Porous hydroxyapatite composite microsphere for activating T cells in vitro as well as preparation method and application of porous hydroxyapatite composite microsphere

Abstract

The invention discloses a porous hydroxyapatite composite microsphere which is prepared by taking liquid paraffin containing an emulsifier as an oil phase and a composite system containing gelatin, a dispersing agent, a polystyrene template and a hydroxyapatite precursor as a water phase in the presence of carbodiimide and N-hydroxysuccinimide ester. The preparation method comprises the following steps: carrying out emulsification cross-linking method curing, freeze drying and high-temperature calcination to form porous hydroxyapatite microspheres, carrying out dopamine hydrochloride self-polymerization reaction on the surfaces of the porous hydroxyapatite microspheres to form first coatings, and loading dendritic cell membrane fragments on the surfaces of the first coatings to prepare the composite microspheres. The composite microsphere simulates a double-signal mechanism of a natural antigen presenting cell, and can significantly improve T cell activation efficiency (CD69 proportion is greater than or equal to 80%) and reduce depletion marker PD-1 expression (less than or equal to 40%). The invention discloses an application of the porous hydroxyapatite composite microsphere in preparation of a reagent for activating T cells in vitro.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical preparations and relates to a porous microsphere and a preparation method and application thereof, and specifically relates to a porous hydroxyapatite composite microsphere capable of activating T cells in vitro and serving as a T cell carrier and a preparation method and application thereof. Background Art

[0002] In vitro activation of T cells plays a key role in cancer immunotherapy, anti-infection and autoimmune disease regulation. In particular, adoptive cell therapy (such as CAR-T) has achieved significant therapeutic effects in the treatment of hematological tumors by expanding and modifying T cells in vitro. However, existing in vitro activation technologies rely on artificial antibodies (such as anti-CD3 / CD28 magnetic beads) or genetic engineering methods, and have problems such as single signal, complex preparation, T cell exhaustion and safety risks. For example, adoptive T cell therapy in clinical practice relies on exogenous cytokines (such as IL-2) to maintain expansion, but long-term stimulation may lead to T cell failure or cytokine storm. In addition, although traditional carriers such as polymer microspheres or nanoparticles can provide physical support, it is difficult to simulate the antigen recognition + co-stimulation "dual signal" mechanism of natural antigen-presenting cells, resulting in low T cell activation efficiency and insufficient proportion of memory subsets.

[0003] In recent years, microsphere carriers have become a research hotspot due to their three-dimensional structure and controllable degradation properties. In order to solve the problem that traditional carriers are difficult to simulate the dual-signal mechanism of natural antigen-presenting cells (APCs), Chinese patent application CN113499322A constructed an injectable microsphere system. This system uses microfluidic technology to prepare methacrylated gelatin (GelMA) porous microspheres, and grafts anti-CD3 / CD28 antibodies on the surface to provide antigen recognition and co-stimulation signals. It also continuously releases IL-15 cytokines through sustained-release nanoparticles to maintain T cell survival and proliferation. In addition, although hydroxyapatite (HAp) microspheres have excellent biocompatibility, traditional HA microsphere preparation methods (such as hydrothermal method and template method) have problems such as uneven particle size distribution and insufficient surface active sites. For example, Chinese patent CN101343054A uses EDTA as a template to prepare high-specific surface area HAp microspheres by a hydrothermal method. The process is cumbersome and it is difficult to introduce a functional coating. This is mainly due to the limited reactivity of the functional groups (mainly -OH) on the HAp surface and the fact that the surface charge state (Zeta potential) is highly dependent on the pH value of the solution. Near physiological pH (7.4), the HAp surface is usually negatively charged. If the coating material is also negatively charged, electrostatic repulsion will be generated, hindering the coating material from approaching and adsorbing to the HAp surface. Summary of the Invention

[0004] The purpose of the present invention is to provide a multifunctional porous hydroxyapatite composite microsphere. The porous hydroxyapatite composite microsphere uses hydroxyapatite as the main raw material and has a first coating and a second coating. The first coating is a polydopamine coating, and the second coating is membrane fragments of mature bone marrow-derived dendritic cells (BMDCs). The porous hydroxyapatite composite microsphere can activate T cells in vitro and serve as a T cell carrier.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] The invention discloses porous hydroxyapatite composite microspheres. The porous hydroxyapatite composite microspheres are prepared by using liquid paraffin containing an emulsifier as the oil phase and a composite system containing gelatin, a dispersant, a polystyrene (PS) template and a hydroxyapatite precursor as the aqueous phase. In the presence of carbodiimide and N-hydroxysuccinimide ester, the porous hydroxyapatite microspheres are solidified by an emulsification cross-linking method, freeze-dried and calcined at high temperature. A first coating is then formed on the surface of the porous hydroxyapatite microspheres through a self-polymerization reaction of dopamine hydrochloride. The membrane fragments of mature bone marrow-derived dendritic cells (BMDCs) are loaded on the surface of the first coating to prepare the composite microspheres.

[0007] The emulsifier is Span-80 or Tween 80.

[0008] The dispersant is citric acid or sodium tartrate.

[0009] The hydroxyapatite precursor is nano-hydroxyapatite.

[0010] The polystyrene template is polystyrene microspheres, and the particle size of the polystyrene microspheres is 20-40 μm, preferably 20 μm.

[0011] The carbodiimide is 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) or its hydrochloride (EDC·HCl).

[0012] After high-temperature calcination, only hydroxyapatite remains. The surface of the porous hydroxyapatite microspheres has a uniformly distributed pore structure with a pore size range of 10-20 μm.

[0013] The first coating is a polydopamine coating.

[0014] The membrane fragments of the mature bone marrow-derived dendritic cells are loaded on the surface of the polydopamine coating through the adhesion and electrostatic adsorption of polydopamine.

[0015] The porous hydroxyapatite composite microspheres are regular spherical in shape, and have a high-density, evenly distributed pore structure on their surface. The pore diameter is 5-20 μm, and the porosity is about 28%.

[0016] Another object of the present invention is to provide a method for preparing porous hydroxyapatite composite microspheres, comprising the following steps:

[0017] Step (1) preparing porous hydroxyapatite microspheres using an emulsification crosslinking method: at a temperature of 50-60°C, gelatin, a dispersant and pure water are prepared into a uniform solution, and then a polystyrene template and a hydroxyapatite precursor are added to obtain a uniform hydroxyapatite composite system; at a temperature of 50-60°C, liquid paraffin and an emulsifier are mixed uniformly to obtain an oil phase containing an emulsifier; the hydroxyapatite composite system is added dropwise to the oil phase containing the emulsifier, stirred and emulsified, cooled to 0-4°C, carbodiimide and N-hydroxysuccinimide ester are added to the system, and the mixture is reacted at 0-4°C for 1.5-2 hours to crosslink and solidify the microspheres; after the reaction is completed, the mixture is allowed to stand and stratify, the precipitate is washed with ethanol and deionized water in sequence, freeze-dried, and calcined at high temperature to obtain porous hydroxyapatite (HAp) microspheres;

[0018] Step (2), using Tris buffer to prepare a dopamine hydrochloride solution, placing the porous HAp microspheres in the dopamine hydrochloride solution, and at a temperature of 35-40° C., a rotation speed of 270-300 rpm, and in the dark, allowing the dopamine hydrochloride to undergo a self-polymerization reaction for 10-15 hours to form a polydopamine (PDA) coating on the surface of the porous HAp microspheres, thereby obtaining hydroxyapatite (HAp) microspheres coated with a polydopamine coating, wherein the HAp microspheres retain a porous structure on the surface;

[0019] In step (3), the hydroxyapatite microspheres coated with the polydopamine coating are co-incubated with mature bone marrow-derived dendritic cell membrane fragments in PBS buffer, and the dendritic cell membrane fragments are loaded on the surface of the polydopamine coating through the adhesion effect and electrostatic interaction of polydopamine to obtain porous hydroxyapatite composite microspheres.

[0020] In step (1), the ratio of gelatin to pure water is 1:10-2:10 g / mL, preferably 1.5:10 g / mL.

[0021] The mass ratio of the gelatin to the dispersant is 1.5:0.1-1.5:0.2, preferably 1.5:0.1.

[0022] The mass ratio of gelatin to hydroxyapatite precursor is 1.5:2-1.8:2, preferably 1.5:2.

[0023] The mass ratio of the hydroxyapatite precursor to the polystyrene template is 2:0.6-2:1, preferably 2:0.8.

[0024] The volume ratio of the liquid paraffin to the emulsifier is 50:0.5-50:1, preferably 50:0.5.

[0025] The dosage ratio of the hydroxyapatite precursor to liquid paraffin is 1.5:50-2.5:50 g / mL, preferably 2:50 g / mL.

[0026] The stirring emulsification is as follows: at a temperature of 50-60°C, the hydroxyapatite composite system is slowly added dropwise to the oil phase containing the emulsifier, and emulsified for 20-30 minutes under stirring at 280-300 rpm, while maintaining the stirring speed, cooling the system to 0-4°C while stirring, and continuing to stir at 0-4°C for 15-20 minutes.

[0027] The mass ratio of the carbodiimide to the hydroxyapatite precursor is 1:2-1.5:2, preferably 1.2:2; the mass ratio of the N-hydroxysuccinimide ester to the hydroxyapatite precursor is 0.02:2-0.06:2, preferably 0.04:2.

[0028] The carbodiimide is added in the form of an aqueous solution, and the concentration of the carbodiimide aqueous solution is 1.2 g / mL; the N-hydroxysuccinimide ester is added in the form of an aqueous solution, and the concentration of the N-hydroxysuccinimide ester aqueous solution is 40 mg / mL.

[0029] The temperature of the high-temperature calcination is 800-1000° C., preferably 800° C.; the time of the high-temperature calcination is 2-3 hours, preferably 2 hours.

[0030] Preferably, the high-temperature calcination is: heating from room temperature to 800-1000°C at a heating rate of 5°C / min, and calcining at 800-1000°C for 2-3 hours.

[0031] More preferably, the high-temperature calcination is: heating from room temperature to 800° C. at a heating rate of 5° C. / min, and calcining at 800° C. for 2 hours.

[0032] In step (2), the pH of the Tris buffer is 8.5.

[0033] The concentration of the dopamine hydrochloride solution is 2 mg / mL.

[0034] Preferably, the temperature of the self-polymerization reaction is 37° C., and the time of the self-polymerization reaction is 12 hours.

[0035] Preferably, after the reaction is completed, the microspheres are collected by centrifugation, washed with pure water, and freeze-dried to obtain hydroxyapatite microspheres coated with a polydopamine coating.

[0036] In step (3), the co-incubation conditions are as follows: 30-50 mg of hydroxyapatite microspheres coated with polydopamine are suspended in 10 mL of pH 7.4 PBS buffer, EDC·HCl and NHS are added to a final concentration of EDC·HCl of 5 mM and a final concentration of NHS of 2.5 mM, and activated by shaking at room temperature for 1 hour; then, a suspension of bone marrow-derived dendritic cell membrane fragments resuspended in 1 mL of pH 7.4 PBS buffer at a concentration of 1 mg / mL is added, and the mixture is co-incubated at a temperature of 0-4°C for 10-12 hours.

[0037] Preferably, after the co-incubation is completed, the mixture is centrifuged, the supernatant is discarded, and the precipitate is washed with PBS to remove unbound membrane fragments to obtain porous hydroxyapatite composite microspheres.

[0038] The present invention achieves stable loading of second coating film fragments through the strong adhesion of the first coating PDA, thereby obtaining multifunctional microspheres with porous structure, immune signal delivery and biocompatibility.

[0039] Another object of the present invention is to provide the use of the porous hydroxyapatite composite microspheres in preparing a reagent for activating T cells in vitro.

[0040] Preferably, the application includes: combining the porous hydroxyapatite composite microspheres with CD3 + T cells were co-cultured in RPMI-1640 complete medium (10% fetal bovine serum, 1% double-antibody) supplemented with 20 ng / mL IL-2 and 50 μM β-mercaptoethanol at 37°C in a 5% CO2 incubator. Dendritic cell membrane fragments on the surface of the composite microspheres provided antigen recognition and co-stimulatory signals, activating T cells and reducing dependence on exogenous cytokines.

[0041] The co-culture ratio of the porous hydroxyapatite composite microspheres and T cells is 10 mg of porous hydroxyapatite composite microspheres: 10,000 T cells, and the activated index CD69 ratio is ≥80%, and the PD-1 ratio is ≤40%.

[0042] Another object of the present invention is to provide the use of the porous hydroxyapatite composite microspheres in the preparation of reagents suitable for adoptive T therapy, tumor immunotherapy or functional regulation of T cells in autoimmune diseases.

[0043] Beneficial effects of the present invention:

[0044] The present invention uses an emulsion cross-linking method combined with a polystyrene template to create microspheres with a uniform porous structure. The microspheres are then sequentially modified with a polydopamine coating and dendritic cell membrane fragments to mimic the "dual signaling" mechanism of natural antigen-presenting cells. These microspheres significantly enhance T cell activation efficiency (CD69 ratio ≥80%) and reduce expression of the exhaustion marker PD-1 (≤40%).

[0045] The present invention ensures that the microspheres have biocompatibility, controllable degradation and stability through preparation process optimization (such as calcination temperature of 800°C and coating reaction time of 12h), and are suitable for adoptive T therapy and tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 : SEM images of hydroxyapatite microspheres prepared with different amounts of pore-forming beads.

[0047] Figure 2 : SEM image of porous hydroxyapatite microspheres.

[0048] Figure 3 : XRD diffraction patterns of HAp microspheres and standard hydroxyapatite.

[0049] Figure 4 : SEM images of PDA coatings produced by reaction with dopamine hydrochloride at different times; from left to right, they are reaction times of 6, 12, and 24 hours.

[0050] Figure 5 : Enlarged SEM image of local pores in the PDA coating after reaction with dopamine hydrochloride for 12 hours.

[0051] Figure 6 : Thermogravimetric analysis of hydroxyapatite microspheres.

[0052] Figure 7 : BCA standard curve (A) and the concentration of BMDC membrane fragments stock solution and the concentration of membrane fragments after incubation with microspheres (B).

[0053] Figure 8 : Images captured under an inverted fluorescence microscope after CFSE-stained T cells were co-cultured with microspheres for 24 hours.

[0054] Figure 9 : Flow cytometric images of CD69 detected after 24 h of stimulation of T cells with blank, PDA single-coated microspheres, double-coated composite microspheres and CD3 / CD28 magnetic beads.

[0055] Figure 10 : Flow cytometric images of PD-1 detected after 72 hours of stimulation of T cells using blank, double-coated composite microspheres, and CD3 / CD28 magnetic beads. DETAILED DESCRIPTION

[0056] The technical solutions of the present invention are further described below through specific examples. However, the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0057] Example 1

[0058] 1. Effect of the number of pore-forming beads on the porous structure of HAp microspheres

[0059] At a temperature of 50-60°C, 1.5g of gelatin was dissolved in 10mL of pure water and stirred continuously for 30 minutes. Then, 100mg of citric acid was added as a dispersant and mixed thoroughly to form a uniform solution. Subsequently, 2g of nano-hydroxyapatite (particle size <100nm, the same below) and different amounts (600mg, 800mg, 1000mg) of polystyrene (PS) microspheres with a particle size of 20μm (pore-forming agent) were added to the solution and stirred until a uniform hydroxyapatite composite system was formed.

[0060] 50 mL of liquid paraffin (CAS: 8042-47-5) was taken as the oil phase, 0.5 mL of Span-80 was added as the emulsifier, and the mixture was stirred and mixed at a temperature of 50° C. to obtain an oil phase containing the emulsifier.

[0061] Porous hydroxyapatite (HAp) microspheres were generated by an emulsion cross-linking method: the HA composite was slowly added dropwise to an oil phase containing an emulsifier, stirred at 300 rpm for 20 minutes, and then cooled to 4°C and stirred for 15 minutes to stabilize the emulsion. A 1.2 g / mL aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and a 40 mg / mL aqueous solution of N-hydroxysuccinimide ester (NHS) were added to the mixture, and the mixture was allowed to react at 4°C for 1.5 hours to cross-link and solidify the microspheres. After the reaction, the precipitate was allowed to stand and separate. The precipitate was washed with anhydrous ethanol and deionized water, freeze-dried, and placed in a muffle furnace. The temperature was increased to 800°C at a rate of 5°C / min and calcined at 800°C for 2 hours to obtain porous HAp microspheres.

[0062] Porous microspheres were generated by emulsion crosslinking (liquid paraffin as the oil phase, gelatin solution as the water phase, stirring at 50°C and 300 rpm). Scanning electron microscopy (SEM) analysis showed that when the addition amount of PS microspheres was 600 mg, the pores on the surface of HAp microspheres were unevenly distributed, with significant blank areas ( Figure 1 When 1000 mg was added, the pore-forming agent was excessively aggregated due to electrostatic adhesion of PS microspheres, and the pores were mainly concentrated inside the microspheres ( Figure 1 When the addition amount is increased to 800 mg, a uniform and dense pore structure is formed on the surface of the microspheres, completely covering the spherical surface ( Figure 2The results show that 800 mg PS microspheres is the optimal amount of pore-forming agent, which can form a high-density and uniformly distributed pore structure on the surface of HAp microspheres.

[0063] Hydroxyapatite (HAp) microspheres were prepared according to the above preparation method using the optimal pore-forming agent addition amount of 800 mg PS microspheres. The microsphere structure and composition were characterized. Scanning electron microscopy images show that the HAp microspheres are regular spherical (diameter 400-500 μm) with a porous structure of 10-20 μm pores evenly distributed on the surface ( Figure 2 The X-ray diffraction (XRD) pattern completely matches that of standard hydroxyapatite, and the peak positions (2θ=25.9°, 31.8°, 39.8°) are free of impurity peaks, confirming that the microspheres after calcination are composed of a single crystalline phase of HAp with a purity of 95% ( Figure 3 ).

[0064] 2. Effect of reaction time on polydopamine coating

[0065] Hydroxyapatite (HAp) microspheres were prepared by using the optimal pore-forming agent addition amount of 800 mg PS microspheres according to the preparation method of "I. Effect of the number of pore-forming beads on the porous structure of HAp microspheres".

[0066] A 2 mg / mL dopamine hydrochloride solution was prepared using pH 8.5 Tris buffer. Hydroxyapatite (HAp) microspheres were immersed in the 2 mg / mL dopamine hydrochloride solution and reacted in a constant temperature shaker at 37°C and 270 rpm for 6, 12, and 24 hours, respectively, to construct a polydopamine (PDA) coating. After the reaction, the microspheres were washed three times with pure water and freeze-dried to obtain hydroxyapatite microspheres coated with polydopamine.

[0067] The morphology of the coatings prepared at different reaction times was analyzed by scanning electron microscopy (SEM). Figure 4 、 Figure 5 As shown, it can be seen that: after 6 hours of reaction, the surface of the microspheres is only partially covered with the PDA coating, and the HAp structure is still partially exposed (porosity of about 35%); after 12 hours of reaction, the entire surface of the microspheres is evenly coated with the PDA coating, and the surface pore structure is completely retained (porosity of about 28%); after 24 hours of reaction, although the entire surface of the microspheres is covered with the PDA layer, the thickness of the PDA layer is uneven, and the porosity is significantly reduced to below 15%. At the same time, after 12 hours of reaction, the pore diameter (5-20μm) is the smallest difference from the uncoated HAp microspheres (10-20μm). Taking into account the coating integrity and structural fidelity, 12 hours was determined to be the optimal reaction time to ensure a balance between the efficient adhesion function of the PDA coating and the porous properties of the microspheres.

[0068] Example 2

[0069] A porous hydroxyapatite composite microsphere is prepared by the following method, the steps of which are as follows:

[0070] At a temperature of 50°C, 1.5g of gelatin was dissolved in 10mL of pure water and stirred continuously for 30 minutes. Then, 100mg of citric acid was added as a dispersant and mixed thoroughly to form a uniform solution. Subsequently, 2g of nanohydroxyapatite (HAp) and 800mg of polystyrene (PS) microspheres with a particle size of 20μm were added to the solution and stirred until a uniform hydroxyapatite composite system was formed.

[0071] Take 50 mL of liquid paraffin as the oil phase, add 0.5 mL of Span-80 as an emulsifier, and stir and mix at a temperature of 50° C. to obtain an oil phase containing the emulsifier.

[0072] At 50°C and 300 rpm, the hydroxyapatite composite was slowly added dropwise to the oil phase containing the emulsifier while stirring. After the addition was complete, the mixture was stirred at 300 rpm for 20 minutes. The mixture was then cooled to 4°C while stirring and continued at 4°C for 15 minutes to stabilize the emulsion. Then, 1 mL of a 1.2 g / mL aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 1 mL of a 40 mg / mL aqueous solution of N-hydroxysuccinimide ester (NHS) were added to the mixture. The mixture was reacted at 4°C for 1.5 hours to crosslink and solidify the microspheres. After the reaction, the mixture was allowed to stand for separation. The precipitate was washed with anhydrous ethanol and deionized water, followed by freeze-drying. The mixture was then heated in a muffle furnace from room temperature to 800°C at a rate of 5°C / min and calcined at 800°C for 2 hours to obtain hydroxyapatite (HAp) microspheres.

[0073] Determination of calcination temperature

[0074] Thermogravimetric analysis (TGA) results are shown in Figure 6 , showing that: when the temperature rises to 800 °C, the mass loss of the microspheres tends to be stable (the residual mass accounts for 90%), indicating that the organic components (gelatin, PS microspheres) have been completely decomposed, so 800-1000 °C is selected as the final calcination temperature.

[0075] A 2 mg / mL dopamine hydrochloride solution was prepared in Tris buffer (pH 8.5). Hydroxyapatite microspheres were immersed in the 2 mg / mL dopamine hydrochloride solution and reacted for 12 hours at 37°C and 270 rpm on a constant temperature shaker to form a polydopamine (PDA) coating. After the reaction, the microspheres were collected by centrifugation at 1000 rpm for 3 minutes, washed three times with pure water, and freeze-dried to obtain polydopamine-coated hydroxyapatite microspheres (denoted as HAp-PDA microspheres).

[0076] Preparation of bone marrow-derived dendritic cell (BMDC) membrane fragments: BALB / c mice aged 4-5 weeks were killed by cervical dislocation and the body surface was disinfected by soaking in 75% ethanol for 5 minutes. The hind legs were aseptically removed in a clean bench, and the muscles around the femur and tibia were stripped (to avoid damaging the bones). The bones were disinfected by soaking in 75% ethanol for 1 minute and washed twice with PBS. The two ends of the bones were cut off, and the bone marrow cavity was repeatedly rinsed with PBS until it turned white. The bone marrow fluid was collected and the cell clumps were blown off with a pipette. The bone marrow suspension was filtered through a 70μm filter and centrifuged at 1500rpm for 5 minutes. The supernatant was discarded; 1mL of red blood cell lysis buffer was added for 5 minutes, and the cells were neutralized by adding 5mL of PBS and centrifuged. The supernatant was discarded; the cells were washed once with PBS and resuspended in RPMI-1640 complete medium containing 10% FBS, 1% penicillin-streptomycin, 20ng / mL IL-4 and 10ng / mL GM-CSF to adjust the concentration to 1×10 6 Cells were cultured at 37°C in a 5% CO2 incubator. Half of the medium was exchanged on the second and fourth days: half of the culture medium was collected, centrifuged, the supernatant was discarded, and the same volume of fresh RPMI-1640 complete medium was added to resuspend the cells. The culture dish was washed with PBS precooled to 4°C, and the cell suspension was added back to the culture dish. The cells were collected on the seventh day of culture. Cell membrane fragments were extracted using a membrane protein extraction kit (Biyuntian): the cell pellet (approximately 1×10 8 cells), 1 mL of the cell suspension was transferred to a centrifuge tube, 900 μL of Reagent A (membrane protein extraction reagent) and 100 μL of PMSF (100 mM) were added, the mixture was mixed, and the tube was transferred to ice for incubation for 40 min. After the incubation, the sample was quickly frozen in liquid nitrogen and thawed at room temperature, and this freeze-thaw cycle was repeated three times. Subsequently, the tube was centrifuged at 4°C and 800 rpm for 10 min, and the supernatant was collected. The supernatant was further centrifuged at 4°C and 12,000 rpm for 30 min, and the supernatant was discarded. The resulting precipitate was the bone marrow-derived dendritic cell membrane fragments. The bone marrow-derived dendritic cell membrane fragments were resuspended in PBS buffer (pH 7.4) to a concentration of 1 mg / mL, and the resuspended bone marrow-derived dendritic cell membrane fragments were stored at -20°C.

[0077] Coupling of BMDC membrane fragments to polydopamine-coated hydroxyapatite microspheres

[0078] 30 mg of HAp-PDA microspheres were resuspended in 10 mL of pH 7.4 PBS buffer, and EDC·HCl and NHS were added sequentially to final concentrations of 5 mM and 2.5 mM, respectively, and activated at room temperature with shaking for 1 h. 1 mL of 1 mg / mL bone marrow-derived dendritic cell membrane fragment resuspension was then added, and the reaction was shaken at 4°C for 12 h for coupling. After the reaction, the microspheres were centrifuged at 1000 rpm for 5 min, and the supernatant was used as the membrane solution of experimental group 2. The precipitate was washed three times with PBS to remove unbound membrane fragments, resulting in double-coated hydroxyapatite composite microspheres, which were resuspended in PBS and stored at 4°C in the dark.

[0079] Confirmation of BMDC membrane fragment coupling concentration: The concentration of BMDC membrane protein was quantified using a BCA protein concentration detection kit. The steps are as follows: The bovine serum albumin (BSA) standard was diluted with PBS buffer in a gradient (0-2000 μg / mL). Experimental group 1 was the BMDC membrane mother solution (i.e., a resuspension of bone marrow-derived dendritic cell membrane fragments at a concentration of 1 mg / mL), and experimental group 2 was the membrane solution collected by centrifugation at 500g after coupling with hydroxyapatite microspheres coated with polydopamine for 12 hours. Take 25 μL of BSA standard or sample and add it to a 96-well plate. Add 200 μL of freshly prepared BCA working solution (volume ratio of reagent A:B = 50:1) to each well, blow gently to mix well, incubate at 37°C in the dark for 30 minutes, and measure the absorbance (OD value) at a wavelength of 562 nm using a microplate reader. Fit the standard curve (R) with the BSA standard concentration (X) and the OD value (Y) after deducting the blank well. 2 >0.99), and the sample protein concentration was calculated according to the regression equation. Figure 7 As shown in the figure, the concentration of BMDC mother solution before co-incubation was about 954 μg / mL, and the concentration dropped to 443 μg / mL after co-incubation with microspheres, indicating that BMDC membranes were successfully coupled to the hydroxyapatite microspheres covered with polydopamine coating.

[0080] Example 3

[0081] Porous HAp composite microspheres as T cell carriers

[0082] The porous hydroxyapatite (HAp) composite microspheres prepared in Example 2 were co-cultured with CFSE (carboxyfluorescein succinimidyl ester)-stained T cells in RPMI-1640 medium containing 10% fetal bovine serum for 48 hours and observed under an inverted fluorescence microscope (eg, Figure 8 As shown in the figure, a large number of fluorescently labeled T cells were tightly adsorbed on the surface of the microspheres and the surrounding pore areas, forming an obvious aggregation phenomenon, indicating that the microspheres have excellent cell adhesion ability and provide an efficient and stable carrier platform for in vitro T cell expansion.

[0083] Example 4

[0084] Effects of porous HAp composite microspheres on T cell activation in vitro

[0085] BALB / c mice aged 4-5 weeks were taken and killed by cervical dislocation. The mice were immersed in 75% ethanol for 5 minutes for disinfection and fixed on a sterile operating table. The spleen was aseptically removed and placed in PBS pre-cooled to 4°C. After stripping the capsule, the spleen was transferred to a 70μm cell sieve, gently ground and rinsed with PBS to collect the cell suspension; the cell suspension was centrifuged at 1500rpm for 5 minutes, the supernatant was discarded, 2mL of red blood cell lysis buffer was added to the pellet and pipetted to mix, lysed at room temperature for 10 minutes, 10mL of PBS was added to terminate the reaction, centrifuged again, the supernatant was discarded, and the cells were resuspended in 1mL FACS buffer. CD3 + T cell sorting kit was used for magnetic bead sorting, and the purified CD3 + T cells were resuspended in RPMI-1640 complete medium (containing 10% fetal bovine serum and 1% double antibody) containing 20 ng / mL IL-2 and 50 μM β-mercaptoethanol, and expanded in a 37° C., 5% CO 2 incubator until ready for use.

[0086] CD3 + T cells were cultured in RPMI-1640 complete medium (10% fetal bovine serum, 1% double-antibody) supplemented with 20 ng / mL IL-2 and 50 μM β-mercaptoethanol in a 37°C, 5% CO2 incubator. The amount of microspheres added to each group was 10 mg, and the number of T cells was approximately 1×10 5 Specifically: Experimental Group 1: Hydroxyapatite microspheres coated with polydopamine (referred to as HAp-PDA microspheres) prepared in Example 2 were mixed with CD3 isolated from mouse spleen. + T cells were co-cultured for 24 hours; Experimental group 2: The hydroxyapatite composite microspheres with double coating prepared in Example 2 were co-cultured with CD3 T cells isolated from mouse spleen. + T cells were co-cultured for 24 hours; positive control group (anti-CD3 / CD28 magnetic bead activation group): anti-CD3 / CD28 magnetic beads were co-cultured with CD3 isolated from mouse spleen. + T cells were co-cultured for 24 hours; blank group: no reagent was added, CD3 isolated from mouse spleen + T cells were cultured for 24 hours; the expression of early activation marker CD69 was detected by flow cytometry. Figure 9, showing that: the proportion of CD69 in experimental group 1 was 3.22%, slightly higher than 1.09% in the blank group; the proportion of CD69 in experimental group 2 was 88.2%, slightly higher than 76.0% in the positive control group and significantly higher than 1.09% in the blank group, indicating that the HAp composite microspheres with double coatings have efficient T cell activation ability. After further culture for 72 hours, the expression of PD-1, a T cell exhaustion marker, was detected. The results are shown in Figure 10 , showing that the PD-1 proportion of experimental group 2 was 39.5%, which was lower than 44.7.3% of the positive control group and 55.2% of the blank group, confirming that the process of porous HAp composite microspheres activating T cells through the bionic "dual signal" mechanism is more gentle, which can effectively delay cell exhaustion and provide a new carrier with both high efficiency and safety for in vitro T cell expansion.

Claims

1. A porous hydroxyapatite composite microsphere, characterized in that: The method uses liquid paraffin containing an emulsifier as the oil phase and a composite system containing gelatin, a dispersant, a polystyrene template and a hydroxyapatite precursor as the water phase. In the presence of carbodiimide and N-hydroxysuccinimide ester, porous hydroxyapatite microspheres are formed by emulsification cross-linking solidification, freeze-drying and high-temperature calcination. A first coating is then formed on the surface of the porous hydroxyapatite microspheres through the self-polymerization reaction of dopamine hydrochloride, and membrane fragments of dendritic cells are loaded on the surface of the first coating to prepare composite microspheres.

2. The porous hydroxyapatite composite microspheres according to claim 1, characterized in that: The emulsifier is Span 80 or Tween 80; the dispersant is citric acid or sodium tartrate; the hydroxyapatite precursor is nano-hydroxyapatite; the polystyrene template is polystyrene microspheres; and the carbodiimide is 1-ethyl-(3-dimethylaminopropyl)carbodiimide or its hydrochloride.

3. The porous hydroxyapatite composite microspheres according to claim 2, characterized in that: The particle size of the polystyrene microspheres is 20-40 μm, preferably 20 μm.

4. The porous hydroxyapatite composite microspheres according to claim 1, characterized in that: The porous hydroxyapatite composite microspheres are regular spherical in shape, have a uniformly distributed pore structure on the surface, and the pore diameter is 5-20 μm.

5. A method for preparing the porous hydroxyapatite composite microspheres according to claim 1, characterized in that: The following steps are involved: Step (1) preparing porous hydroxyapatite microspheres using an emulsion crosslinking method: gelatin, a dispersant, and pure water are prepared into a uniform solution at a temperature of 50-60° C., and a polystyrene template and a hydroxyapatite precursor are added to obtain a uniform hydroxyapatite composite system; At a temperature of 50-60°C, liquid paraffin and an emulsifier are uniformly mixed to obtain an oil phase containing the emulsifier; a hydroxyapatite composite system is dropwise added to the oil phase containing the emulsifier, stirred and emulsified, and the temperature is lowered to 0-4°C, carbodiimide and N-hydroxysuccinimide ester are added to the system, and the reaction is carried out at 0-4°C for 1.5-2 hours to cross-link and solidify the microspheres; after the reaction is completed, the system is allowed to stand and separate, and the precipitate is washed with ethanol and deionized water in sequence, freeze-dried, and calcined at high temperature to obtain porous hydroxyapatite microspheres; Step (2), using Tris buffer to prepare a dopamine hydrochloride solution, placing the porous hydroxyapatite microspheres in the dopamine hydrochloride solution, and allowing the dopamine hydrochloride to undergo a self-polymerization reaction for 10-15 hours at a temperature of 35-40° C., a rotation speed of 270-300 rpm, and in the dark, to form a polydopamine coating on the surface of the porous hydroxyapatite microspheres, thereby obtaining hydroxyapatite microspheres coated with the polydopamine coating; In step (3), the hydroxyapatite microspheres coated with the polydopamine coating are co-incubated with mature bone marrow-derived dendritic cell membrane fragments in PBS buffer, and the dendritic cell membrane fragments are loaded on the surface of the polydopamine coating through the adhesion effect and electrostatic interaction of polydopamine to obtain porous hydroxyapatite composite microspheres.

6. The method for preparing porous hydroxyapatite composite microspheres according to claim 5, characterized in that: In step (1), the ratio of gelatin to pure water is 1:10-2:10 g / mL, preferably 1.5:10 g / mL; The mass ratio of gelatin to dispersant is 1.5:0.1-1.5:0.2, preferably 1.5:0.1; The mass ratio of gelatin to hydroxyapatite precursor is 1.5:2-1.8:2, preferably 1.5:2; The mass ratio of the hydroxyapatite precursor to the polystyrene template is 2:0.6-2:1, preferably 2:0.8; The volume ratio of the liquid paraffin to the emulsifier is 50:0.5-50:1, preferably 50:0.5; The dosage ratio of the hydroxyapatite precursor and liquid paraffin is 1.5:50-2.5:50 g / mL, preferably 2:50 g / mL; The mass ratio of the carbodiimide to the hydroxyapatite precursor is 1:2-1.5:2, preferably 1.2:2; the mass ratio of the N-hydroxysuccinimide ester to the hydroxyapatite precursor is 0.02:2-0.06:2, preferably 0.04:

2.

7. The method for preparing porous hydroxyapatite composite microspheres according to claim 5, characterized in that: In step (1), the stirring and emulsification: at a temperature of 50-60°C, the hydroxyapatite composite system is slowly added dropwise to the oil phase containing the emulsifier, and emulsified for 20-30 minutes under stirring at 280-300 rpm, while maintaining the stirring speed, cooling the system to 0-4°C while stirring, and continuing to stir at 0-4°C for 15-20 minutes; the temperature of the high-temperature calcination is 800-1000°C, preferably 800°C; the time of the high-temperature calcination is 2-3 hours, preferably 2 hours.

8. The method for preparing porous hydroxyapatite composite microspheres according to claim 5, characterized in that: In step (2), the pH of the Tris buffer is 8.5; the concentration of the dopamine hydrochloride solution is 2 mg / mL; the temperature of the self-polymerization reaction is 37° C., and the time of the self-polymerization reaction is 12 h; In step (3), the co-incubation conditions are as follows: 30-50 mg of hydroxyapatite microspheres coated with polydopamine are suspended in 10 mL of pH 7.4 PBS buffer, EDC·HCl and NHS are added to a final concentration of EDC·HCl of 5 mM and a final concentration of NHS of 2.5 mM, and activated by shaking at room temperature for 1 hour; then, a suspension of bone marrow-derived dendritic cell membrane fragments resuspended in 1 mL of pH 7.4 PBS buffer at a concentration of 1 mg / mL is added, and the mixture is co-incubated at a temperature of 0-4°C for 10-12 hours.

9. Use of the porous hydroxyapatite composite microspheres according to claim 1 in preparing a reagent for activating T cells in vitro.

10. Use of the porous hydroxyapatite composite microspheres according to claim 1 in the preparation of a reagent suitable for adoptive T therapy, tumor immunotherapy or functional regulation of T cells in autoimmune diseases.

Images