Method for preparing a dendritic cell membrane protein hybridized nanovaccine

Abstract

This invention discloses a method for preparing a dendritic cell membrane protein hybrid nanovaccine, comprising the following steps: S1: preparation of primary dendritic cell membrane proteins; S2: preparation of zinc hydroxyphosphate nanoparticles co-loaded with antigen peptides and photosensitizer melanin; S3: optimization of the amount of dendritic cell membrane proteins; S4: preparation of the dendritic cell membrane protein hybrid nanovaccine; the homologous targeting properties of dendritic cell membrane proteins can be utilized to achieve efficient capture of the vaccine by dendritic cells. Combined with the mild photothermal effect generated by near-infrared irradiation of melanin, compared with the killing effect of traditional high heat on immune cells, the mild photothermal effect has good biocompatibility and can induce an effective anti-tumor immune response.

Description

Technical Field

[0001] This invention relates to the field of nanovaccine technology, and in particular to a method for preparing a nanovaccine with dendritic cell membrane protein hybridization. Background Technology

[0002] Dendritic cells (DCs) play a crucial role in tumor immunotherapy because they initiate, regulate, and maintain specific T-cell immune responses. In 2010, the U.S. Food and Drug Administration approved Provenge, the first dendritic cell vaccine in human history, for the treatment of prostate cancer. However, the production process for this DC vaccine is complex and expensive, costing up to $93,000 for a single treatment cycle.

[0003] Furthermore, dendritic cells (DCs) are prone to inactivation or even death during transplantation, resulting in a low number of viable cells actually reaching the lymph nodes, thus limiting the clinical efficacy of the vaccine. Compared to DC vaccines produced in vitro, immunologically active subunit vaccines can deliver tumor antigens to DCs in vivo to achieve in situ DC maturation and induce a durable and efficient specific T-cell response. With advancements in vaccine preparation technology, it is now possible to obtain tumor-specific antigen peptides through biological or chemical means. These peptides have advantages such as high purity, absence of toxic or infectious agents, and direct presentation by DCs without intracellular processing. Nanotechnology-based carrier systems can effectively load antigen peptides, protecting them from degradation in vivo and extending their half-life. Introducing DC cell membrane proteins into nanosystems to construct hybrid systems can utilize the homologous targeting effect of cell membranes to promote DC antigen capture and achieve precise vaccine delivery.

[0004] After dendritic cells (DCs) capture antigens, they need to process the antigens and migrate to lymphoid organs to present them to T cells. Near-infrared (NIR) irradiation of the photosensitizer, compared to the high heat (>42°C) used in traditional methods that kill immune cells, produces a milder photothermal effect (38–42°C), demonstrating good biocompatibility and promoting antigen processing and return to lymphoid tissues. Therefore, it is necessary to develop novel dendritic cell membrane protein hybrid nanovaccines to solve the aforementioned technical problems. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing a dendritic cell membrane protein hybrid nanovaccine, which can utilize the homologous targeting properties of dendritic cell membrane proteins to achieve efficient capture of the vaccine by dendritic cells. Combined with the mild photothermal effect generated by near-infrared irradiation of melanin, compared with the killing effect of traditional high heat on immune cells, the mild photothermal effect has good biosafety and can induce an effective anti-tumor immune response.

[0006] To achieve the above objectives, the present invention provides a method for preparing a dendritic cell membrane protein hybrid nanovaccine, comprising the following steps:

[0007] S1: Preparation of primary dendritic cell membrane proteins;

[0008] S2: Preparation of zinc hydroxyphosphate nanoparticles co-loaded with antigenic peptides and photosensitizer melanin;

[0009] S3: Optimize dendritic cell membrane protein levels;

[0010] S4: A dendritic cell membrane protein hybrid nanovaccine was prepared.

[0011] Preferably, the preparation process of zinc hydroxyphosphate nanoparticles co-loaded with antigen peptides and photosensitizer melanin in step S2 is as follows:

[0012] The antigenic polypeptide was weighed and dissolved in Zn(NO3)2 to prepare a polypeptide stock solution. The polypeptide stock solution was then added to the oil phase composed of cyclohexane and Igepal CO-520.

[0013] Melanin and DOPA (dioleoylphosphatidic acid) were added to the oil phase to prepare a drug-containing Zn phase proemulsion.

[0014] The P-phase primary emulsion was prepared by adding the oil phase and dioleoyl phosphatidic acid to the Na2HPO4 solution. The P-phase primary emulsion was added dropwise to the Zn-phase primary emulsion and reacted at room temperature. After demulsification and washing with anhydrous ethanol, the drug-loaded phospholipid-coated zinc hydroxyphosphate nanoparticles were collected and redispersed with chloroform.

[0015] Preferably, the specific process for optimizing dendritic cell membrane proteins in step S3 is as follows:

[0016] The chloroform was removed by spin-coating unloaded phospholipid-coated zinc hydroxyphosphate nanoparticles together with dioleoylphosphatidylcholine, cholesterol and DSPE-PEG2000.

[0017] Dendritic cell membrane proteins were added to phosphate buffer at different protein / phospholipid weight ratios. The phosphate buffer was then added to a rotary evaporator and vortexed vigorously. The mixture was then subjected to ultrasonic treatment in a water bath and incubated in a constant temperature water bath to obtain dendritic cell membrane protein hybrid hydroxy zinc phosphate nanoparticles.

[0018] Dendritic cell membrane protein hybrid zinc hydroxyphosphate nanoparticles were obtained by varying amounts of dendritic cell membrane proteins.

[0019] Preferably, the specific process for preparing the dendritic cell membrane protein hybrid nanovaccine in step S4 is as follows: take the polypeptide stock solution and add it to the oil phase composed of cyclohexane and Igepal CO-520, add melanin and dioleoyl phosphatidic acid to the oil phase to prepare the drug-containing Zn phase proemulsion.

[0020] A P-phase primary emulsion was prepared by adding the oil phase and dioleoyl phosphatidic acid to a Na₂HPO₄ solution. After 30-60 minutes, the P-phase primary emulsion was added dropwise to the Zn-phase primary emulsion and reacted for 2-3 hours. After demulsification and washing with anhydrous ethanol, the drug-loaded phospholipid-coated zinc hydroxyphosphate nanoparticles were collected by high-speed centrifugation and redispersed with chloroform.

[0021] Dispersed drug-loaded phospholipid-coated zinc hydroxyphosphate nanoparticles were combined with dioleoylphosphatidylcholine, cholesterol, and DSPE-PEG2000 to remove chloroform by spin coating.

[0022] Dendritic cell membrane proteins were added to phosphate buffer at a protein / phospholipid weight ratio of 1:300, then added to a rotary evaporator and vortexed vigorously for 3-5 minutes. The mixture was then sonicated in a water bath for 3-5 minutes and incubated in a constant temperature water bath at 37°C for 30-60 minutes to obtain a dendritic cell membrane protein hybrid nanovaccine.

[0023] Preferably, the molar ratio of dioleoylphosphatidylcholine, cholesterol, and DSPE-PEG2000 is 4:4:1.

[0024] Preferably, the volume ratio of cyclohexane to Igepal CO-520 in step S2 is 71:29.

[0025] Preferably, the ultrasonic treatment in the water bath in step S3 takes 3-5 minutes, and the constant temperature water bath incubation takes 30-60 minutes.

[0026] Preferably, in step S4, the P-phase colostrum is added dropwise to the Zn-phase colostrum and reacted for 2-3 hours, the rotary evaporator is vortexed vigorously for 3-5 minutes, the water bath is ultrasonically treated for 3-5 minutes, and the constant temperature water bath is incubated for 30-60 minutes.

[0027] Compared with the prior art, the beneficial effects are as follows: The dendritic cell membrane protein hybrid nanovaccine provided in the embodiments of the present invention successfully introduces dendritic cell membrane proteins into the nano system and successfully co-loads antigenic peptides and photosensitizer melanin. Utilizing the unique chelating effect of zinc phosphate nanoparticles, it co-loads antigenic peptide Adpgk and photosensitizer melanin, and further introduces a dendritic cell membrane protein hybrid phospholipid layer on the surface of the nanoparticles.

[0028] This dendritic cell membrane protein hybrid nanovaccine utilizes the homology-targeting properties of dendritic cell membrane proteins to achieve efficient vaccine capture by dendritic cells. Combined with the gentle photothermal effect generated by near-infrared irradiation of melanin, compared to the killing effect of traditional high heat on immune cells, the thermo-induced immune response can effectively inhibit tumor growth and demonstrates good biocompatibility. Furthermore, due to the small size effect of the nanoparticles, the vaccine can directly flow back to the lymph nodes, further enhancing the anti-tumor immune response. Attached Figure Description

[0029] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 Morphology images of phospholipid-coated zinc hydroxyphosphate nanoparticles with different amounts of melanin.

[0031] Figure 2 DSC curves of nanovaccines obtained with different dendritic cell membrane protein amounts.

[0032] Figure 3 The image shows the morphology of dendritic cell membrane protein hybrid zinc hydroxyphosphate nanoparticles according to an embodiment of the present invention under a transmission electron microscope.

[0033] Figure 4 A comparative diagram showing the effect of phospholipid-coated zinc hydroxyphosphate nanoparticles and dendritic cell membrane protein hybrid zinc hydroxyphosphate nanoparticles on uptake by primary dendritic cells.

[0034] Figure 5 Comparative diagram of the distribution of phospholipid-coated zinc hydroxyphosphate nanoparticles and dendritic cell membrane protein hybrid zinc hydroxyphosphate nanoparticles in axillary lymph nodes (ALN) and inguinal lymph nodes (ILN).

[0035] Figure 6 The images show line graphs of tumor changes and immune cell changes in mice obtained after tumor transplantation in different drug administration groups in Example 3 of this invention. Detailed Implementation

[0036] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described in this specification are merely for explaining the invention and are not intended to limit the invention.

[0037] Please see Figure 1 This invention provides a method for preparing a dendritic cell membrane protein hybrid nanovaccine, comprising the following steps:

[0038] S1: Preparation of primary dendritic cell membrane proteins;

[0039] Specifically, mouse bone marrow progenitor cells were aseptically collected and resuspended in a complete culture medium containing granulocyte-macrophage colony-stimulating factor and interleukin-4.

[0040] Seven days later, suspension and semi-adherent cells (i.e., primary dendritic cells) were collected. The semi-adherent cells were then scraped off with a cell scraper, centrifuged at 1500g for 5-10 minutes, washed twice with PBS (phosphate buffer), and the DC cell membrane proteins were extracted using a membrane protein extraction kit.

[0041] S2: Preparation of zinc hydroxyphosphate nanoparticles co-loaded with antigenic peptides and photosensitizer melanin;

[0042] Specifically, weigh out the antigenic polypeptide and dissolve it in Zn(NO3)2 (500mM) to prepare a polypeptide stock solution of 2mg / mL. Take 125μL of the polypeptide stock solution and add it to the oil phase composed of 5mL of cyclohexane and Igepal CO-520 (volume ratio of 71:29).

[0043] 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, and 0.5 mg of melanin (20 mg / mL dissolved in concentrated ammonia) were added to the oil phase, respectively, along with 62.5 μL of 20 mg / mL DOPA (dioleoylphosphatidic acid), to prepare a drug-containing Zn-phase proemulsion.

[0044] A P-phase primary emulsion was prepared by adding the oil phase and DOPA to 125 μL of Na2HPO4 solution (100 mM). After 30-60 minutes, the P-phase primary emulsion was added dropwise to the Zn-phase primary emulsion and reacted for 2-3 hours. After demulsification and washing with anhydrous ethanol, the drug-loaded phospholipid-coated zinc hydroxyphosphate nanoparticles were collected by high-speed centrifugation and redispersed with chloroform. The nanoparticles with the best morphology were obtained when the amount of melanin added was 0.4 mg (e.g., ...). Figure 1 (As shown).

[0045] S3: Optimize the amount of dendritic cell membrane protein added;

[0046] Specifically, unloaded phospholipid-coated zinc hydroxyphosphate nanoparticles were combined with DOPC (dioleoylphosphatidylcholine), cholesterol, and DSPE-PEG2000 (distearate-phosphatidylethanolamine-polyethylene glycol 2000) and chloroform was removed by spin coating, wherein the molar ratio of DOPC, cholesterol, and DSPE-PEG2000 was 4:4:1.

[0047] Dendritic cell membrane proteins were added to phosphate-buffered saline (PBS) at different protein / phospholipid weight ratios (1:0, 1:100, 1:200, 1:300, 1:400). The PBS was then added to a rotary evaporator and vortexed vigorously for 3-5 minutes. The mixture was then sonicated in a water bath for 3-5 minutes and incubated in a constant temperature water bath at 37°C for 30-60 minutes to obtain dendritic cell membrane protein hybrid zinc hydroxyphosphate nanoparticles.

[0048] Dendritic cell membrane protein hybrid zinc hydroxyphosphate nanoparticles obtained with different amounts of dendritic cell membrane proteins were freeze-dried, weighed, and their DSC curves were obtained using differential scanning calorimetry (DSC). Figure 2 (as shown);

[0049] like Figure 2 As shown, the nanosystem exhibits a single peak at protein / phospholipid weight ratios of 1:300 and 1:400, indicating that cell membrane proteins are uniformly inserted into the phospholipid layer on the surface of the nanoparticles. At a ratio of 1:300, a larger amount of dendritic cell membrane proteins can be inserted. Therefore, dendritic cell membrane protein hybrid zinc hydroxyphosphate nanoparticles with a protein / phospholipid weight ratio of 1:300 are selected to prepare dendritic cell membrane protein hybrid nanovaccines.

[0050] S4: A dendritic cell membrane protein hybrid nanovaccine was prepared.

[0051] Specifically, 125 μL of peptide stock solution was added to an oil phase consisting of 5 mL of cyclohexane and Igepal CO-520. 0.4 mg of melanin and 62.5 μL of DOPA (dioleoylphosphatidic acid) were added to the oil phase to prepare a drug-containing Zn-phase proemulsion.

[0052] A P-phase primary emulsion was prepared by adding the oil phase and DOPA (dioleoylphosphatidic acid) to 125 μL of Na2HPO4 solution (100 mM). After 30-60 minutes, the P-phase primary emulsion was added dropwise to the Zn-phase primary emulsion and reacted for 2-3 hours. After demulsification and washing with anhydrous ethanol, the drug-loaded phospholipid-coated zinc hydroxyphosphate nanoparticles were collected by high-speed centrifugation and redispersed with chloroform.

[0053] Dispersed drug-loaded phospholipid-coated zinc hydroxyphosphate nanoparticles were combined with DOPC (dioleoylphosphatidylcholine), cholesterol, and DSPE-PEG2000 (molar ratio 4:4:1) and chloroform was removed by spin coating.

[0054] Dendritic cell membrane proteins were added to PBS (phosphate-buffered saline) at a protein / phospholipid weight ratio of 1:300, then added to a rotary evaporator and vortexed vigorously for 3-5 minutes. The mixture was then sonicated in a water bath for 3-5 minutes and incubated at 37°C for 30-60 minutes to obtain a dendritic cell membrane protein hybrid nanovaccine. The encapsulation efficiencies of this nanovaccine for melanin and antigenic peptides were 87.6±4.6% and 57.2±10.0%, respectively. Its morphology was observed under a transmission electron microscope (e.g., [image of melanin]). Figure 3 (As shown).

[0055] Example

[0056] Example 1: Homologous targeting of dendritic cell membrane protein hybrid nanovaccines;

[0057] Drug-loaded phospholipid-coated zinc hydroxyphosphate nanoparticles and dendritic cell membrane protein-hybridized zinc hydroxyphosphate nanoparticles were labeled with the fluorescent dye DiI. After being treated with primary dendritic cells for 0.5, 1, 2, 4 and 6 hours, the cells were collected, and the targeting effect of the nanoparticles on homologous dendritic cells was detected by laser confocal microscopy and flow cytometry.

[0058] like Figure 4 As shown, the uptake of drug-loaded phospholipid-coated zinc hydroxyphosphate nanoparticles and dendritic cell membrane protein hybrid zinc hydroxyphosphate nanoparticles by dendritic cells is time-dependent. In the short treatment time (0.5 and 1 hour), the homologous targeting effect of dendritic cell membrane protein hybrid zinc hydroxyphosphate nanoparticles is not obvious. With the extension of treatment time, the uptake of dendritic cells by dendritic cells by dendritic cell membrane protein hybrid zinc hydroxyphosphate nanoparticles is significantly stronger than that of drug-loaded phospholipid-coated zinc hydroxyphosphate nanoparticles.

[0059] Example 2: Lymphatic reflux effect of dendritic cell membrane protein hybrid nanovaccine;

[0060] Drug-loaded phospholipid-coated zinc hydroxyphosphate nanoparticles and dendritic cell membrane protein hybrid zinc hydroxyphosphate nanoparticles were labeled with the fluorescent dye DiI and then subcutaneously injected into the axilla and groin of mice, respectively. After 24 hours, the mice were sacrificed, and the axillary lymph nodes (ALN) and groin lymph nodes (ILN) of the mice were dissected, frozen sections were prepared, cell nuclei were stained with DAPI, and observed under a confocal microscope.

[0061] like Figure 5 As shown, compared with drug-loaded phospholipid-coated zinc hydroxyphosphate nanoparticles, dendritic cell membrane protein hybrid zinc hydroxyphosphate nanoparticles have stronger distribution in both ALN and ILN, and can effectively return to the lymph nodes.

[0062] Example 3: Antitumor effect of dendritic cell membrane protein hybrid nanovaccine.

[0063] Please refer to Figure 6 AB was administered to mice via subcutaneous injection of 1.25 × 10⁻⁶ ppm. 5 MC-38 cells were transplanted into mice and then administered the following treatments: phosphate-buffered saline (PBS), PBS + near-infrared irradiation (NIR), free melanin + antigenic peptide (Free M+A), free melanin + antigenic peptide + near-infrared irradiation (Free M+A + NIR), dendritic cell membrane protein hybrid hydroxyzinc phosphate nanoparticles carrying melanin + near-infrared irradiation, dendritic cell membrane protein hybrid hydroxyzinc phosphate nanoparticles carrying antigenic peptide, and dendritic cell membrane protein hybrid hydroxyzinc phosphate nanoparticles carrying both melanin and antigenic peptide + NIR.

[0064] During treatment, the mice's mental and physiological condition was observed, and their weight and tumor growth were monitored. Figure 6As shown in AB, the dendritic cell membrane protein hybrid nanovaccine can effectively inhibit tumor growth and has almost no effect on mouse body weight during the treatment period, demonstrating good biocompatibility.

[0065] Further analysis of tumor-draining lymph nodes and the tumor microenvironment revealed that dendritic cell membrane protein hybrid nanovaccines can effectively promote the maturation of dendritic cells in draining lymph nodes and the number of effector T cells in the tumor microenvironment (e.g., Figure 6 As shown in CD, this nanovaccine can induce a significant anti-tumor immune response.

[0066] The melanin used in the dendritic cell membrane protein hybrid nanovaccine prepared in this embodiment of the invention has strong absorption of near-infrared light and can realize photothermal conversion. At the same time, it also has high biocompatibility and metal ion chelating ability. The Zn ions in the zinc phosphate nanoparticles have unique chelating ability and can effectively chelate multiple drugs to achieve drug loading.

[0067] Utilizing the unique chelating properties of zinc phosphate nanoparticles, antigenic peptides and photosensitizer melanin were co-loaded, and a dendritic cell membrane protein hybrid phospholipid layer was further introduced onto the nanoparticle surface to construct a dendritic cell membrane protein hybrid nanovaccine. This dendritic cell membrane protein hybrid nanovaccine can leverage the homologous targeting properties of dendritic cell membrane proteins to achieve efficient capture of the vaccine by dendritic cells. Combined with the gentle photothermal effect generated by near-infrared irradiation of melanin, compared to the killing effect of traditional high heat on immune cells, the thermo-induced immune response can effectively inhibit tumor growth and demonstrates good biocompatibility.

[0068] The present invention is not limited to the description in the specification and embodiments, and thus other advantages and modifications can be readily realized by those skilled in the art. Therefore, the present invention is not limited to the specific details, representative devices and examples shown and described herein without departing from the spirit and scope of the general concept as defined by the claims and their equivalents.

Claims

1. A method for preparing a nanovaccine hybridized with dendritic cell membrane proteins, characterized in that, Includes the following steps: S1: Preparation of primary dendritic cell membrane proteins; S2: Preparation of zinc hydroxyphosphate nanoparticles co-loaded with antigenic peptide Adpgk and photosensitizer melanin; The antigenic polypeptide Adpgk was weighed and dissolved in Zn(NO3)2 to prepare a polypeptide stock solution. The polypeptide stock solution was then added to the oil phase composed of cyclohexane and Igepal CO-520. Melanin and dioleoylphosphatidic acid were added to the oil phase to prepare a drug-containing Zn phase proemulsion. The P-phase primary emulsion was prepared by adding oil phase and dioleoyl phosphatidic acid to Na2HPO4 solution. The P-phase primary emulsion was added dropwise to the Zn-phase primary emulsion. After demulsification and washing with anhydrous ethanol, the drug-loaded phospholipid-coated zinc hydroxyphosphate nanoparticles were collected and redispersed with chloroform for later use. S3: Optimize dendritic cell membrane protein levels; The chloroform was removed by spin-coating unloaded phospholipid-coated zinc hydroxyphosphate nanoparticles together with dioleoylphosphatidylcholine, cholesterol and DSPE-PEG2000. Dendritic cell membrane proteins were added to phosphate buffer at different protein / phospholipid weight ratios. The phosphate buffer was then added to a rotary evaporator and vortexed vigorously. The mixture was then subjected to ultrasonic treatment in a water bath and incubated in a constant temperature water bath to obtain dendritic cell membrane protein hybrid hydroxy zinc phosphate nanoparticles. Dendritic cell membrane protein hybrid zinc hydroxyphosphate nanoparticles with different dendritic cell membrane protein amounts were freeze-dried and weighed. S4: Dispersed drug-loaded phospholipid-coated zinc hydroxyphosphate nanoparticles were combined with dioleoylphosphatidylcholine, cholesterol, and DSPE-PEG2000 and chloroform was removed by spin coating. Dendritic cell membrane proteins were added to phosphate buffer at a protein / phospholipid weight ratio, added to a rotary evaporator flask and vortexed. The mixture was then subjected to ultrasonic treatment in a water bath and incubated in a constant temperature water bath to prepare a dendritic cell membrane protein hybrid nanovaccine.

2. The method for preparing the dendritic cell membrane protein hybrid nanovaccine as described in claim 1, characterized in that, The molar ratio of dioleoylphosphatidylcholine, cholesterol, and DSPE-PEG2000 is 4:4:

1.

3. The method for preparing the dendritic cell membrane protein hybrid nanovaccine as described in claim 1, characterized in that, The volume ratio of cyclohexane to Igepal CO-520 in step S2 is 71:

29.

4. The method for preparing the dendritic cell membrane protein hybrid nanovaccine as described in claim 1, characterized in that, The ultrasonic treatment in the water bath described in step S3 takes 3-5 minutes, followed by incubation in a constant temperature water bath for 30-60 minutes.

5. The method for preparing the dendritic cell membrane protein hybrid nanovaccine as described in claim 1, characterized in that, In step S4, the P-phase colostrum is added dropwise to the Zn-phase colostrum and reacted for 2-3 hours. The time for vigorous vortexing in the rotary evaporator is 3-5 minutes, the time for ultrasonic treatment in the water bath is 3-5 minutes, and the time for incubation in the constant temperature water bath is 30-60 minutes.

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