at 60°C for 4 h, then 10 mg of photosensitizer 2959 was added, and stirring was continued for 1 h to obtain reaction system 1;

[0076] (7) After the reaction system 1 is sterilized, it is mixed with the gelatin microspheres of step (5) at a ratio of 1:2 to obtain reaction system 2;

[0077] (8) The DCs are mixed with the reaction system 2 of step (7) to achieve three-dimensional encapsulation of the DCs, thereby obtaining complex 1;

[0078] (9) The complex 1 is placed 5 cm below a 365 nm laser, and irradiated for 1 min to solidify, thereby obtaining a hydrogel three-dimensional culture model with a pore size of 15 μm.

[0079] (10) After the culture model is cultured in an incubator for 24 h, the number of DCs in the supernatant of the culture medium is measured at fixed points every 24 h.

[0080] Example 3

[0081] (1) 0.4 g of gelatin is added to 10 mL of deionized water, and the temperature of the water tank is controlled at 50°C, and the magnetic stirring is performed at 400 rpm until the gelatin is completely dissolved;

[0082] (2) 10 mL of anhydrous ethanol is added to the reaction solution of step (1), and after the addition of the anhydrous ethanol, the reaction system changes from transparent to milky white;

[0083] (3) 0.1 g of poloxamer and 0.04 g of alginic acid are weighed and added to step (2), and after the poloxamer and alginic acid are completely dissolved, the temperature is lowered to 24°C, and the stirring reaction is continued for 8 h;

[0084] (4) 300 g of the solution of step (3) is centrifuged for 5 min to remove the emulsion, thereby obtaining gelatin microgels;

[0085] (5) The product obtained in step (4) is resuspended with PBS, and centrifuged at 1000 g for 5 min, and the operation is repeated three times to remove the residual emulsion. The gelatin microgel precipitate is resuspended with PBS and stored at 4°C for standby;

[0086] (6) 400 mg of GelMA and 32 mg of OHA are weighed in a glass bottle, 4 mL of the basic culture medium is added, and the magnetic stirring is performed at 300 rpm and 60°C for 4 h. Then, 10 mg of photosensitizer 2959 is added, and the stirring is continued for 1 h to obtain reaction system 1;

[0087] (7) After the reaction system 1 is sterilized, it is mixed with the gelatin microspheres of step (5) at a ratio of 1:2 to obtain reaction system 2;

[0088] (8) The DCs are mixed with the reaction system 2 of step (7) to achieve three-dimensional encapsulation of the DCs, thereby obtaining complex 1;

[0089] (9) Place the composite 1 5 cm directly below a 365 nm laser and irradiate for 1 min to solidify, thus obtaining a three-dimensional hydrogel culture model with a pore size of 100 μm.

[0090] (10) After the culture model was placed in the incubator and cultured for 24 hours, the number of DCs in the supernatant of the culture medium was measured at fixed points every 24 hours.

[0091] The temperature-responsive gelatin microspheres prepared in this example are as follows: Figure 1 As shown, by introducing emulsion stabilizers (poloxam and gum arabic) into the emulsion system to control the size of the emulsion droplets, gelatin microspheres of two sizes were prepared. The particle sizes of the two sizes of gelatin microspheres were mainly distributed at 15 μm and 100 μm, respectively. The gelatin microspheres were thoroughly mixed with a matrix adhesive, and the matrix adhesive was cross-linked by UV curing. Subsequently, the temperature was increased until the gelatin microspheres melted to obtain a porous hydrogel. The internal structure of the supramolecular porous hydrogel is shown below. Figure 2 , Figure 3 As shown, the results indicate that the hydrogel possesses a uniformly sized and evenly distributed porous structure both in the wet and lyophilized states. The stiffness of supramolecular hydrogels with different pore sizes was characterized, as shown in the figure. Figure 4 As shown, the stiffness of both the dense hydrogels with small pore sizes (15 μm) and those with large pore sizes (100 μm) is within 1 kPa, indicating that porous hydrogels with consistent stiffness but different pore sizes were successfully prepared. The morphology of the prepared porous hydrogels was characterized after culturing DCs in vitro for 24 h, as shown in the figure. Figure 5 As shown, hydrogels with porous structures are more conducive to the elongation of DC synapses. Figure 6 After 24 hours of three-dimensional culture of dendritic cells (DCs) in a hydrogel, their in vitro release was measured at specific points. The results showed that hydrogels with porous structures were more conducive to DC release than dense hydrogels. The efficient release of DCs lays the foundation for porous hydrogels as cell delivery carriers.

[0092] Conclusion: We have successfully prepared a supramolecular hydrogel with decoupled stiffness and pore size. Compared with traditional hydrogels, this hydrogel has the advantages of controllable pore size, mimicking extracellular matrix components, and a dual-network structure. Furthermore, this hydrogel is convenient to use, simple to operate, and biosafe, making it suitable for applications such as three-dimensional culture and delivery of immune cells, as well as tumor immunotherapy.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered therein.

Claims

1. A method for preparing a biomimetic extracellular matrix porous hydrogel for three-dimensional culture and delivery of dendritic cells, characterized in that: Includes the following steps: (1) Add methacrylic anhydride-modified gelatin and oxidized hyaluronic acid to the basal culture medium and stir for 3-5 h to obtain matrix gel solution 1; wherein the mass percentage of methacrylic anhydride-modified gelatin is 3%-10% and the mass percentage of hyaluronic acid is 0.4%-0.8%; (2) Add photosensitizer 2959 to matrix adhesive solution 1 in step (1) to obtain matrix adhesive solution 2; wherein the mass percentage of photoinitiator is 0.2%~0.5%; (3) Add the prepared temperature-responsive gelatin microspheres to the matrix gel solution 2 in step (2) in proportion, mix by blowing, and obtain a porous hydrogel precursor solution; wherein the ratio of gelatin microspheres to matrix gel solution 2 is 1:1~3:1; (4) Under room temperature conditions, dendritic cells were thoroughly mixed with the hydrogel precursor solution to achieve three-dimensional encapsulation of dendritic cells, resulting in complex 1 with a cell density of 5 × 10⁻⁶ cells / mL. 6 ~1×10 7 Cells / mL sol; (5) Place complex 1 in a cell culture device and cure it under ultraviolet light for 1 min to obtain a three-dimensional culture model of porous hydrogel. After adding complete culture medium, place it in a cell culture box for in vitro culture for 24 h. The method for preparing the temperature-responsive gelatin microspheres includes the following steps: (1) Add gelatin to deionized water and stir at 40~60℃ until completely dissolved to obtain solution 1; (2) Add anhydrous ethanol to solution 1 from step (1). After adding anhydrous ethanol, the reaction system changes from transparent to milky white, resulting in solution 2. The volume ratio of anhydrous ethanol to deionized water is 3:5 to 1:

1. (3) Add poloxamer and gum arabic to solution 2 in step (2) and stir until completely dissolved. Then cool to 20℃~25℃ and stir. After subsequent processing, gelatin microgel is obtained. The mass ratio of gum arabic to gelatin is 0.05~0.1 and the mass ratio of poloxamer to gelatin is 0.125~0.25.

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