Bacterial vaccine, antigen-enriched nanoparticles and corresponding application and preparation method thereof

Abstract

The invention provides a bacterial vaccine for resisting recurrence of bacterial infection, the bacterial vaccine comprises polydopamine and a specific photo-thermal bactericide, the specific photo-thermal bactericide is used for killing bacteria, the polydopamine is used for in-situ enrichment of antigens to trigger immune reaction, and the antigens are killed bacteria. The invention also provides an antigen-enriched nanoparticle which comprises a Prussian blue nanoparticle core, a polydopamine coating and an erythrocyte membrane, the outer surface of the Prussian blue nanoparticle core is coated with polydopamine, so that the polydopamine coating is formed on the outer surface of the Prussian blue nanoparticle, and the erythrocyte membrane is coated with the polydopamine coating. The erythrocyte membrane is coated with the polydopamine coating grafted with the targeting peptide. The invention also provides application of the bacterial vaccine or the antigen-enriched nanoparticles in preparation of a biomedical material for treating skin infection or preparation of a biomedical material for preventing recurrence of bacterial infection and a preparation method of the bacterial vaccine or the antigen-enriched nanoparticles. The nanoparticles can promote elimination of skin infectious abscess and reduce the recurrence risk of MRSA infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of novel multifunctional nano drug treatment, and specifically relates to a bacterial vaccine for resisting recurrence of bacterial infection, antigen-enriched nanoparticles and corresponding applications and preparation methods thereof. Background Art

[0002] The recurrence of bacterial infections has become a huge challenge in the biomedical field. However, with the abuse of antibiotics, the emergence of drug-resistant bacteria has become more frequent, resulting in the continued deterioration of the current treatment of bacterial infections. For example, according to the latest data from the World Health Organization, methicillin-resistant Staphylococcus aureus (MRSA), a multidrug-resistant strain, increases the mortality rate of patients by 64% compared with non-resistant types. In addition, the development of new antibiotics is far behind the speed of the emergence of drug-resistant microorganisms. In order to meet this challenge, more and more basic research is focusing on the application of biomaterials in the treatment of bacterial infections. In this context, photothermal therapy (PTT) has received unprecedented attention due to its non-invasiveness and non-drug resistance. Photothermal agents can convert near-infrared light (NIR) into heat to achieve physical high-temperature killing of bacteria. In recent years, researchers have developed a large number of photothermal agents, such as black phosphorus nanosheets, gold nanoparticles, near-infrared small molecules, Prussian blue nanoparticles (PB), copper sulfide nanoparticles, and other photothermal agents, and have been widely used in antibacterial treatment. However, PTT has shown good efficacy in local infections, but has not yet achieved satisfactory results in systemic infections and recurrence of infections. This is because bacteria can easily metastasize or escape into cells through the blood vessels at the site of infection, leading to incomplete treatment and high recurrence rates, such as 18% to 43% for MRSA. In addition, the local high temperature generated in PTT may damage normal cells or cause adverse inflammatory reactions due to the lack of targeting.

[0003] Immunotherapy, especially bacterial vaccine-mediated immunotherapy, will be an ideal strategy for treating systemic infections and avoiding recurrence of infections. Unfortunately, taking MRSA as an example, no vaccine against MRSA infection has been successfully approved for marketing in the world. The development of bacterial vaccines has not made substantial progress worldwide, and most clinical studies have been terminated or have no follow-up reports. The single and inaccurate selection of vaccine components is the root cause of clinical failure, among which the high variability of bacterial antigen epitopes and the heterogeneity of bacterial infection strains further hinder the clinical progress of subunit vaccines. Therefore, bacterial vaccines based on intact bacteria or lysed parts of dead bacteria have attracted increasing attention from researchers due to their more comprehensive protective effects. However, the short half-life and low uptake efficiency of antigen presenting cells (APCs) seriously affect the immune reactivity and long-term immune memory they induce.

[0004] Photoimmunotherapy (PIT) was initially proposed as a potential anti-tumor therapeutic approach because it can combine phototherapy and immunotherapy to form an integrated therapeutic platform, simultaneously achieving the treatment of in situ tumor cells and acting as an "in situ vaccine" to prevent tumor metastasis and recurrence. It is worth noting that the enhancement of immunity to eliminate cancer cells depends largely on phototherapy-mediated immunogenic cell death (ICD), which may be triggered by the heat (or ROS) production caused by photosensitization. In fact, the main role of the immune system is focused on responding to invading pathogens. Therefore, it is speculated that ICD may also contribute greatly to resisting microbial infections. Similarly, PIT will be an important strategy to overcome bacterial infections by combining rapid photothermal sterilization and immunomodulation. In this context, PTT-mediated bacterial death provides a rich antigen library for immunotherapy, thereby solving the limitations of previous antigen-based single-priming immunotherapy based on vaccine delivery strategies. In addition, immunotherapy, as a complementary approach, makes up for the shortcomings of PTT in combating systemic infections. However, there are currently few reports on the use of PIT for the treatment of bacterial infections, and this area still needs further exploration. According to previous analysis, PIT is a revolutionary in situ bacterial vaccine production strategy that enables the invading pathogen to become the source of the bacterial vaccine. Therefore, it can provide a complete map of pathogen-associated molecular patterns (PAMPs) for recognition, leading to a comprehensive immune response. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a bacterial vaccine for resisting recurrence of bacterial infection, antigen-enriched nanoparticles and their corresponding applications and preparation methods.

[0006] To solve the above technical problems, the first aspect of the present invention provides a bacterial vaccine, the main characteristics of which are that it includes polydopamine and a specific bactericide, the specific bactericide is used to kill bacteria, the polydopamine is used to enrich antigens in situ to induce an immune response, and the antigens are killed bacteria.

[0007] Preferably, the specific bactericidal agent is Prussian blue nanoparticles.

[0008] Preferably, the polydopamine is wrapped around the outer surface of the Prussian blue nanoparticles to form a polydopamine coating on the outer surface of the Prussian blue nanoparticles, and the polydopamine coating is grafted with a targeting peptide and coated by a red blood cell membrane.

[0009] Preferably, the targeting peptide targets MRSA, and the targeting peptide targets the surface of MRSA bacteria, and the red blood cell membrane can adsorb and respond to toxins.

[0010] The second aspect of the present invention provides an antigen-enriched nanoparticle, whose main feature is that the nanoparticle includes a Prussian blue nanoparticle core, a polydopamine coating, and an erythrocyte membrane, polydopamine is wrapped on the outer surface of the Prussian blue nanoparticle core to form the polydopamine coating on the outer surface of the Prussian blue nanoparticle, and the erythrocyte membrane is coated with the polydopamine coating grafted with a targeting peptide.

[0011] The third aspect of the present invention provides the use of the bacterial vaccine or the antigen-enriched nanoparticles in the preparation of a biomedical material for treating skin infection or in the preparation of a biomedical material for preventing recurrence of bacterial infection.

[0012] Preferably, the skin infection is a MRSA skin infection, and the bacterial infection recurrence is a MRSA infection recurrence.

[0013] The fourth aspect of the present invention provides a method for preparing the antigen-enriched nanoparticles, which mainly comprises the following steps:

[0014] S1, dissolving potassium ferrocyanide and polyvinylpyrrolidone (PVP) in deionized water, stirring, then adding hydrochloric acid and stirring until the solution becomes transparent, dropping H2PtCl·6H2O and stirring, reacting in an oil bath, and collecting after centrifugation to obtain the Prussian blue nanoparticle core;

[0015] S2, washing the core of the Prussian blue nanoparticles in S1, mixing with dopamine hydrochloride in Tris-HCl buffer and stirring, and collecting the polydopamine PDA-coated nanoparticles PPB by centrifugation;

[0016] S3, dissolving the PPB nanoparticles and MRSA targeting peptide in S2 in Tris-HCl buffer, stirring the mixture in the dark, and collecting the nanoparticles PPPB grafted with the targeting peptide by centrifugation;

[0017] S4. The PPPB nanoparticles in S3 and the red blood cell membranes are mixed in a PBS buffer solution, and then coated with the aid of ultrasound. After centrifugation and washing, the antigen-enriched nanoparticles RBCM@PPPB nanoparticles are obtained.

[0018] Preferably, in the step S1, the final concentration of H2PtCl·6H2O in the solution is 38.6 mM.

[0019] Preferably, the step S2 is specifically as follows: 1 mg / mL Prussian blue nanoparticle core and 0.5 mg / mL dopamine hydrochloride are mixed in a pH=8.5, 10 mM Tris-HCl buffer and stirred overnight, then centrifuged at 12000 rpm for 30 min to collect, and washed three times with deionized water and ethanol respectively to obtain PPB nanoparticles;

[0020] The step S3 is specifically as follows: dissolving the MRSA targeting peptide 5-TAMRA-CARGG-NH2 and 1 mg / mL PPB nanoparticles in a pH=8.5, 10 mM Tris-HCl buffer, placing the mixture in a dark place at 37°C and stirring overnight, and finally centrifuging at a speed of 12000 rpm for 30 min, washing three times with deionized water and ethanol respectively, to obtain PPPB nanoparticles, wherein the final concentration of the targeting peptide in the mixed solution is 50 μg / mL;

[0021] The step S4 is specifically as follows: RBCM coating is performed under ultrasound-assisted conditions, 5 mL of 1 mg / mL PPPB nanoparticles and RBCM are coated under ultrasound-assisted conditions for 10 min, and then the product is collected by centrifugation at 12000 rpm for 30 min, and washed three times with phosphate buffer to obtain RBCM@PPPB nanoparticles.

[0022] The invention provides a preparation method of "antigen-enriched" nanoparticles for resisting recurrence of bacterial infection. The method comprises the following steps: after potassium ferrocyanide and polyvinylpyrrolidone (PVP) are completely dissolved, H2PtCl·6H2O is added dropwise and stirred to obtain Prussian blue nanoparticles; dopamine hydrochloride is mixed with the Prussian blue nanoparticles in a Tris-HCl buffer and stirred overnight to obtain nanoparticles PPB wrapped by dopamine; the PPB nanoparticles and a MRSA targeting peptide are dissolved in a Tris-HCl buffer, the mixture is placed in a dark place at 37°C and stirred overnight, and the nanoparticles PPPB modified with the targeting peptide are collected by centrifugation; the PPPB nanoparticles and a red blood cell membrane (RBCM) are mixed in a PBS buffer solution, coated under the assistance of ultrasound, and collected by centrifugation and washed to obtain "antigen-enriched" photoimmunotherapy nanoparticles RBCM@PPPB.

[0023] The synthesis steps of Prussian blue nanoparticles specifically include: dissolving potassium ferrocyanide and PVP in 490 mL of deionized water, stirring for 1 hour, then adding 10 mL of 1M hydrochloric acid to the above solution, and continuing to stir until the color is transparent. Add 7 mL of H2PtCl·6H2O and stir for 30 minutes, and finally continue to react for 24 hours under 80°C oil bath heating conditions. Centrifuge the above reaction solution at 12000 rpm for 30 minutes and collect the Prussian blue nanoparticles.

[0024] In the above-mentioned synthesis method of Prussian blue, 1.3 g of potassium ferrocyanide and 38 g of PVP were added, and the final concentration of H2PtCl·6H2O added in the solution was 38.6 mM.

[0025] The synthesis steps of PPB nanoparticles specifically include: mixing Prussian blue nanoparticles and dopamine hydrochloride in a Tris-HCl buffer solution and stirring overnight, then centrifuging for 30 minutes to collect the nanoparticles, and washing them three times with deionized water and ethanol respectively to obtain PPB nanoparticles.

[0026] In the above-mentioned method for synthesizing PPB nanoparticles, the concentration ratio of the added Prussian blue nanoparticles and dopamine hydrochloride is 2:1, the added Tris-HCl buffer has a pH of 8.5 and a concentration of 10 mM, and the rotation speed used for centrifugation is 12000 rpm.

[0027] The synthesis steps of PPPB nanoparticles specifically include: dissolving MRSA targeting peptide and PPB nanoparticles (1 mg / mL) in Tris-HCl buffer, placing the mixture in the dark at 37°C and stirring overnight, and finally centrifuging for 30 minutes, washing three times with deionized water and ethanol, respectively, to obtain PPPB nanoparticles. The targeting peptide sequence used is 5-TAMRA-CARGG-NH2, the final concentration of the targeting peptide is 50 μg / mL, the pH of the added Tris-HCl buffer is 8.5, the concentration is 10 mM, and the centrifugal speed is 12000 rpm.

[0028] RBCM coating was performed under ultrasound-assisted conditions. The specific steps were as follows: 5 mL of PPPB nanoparticles (1 mg / mL) and RBCM (red blood cell membrane extracted from 1 mL of blood) were coated under ultrasound-assisted conditions for 10 min. The product was then collected by centrifugation at 12,000 rpm for 30 min and washed three times with phosphate buffer (1×PBS) to obtain RBCM@PPPB nanoparticles.

[0029] The extraction method of RBCM used is as follows: 1mL of mouse blood is centrifuged at 3000rpm, the red blood cells at the bottom are collected, and resuspended with 1×PBS. This operation is repeated 5 times to obtain red blood cells with higher purity. Then, these red blood cells are resuspended in 0.25×PBS buffer and incubated at 4°C for 20min to ensure complete lysis of red blood cells. The above sample is then centrifuged at 17000rpm for 5min to collect RBCM and resuspended in 1×PBS buffer. After washing 3 times with 1×PBS buffer, pure RBCM is obtained and stored at -20°C before use.

[0030] The present invention develops PIT nanoparticles enriched with exotoxin-responsive "antigen library" made of PDA-modified PB nanoparticles and external MRSA targeting peptides and erythrocyte membranes, which can form the "antigen library" of the vaccine in situ, further triggering APC-enhanced antigen presentation and processing capabilities and subsequent adaptive immune responses, as well as immune memory, effectively preventing the recurrence of infection, and realizing in situ vaccination and adaptive immune enhancement to treat systemic bacterial infections and recurrence of infections.

[0031] The present invention utilizes the targeted cumulative responsiveness of targeting peptides and red blood cell membranes to specifically target the invading MRSA surface, the photothermal responsiveness of Prussian blue to accurately kill the infected bacteria in the infected area, and the antigen collection ability of the PDA coating, and uses Prussian blue-polydopamine-targeting peptide as a carrier to construct "antigen-enriched" photoimmunotherapy nanoparticles for preventing recurrence of bacterial infection.

[0032] The RBCM@PPPB nanoparticles prepared by the present invention are composed of polydopamine (PDA)-modified Prussian blue (PB) nanoparticles, MRSA targeting peptides and erythrocyte membrane coatings. The nanoparticles disguised by erythrocyte membranes can effectively avoid being phagocytosed prematurely, and can achieve exposure of the targeting peptide and targeted binding on the bacterial surface with the help of the exotoxin response secreted by MRSA. This precise binding with MRSA is conducive to the PTT-mediated antibacterial performance, which provides a rich source of bacterial antigens for subsequent immunotherapy, and the PDA coating on the nanoparticles enhances their antigen enrichment ability and induces further immune responses. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The synthesis process of RBCM@PPPB nanoparticles;

[0034] Figure 2 TEM images of different nanoparticles;

[0035] Figure 3 Field emission scanning electron microscopy and elemental analysis images of RBCM@PPPB nanoparticles;

[0036] Figure 4 is the hydration kinetic diameter of different nanoparticles;

[0037] Figure 5 The CCK-8 method was used to determine the effects of different concentrations of RBCM@PPPB nanoparticles and different types of nanoparticles on the viability of L-929 cells;

[0038] Figure 6 The hemolysis images and hemolysis rates of RBCM@PPPB nanoparticles at different concentrations and different types of nanoparticles at different concentrations;

[0039] Figure 7H&E staining of tissue sections of major organs after 15 days of treatment with different nanoparticles;

[0040] Figure 8 Analysis of blood biochemical indexes of mice after treatment with different nanoparticles on the 15th day;

[0041] Fig. 9 TEM image of RBCM@PPPB nanoparticles after co-cultivation with α-hemolysin;

[0042] Fig.10 TEM images of RBCM@PPPB (PPPB nanoparticles, control) after incubation with MRSA strains for 4 h;

[0043] Fig.11 The photothermal curves of different nanoparticles and RBCM@PPPB nanoparticles with different concentrations;

[0044] Fig.12 The photothermal curve of RBCM@PPPB nanoparticles obtained by near-infrared laser irradiation and the linear curve of RBCM@PPPB nanoparticles obtained during cooling (Note: η is the photothermal conversion efficiency);

[0045] Fig.13 Plate counts and quantitative data of MRSA survival after co-incubation of different nanoparticles with MRSA and irradiation with 808 nm near-infrared light;

[0046] Fig.14 Expression analysis of HSPs-related genes in MRSA after different treatments was recorded by RNA sequencing and by real-time quantitative PCR;

[0047] Fig.15 SEM images of different nanoparticles co-incubated with MRSA and irradiated with 808 nm near-infrared light;

[0048] Fig.16 The BCA protein quantification results of the supernatant after PSMα3 was incubated with different nanoparticles for 4 h and centrifuged;

[0049] Fig.17 The changes of hydration kinetic diameters of different nanoparticles before and after treatment with α-hemolysin and MRSA;

[0050] Fig.18 Typical flow cytometry data of bone marrow-derived dendritic cells (BMDCs) co-incubated with various antigen-enriched nanoparticles for 24 hours;

[0051] Fig.19 KEGG enrichment analysis showed the top 20 signaling pathways regulated by RBCM@PPPB with statistical significance (Q<0.05);

[0052] Fig. 20 For Fig.19 Gene set enrichment analysis of representative immune signaling pathways in DC2.4 cells after RBCM@PPPB treatment;

[0053] Fig.21 Schematic diagram of the construction of mouse skin abscess model and the process of nanoparticle-mediated PIT treatment of abscess;

[0054] Fig. 22 Representative photos of the abscess area taken on days 1, 5, 10, and 15 of the MRSA-infected skin abscess mouse model after different treatments, and the area change curve of the abscess area in mice;

[0055] Fig.23 Results of H&E staining and Giemsa staining of the abscess area in each treatment group 15 days after different treatments (scale bar: 100 μm);

[0056] Fig.24 Near-infrared imaging of bilateral abscess mice after different treatments and the corresponding 6-hour total radiation efficiency data (Note: the left side of the bilateral abscess mice was injected with MRSA, and the right side was injected with E.coli or Δhla MRSA);

[0057] Fig.25 Schematic diagram of reinfection of mice with bacteria after PIT treatment and corresponding flow cytometric analysis;

[0058] Fig.26 Representative images of abscess areas in cured mice after MRSA reinfection.

[0059] Fig. 27 CD4+ / CD8+ memory T cells (CD44+CD62L-) in the spleen of mice 20 days after MRSA reinfection;

[0060] Fig.28 20 days after reinfection with MRSA, B220 in the lymph of mice + CD4 + Memory B cells (IgG + IgD - ) and CD80 + IgG + Memory B cells (CD80 + IgG + IgD - ). DETAILED DESCRIPTION

[0061] In order to make the technical problem solved by the present invention clearer, the present invention is further described below in conjunction with embodiments and drawings. The specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0062] like Figure 1 , the present invention develops PIT nanoparticles enriched with exotoxin-responsive "antigen library" made of PDA-modified PB nanoparticles and external MRSA targeting peptides and erythrocyte membranes. With the help of targeting peptides and erythrocyte membranes, when the nanoparticles encounter toxins secreted by MRSA, they will first complete the toxic response process, causing the lysis of the erythrocyte membrane and the exposure of the targeting peptide, and then cause the nanoparticles to effectively accumulate in the bacterial infection area. The accumulated nanoparticles can specifically target the invading MRSA surface, and after near-infrared irradiation, the local infection strains are accurately killed. These dead bacteria provide a broad spectrum of PAMPs, which can be collected by PDA-coated nanoparticles, thereby forming an "antigen library" for in situ vaccines, further triggering APC-enhanced antigen presentation and processing capabilities and subsequent adaptive immune responses, as well as immune memory, effectively preventing the recurrence of infection, and realizing in situ vaccination and adaptive immune enhancement to treat systemic bacterial infections and recurrence of infections.

[0063] like Figure 1 As shown, the preparation method of antigen-enriched nanoparticles for resisting recurrence of bacterial infection of the present invention is prepared by a four-step method, which is divided into four parts: Prussian blue nanoparticles (PB), polydopamine-coated PPB nanoparticles, targeting peptide-modified PPPB nanoparticles, and erythrocyte membrane-coated BCM@PPPB nanoparticles. Spherical Prussian blue particles are synthesized by chloroauric acid etching-assisted hydrothermal method, and dopamine is oxidized and polymerized on the surface of PB particles to form a PDA coating to obtain PPB nanoparticles. Subsequently, the prepared PPB nanoparticles are wrapped by MRSA targeting peptide (5-TRAMA-CARGG-NH2) through physical adsorption to form PPPB nanoparticles, and then erythrocyte membranes (RBCM) are used for camouflage to finally obtain RBCM@PPPB nanoparticles.

[0064] Example 1

[0065] A preparation process of "antigen-enriched" photoimmunotherapy nanoparticles for anti-recurrence of bacterial infection is as follows:

[0066] S1, spherical PB particles were synthesized by chloroauric acid etching-assisted hydrothermal method. Potassium ferrocyanide (1.3 g) and PVP (38 g) were dissolved in 490 mL of deionized water and stirred for 1 h. Then 10 mL of 1 M hydrochloric acid was added to the above solution and stirred until the color was transparent. 7 mL of H2PtCl·6H2O (final concentration of 38.6 mM) was added dropwise and stirred for 30 min. Finally, the reaction was continued under 80 ° C oil bath heating conditions for 24 h. The above reaction solution was centrifuged at 12000 rpm for 30 min and the Prussian blue nanoparticles were collected and washed three times with deionized water and ethanol before use.

[0067] S2, dopamine is oxidized and polymerized on the surface of PB particles to form a PDA coating to obtain PPB nanoparticles. The prepared Prussian blue nanoparticles (1 mg / mL) and dopamine hydrochloride (0.5 mg / mL) are mixed in Tris-HCl buffer (pH=8.5, 10 mM) and stirred overnight, and then the nanoparticles are collected by centrifugation at 12000 rpm for 30 min, and washed three times with deionized water and ethanol respectively. The final modification product of this process is called PPB nanoparticles.

[0068] S3, MRSA targeting peptide encapsulates PPB nanoparticles to form PPPB nanoparticles by physical adsorption, MRSA targeting peptide (5-TAMRA-CARGG-NH2) (the targeting peptide used in this example is from the literature: Hussain S, Joo J, Kang J, Kim B, Braun GB, She ZG, et al. Antibiotic-loaded nanoparticles targeted to the site of infection enhance antibacterial efficacy. Nat Biomed Eng 2018, 2 (2): 95-103) and PPB nanoparticles (1 mg / mL) are dissolved in Tris-HCl buffer (pH = 8.5, 10mM), and the final concentration of the peptide is 50μg / mL. The mixture is placed in the dark at 37 ° C and stirred overnight, and finally centrifuged at 12000rpm for 30min, and washed three times with deionized water and ethanol respectively. The product of this step is named PPPB nanoparticles.

[0069] S4, PPPB nanoparticles were camouflaged and wrapped with red blood cell membrane (RBCM), and finally RBCM@PPPB nanoparticles were obtained, and RBCM coating was performed under ultrasound-assisted conditions. Specifically, 5mL PPPB nanoparticles (1mg / mL) and the above-mentioned RBCM (red blood cell membrane extracted from 1mL blood) were coated with ultrasound for 10min. The product was then collected by centrifugation at 12000rpm for 30min and washed three times with PBS buffer. The product of this step was named RBCM@PPPB nanoparticles.

[0070] S5, extraction of red blood cell membrane, 1mL mouse blood was centrifuged at 3000rpm, the red blood cells at the bottom were collected, and resuspended with phosphate buffer (1×PBS). This operation was repeated 5 times to obtain red blood cells with higher purity. Then, the red blood cells were resuspended in 0.25×PBS buffer and incubated at 4°C for 20min to ensure complete lysis of the red blood cells. The above sample was then centrifuged at 17000rpm for 5min to collect RBCM and resuspended in 1×PBS buffer. Pure RBCM was obtained after washing 3 times with 1×PBS buffer and stored at -20°C before use.

[0071] Example 2

[0072] Morphology of RBCM@PPPB nanoparticles

[0073] Take 10 μL of PB, PPB, PPPB, and RBCM@PPPB nanoparticle solutions in Example 1 and drop them on a copper mesh, let them air dry overnight, then use 1% (w / v) phosphotungstic acid solution for negative staining and characterize their morphology under a transmission electron microscope; take 20 μL of PB, PPB, PPPB, and RBCM@PPPB nanoparticle solutions and drop them on a silicon wafer, let them dry overnight, and then use a GeminiSEM 500 field emission scanning electron microscope to obtain the corresponding element mapping; similarly, take 20 μL of PB, PPB, PPPB, and RBCM@PPPB nanoparticle solutions and drop them on the surface of a mica sheet, gently rinse the salt body with deionized water, and then characterize it under the tapping mode of AFM. Figure 2 As shown, compared with PB nanoparticles, there is an obvious organic coating with lower contrast in PPB nanoparticles, and a denser membrane coating in RBCM@PPPB nanoparticles. This result indicates that the surface of PB nanoparticles was successfully coated with PDA and RBCM. Figure 3 The results of elemental analysis showed that the common C, O, and N elements in PDA, the representative P element of cell membrane, and the Fe element in PB nanoparticles were all present in RBCM@PPPB nanoparticles, further confirming that the designed nanosystem was successfully prepared. Figure 4Dynamic light scattering (DLS) data showed that the hydrodynamic diameter of the nanoparticles gradually increased with the modification process. The PDA coating increased the hydrodynamic diameter of the nanoparticles by about 24 nm, while the RBCM coating increased the hydrodynamic diameter of the nanoparticles by about 30 nm.

[0074] Example 3

[0075] In vitro cytotoxicity of RBCM@PPPB nanoparticles

[0076] Mouse fibroblast L-929 cells and CCK-8 assay were used to determine the potential cytotoxicity of the nanoparticles. L-929 cells were cultured at 1x10 4 cells / well were cultured in a 96-well plate, and PBS buffer (negative group), 0.1% Triton X-100 (positive group), different concentrations of RBCM@PPPB nanoparticles and different types of nanoparticles (PB, PPB, PPPB, final concentration of 100 μg / mL) were added to the above culture dishes and incubated for 1 day. Then the previous culture medium was discarded, and 100 μL of fresh DMEM containing 10% CCK-8 was added to each well. After the sample was incubated at 37°C for 2 hours, 100 μL was transferred to a new 96-well plate, and its absorbance was measured with an enzyme reader at a wavelength of 450 nm. Figure 5 When the concentration of RBCM@PPPB nanoparticles reached 200 μg / mL, the co-incubated L-929 cells still showed more than 90% cell viability. In addition, the cell viability of all different types of nanoparticles at a concentration of 100 μg / mL was higher than 90%. These results confirmed that the nanosystem has good in vitro cell compatibility.

[0077] Example 4

[0078] Blood compatibility of RBCM@PPPB nanoparticles

[0079] For blood compatibility determination, 1 mL of fresh blood was collected from the retroorbital venous plexus of mice and diluted to 10 mL with PBS. Centrifuge at 4°C, 2500 rpm for 5 min, and repeat the above process five times until the supernatant is transparent. After discarding the supernatant, add 10 mL of PBS to resuspend the red blood cells, take 900 μL of red blood cell solution and add 100 μL of 1% TritonX-100 (positive control) and PBS (negative control), add different concentrations of RBCM@PPPB nanoparticles and different types of nanoparticles (PB, PPB, PPPB, final concentration of 100 μg / mL) to the prepared test tubes, and incubate at room temperature for 4 hours. After the end, centrifuge at 10000 rpm for 5 min, take 100 μL of the supernatant in a 96-well plate, and measure the absorbance at 540 nm. The formula for calculating the hemolysis rate is: Hemolysis rate (%) = (OD 样品-OD PBS ) / (OD Triton X-100 –OD PBS ), where OD is the absorbance. The results are as follows Figure 6 As shown in the figure, the hemolysis rate of RBCM@PPPB nanoparticles at different concentrations (10 μg / mL to 300 μg / mL) and the hemolysis rate of different types of nanoparticles (100 μg / mL) are all lower than 5%, which is the highest threshold required for blood compatibility. These results indicate that the nanoparticles have good blood compatibility.

[0080] Example 5

[0081] In vivo biosafety evaluation of RBCM@PPPB

[0082] In order to further evaluate the biosafety of the present invention, we evaluated the major organs and blood biochemistry after treatment. Figure 7-8 As shown, each group of nanoparticle components did not exhibit obvious organ toxicity, indicating their biosafety in actual medical applications.

[0083] Example 6

[0084] In vitro toxin response and bacterial targeting ability of RBCM@PPPB nanoparticles

[0085] The toxin-responsive behavior of RBCM@PPPB was characterized by transmission electron microscopy. First, 100 μg / mL RBCM@PPPB nanoparticles were mixed with 100 μg / mL α-hemolysin and incubated for 2 h. Samples were taken at specific time points (0 h, 0.5 h, 1 h, 2 h) and characterized by transmission electron microscopy. In addition, the PPPB nanoparticle treatment group served as the positive control group. 6 CFU / mL MRSA (wild-type and Δhla MRSA) were incubated for 4 h to confirm their responsiveness to bacterial toxins. Fig. 9 When RBCM@PPPB nanoparticles and α-hemolysin were co-incubated for 2 hours, the results showed that the RBCM coating became more and more damaged as the incubation time increased, which means that only α-hemolysin can indeed perforate and dissolve the RBCM coating on the membrane. The host bacteria MRSA that secretes α-hemolysin was further used to simulate a more realistic infection microenvironment to verify the toxin response ability of RBCM@PPPB. Fig.10 As shown, the results not only prove that RBCM@PPPB nanoparticles can achieve α-hemolysin-mediated toxin responsiveness, but also confirm its ability to actively target the bacterial surface after the response. The nanoparticles have the ability to selectively target MRSA, which provides a good basis for photothermal sterilization.

[0086] Example 7

[0087] Photothermal properties of RBCM@PPPB nanoparticles

[0088] The photothermal conversion performance of the nanosystem is an important factor affecting the effect of photoimmunotherapy (PIT). In order to confirm the effect of subsequent modification steps on the original photothermal conversion properties of PB, the in vitro photothermal performance of various nanoparticles (PB, PPB, PPPB and RBCM@PPPB) under 808nm near-infrared light irradiation was characterized by the Fotric X thermal imaging instrument. Fig.11 As shown in the figure, the modification steps of targeting peptide and RBCM coating reduced the photothermal conversion ability to a certain extent, but their maximum temperatures could reach ~74℃ (PPPB) and ~73℃ (RBCM@PPPB), respectively. And the photothermal conversion ability of RBCM@PPPB showed concentration dependence, which was 2 Under laser irradiation and a concentration of 100 μg / mL, the temperature can reach about 60°C, which is a reasonable temperature for bacterial killing and does not cause significant damage to normal tissues. In addition, the photothermal conversion efficiency of RBCM@PPPB still reaches 39.38% ( Fig.12 ), which is comparable to the conversion efficiency reported for Prussian blue nanoparticles. These results also suggest that the nanoparticles have the potential to serve as a PIT photothermal platform.

[0089] Example 8

[0090] In vitro antibacterial activity of RBCM@PPPB nanoparticles

[0091] The present invention selects MRSA as the target strain and uses a combination of plate coating method and scanning electron microscopy characterization to evaluate the antibacterial activity of the nanosystem. Specifically, MRSA is first cultured to the logarithmic phase (10 7 CFU / mL), and then different types of nanoparticles were added for NIR treatment. After treatment, the plates were coated and cultured for 24 hours. The group without nanoparticles and the group without NIR treatment were used as the control group. Fig.13 As shown in the figure, compared with the control group without NIR irradiation, the inhibition rate of the NIR-treated group was very significant, reaching more than 85%. 7 CFU / mL) has the highest sterilization efficiency (93.84%), and the MRSA surface has obvious shrinkage and deformation. The RBCM@PPPB nanoparticles prepared by the present invention have good in vitro MRSA killing ability after NIR treatment.

[0092] Statistical analysis of the differentially expressed genes (DEGs) of MRSA was performed to elucidate the potential response of bacteria to nanoparticle-mediated PTT. Many heat shock stress-related DEGs were detected to be upregulated before and after PTT, and these DEGs were mainly related to the production of heat shock proteins (HSPs) ( Fig.14 ). Similarly, real-time quantitative PCR data also confirmed the upregulation of these genes. The upregulation of these HSP-related genes suggests that RBCM@PPPB nanoparticle-mediated PTT may induce the release of a large number of HSP signals from dead MRSA, thereby enhancing the immunogenicity and antigen presentation process, which will be beneficial for the subsequent activation of immune enhancement and memory. On the other hand, these results also highlight the excellent effect of RBCM@PPPB PTT in eliminating bacteria.

[0093] Example 9

[0094] In vitro antigen enrichment ability of RBCM@PPPB nanoparticles

[0095] PSMα3 was selected as the target protein to evaluate the antigen enrichment ability of RBCM@PPPB. PSMα3 (100 μg / mL) with or without FITC labeling was mixed with different types of nanoparticles (100 μg / mL) and incubated for 4 h. After centrifugation at 12000 rpm for 30 min, the amount of unbound protein in the supernatant was detected by BCA method and fluorescence spectroscopy. Figure 15-16 As shown in the figure, PB nanoparticles adsorbed 58.76% of PSMα3 toxin, and PPB nanoparticles had the highest collection rate (93.08%), which may be because PSMα3 is positively charged in PBS buffer (pH = 7.4) and can bind to the negatively charged PB surface through electrostatic interaction, while the PDA coating has a higher negative charge and catechol-mediated adhesion properties. In addition, the in vitro antigen collection efficiency of PPPB and RBCM@PPPB nanoparticles was 89.02% and 89.64%, respectively. In comparison, after the rest of the modifications on the PDA surface, their enrichment efficiency was lower than that of PPB nanoparticles, but they also showed a very high bacterial antigen enrichment efficiency, achieving effective absorption and neutralization of PSMα3 toxin.

[0096] To further verify the antigen enrichment ability of the nanosystem, the study measured the changes in the hydration kinetic diameter of the nanoparticles before and after toxin / bacteria (NIR treatment). Fig.17As shown, after treatment with toxin (α-hemolysin) and bacteria (MRSA), the hydration kinetic diameter of the nanoparticles increased significantly, which was observed in all groups. Unexpectedly, the diameter of the PPB nanoparticles after incubation with toxins was larger than that of any other nanoparticles, which was consistent with the previous collection rate results. The diameter of the collected RBCM@PPPB nanoparticles (~900nm) after co-incubation with bacteria and NIR treatment was about 4.5 times that of the original nanoparticles (~200nm), the largest diameter among all groups, which may be related to the toxin neutralization performance of the RBCM coating and the antigen enrichment contributed by PDA.

[0097] In summary, these results confirm that the prepared nanosystem has good in vitro antibacterial activity and the ability to enrich relevant antigens from dead bacteria, which are mainly attributed to the antigen accumulation mediated by the catechol structure of the PDA coating and the toxin neutralization effect of the RBCM coating. These evidences further support the antigen presentation process in subsequent immunotherapy.

[0098] Example 10

[0099] RBCM@PPPB enhances antigen presentation

[0100] In Example 9, RBCM@PPPB nanoparticles achieved effective enrichment of dead bacterial antigens, but so far, how they further trigger the antigen presentation process of immune cells remains unknown. Dendritic cells (DCs) are the most prominent professional antigen presenting cells (APCs) in the immune system. Immature DC cells with migration ability can track bacterial infection areas by recognizing PAMPs released by dead bacteria, and transform into mature DC cells after antigen processing and presentation, thereby activating subsequent immune responses. Therefore, the maturation of DC cells is a key marker of the antigen presentation process. CD11c is a specific marker for DC cells and can serve as a key clue to distinguish DC cells from other cells.

[0101] Bone marrow-derived dendritic cells (BMDCs) were used to investigate the effects of different nanoparticles on the recognition and maturation of antigen-presenting cells represented by DCs, thereby enhancing the antigen presentation process. To verify the enhanced antigen presentation mediated by nanoparticles, freshly prepared nanoparticles were first incubated with MRSA strains and treated with 808nm NIR laser for 20min, and then the antigen-captured nanoparticles were incubated with DCs for another 24h. Fig.18 As shown, RBCM@PPPB nanoparticles had the highest DC cell maturation rate among all treatment groups, promoting the recognition and maturation of antigen-presenting cells represented by DC cells, and further enhancing the antigen presentation process.

[0102] In addition, DC2.4 cells treated with RBCM@PPPB nanoparticles were collected for RNA sequencing. From the perspective of GO enrichment ( Fig.19 ) analysis, multiple signaling pathways in DC cells were also significantly activated, including molecular pattern recognition receptors, antigen processing and presentation ( Fig. 20 ), T cell receptors, Toll-like receptors, NOD-like receptors, and IL-17 signaling pathways, etc. This shows that the types of antigens enriched in RBCM@PPPB nanoparticles cover a wider range, and thus can fully activate the relevant pathways of DC cells, among which the enhancement of signaling pathways related to molecular pattern recognition and antigen presentation is the most significant. DC2.4 cells can effectively respond to different antigens enriched in RBCM@PPPB nanoparticles, thereby enhancing their antigen presentation function and effectively activating subsequent adaptive immune processes. These data demonstrate the key connecting role of the nanoparticles in photothermal therapy and immunotherapy.

[0103] Embodiment 11

[0104] Establishment of MRSA infection mouse abscess model

[0105] Six-week-old healthy BALB / c mice were used to construct the model. Fig.21 As shown. Before animal experiments, bacteria were cultured on sheep blood agar plates at 37°C overnight. Single colonies of each strain were collected and incubated in 4 mL TSB at 37°C overnight. After washing twice with sterile PBS (10 mM, pH = 7.4), the bacteria were diluted to 10 7 The concentration of CFU / mL was used for subsequent experiments. First, the mice were anesthetized with 1% sodium pentobarbital and the hair on the back was cut. 50 μL of the prepared MRSA suspension (1×10 7 CFU / mL) were injected into the upper back of mice one day after inoculation to form an abscess infection.

[0106] Example 12

[0107] In vivo evaluation of RBCM@PPPB-mediated PIT against MRSA-infected skin abscesses

[0108] After constructing the abscess infection model mice, 50 μL of 1 mg / mL prepared nanoparticles (PB, PPB, PPPB and RBCM@PPPB) were injected into the abscess area of ​​the mice the next day. After 4 hours, an 808 nm laser (1 W) was irradiated on the abscess area of ​​the mice for 20 minutes. Photos of the abscess were taken in the following days, and the abscess area was recorded every 5 days. In order to observe the changes in the number of bacteria in the abscess, bioluminescence imaging of the infected GFP-expressing MRSA was performed every 5 days. On the 15th day, the abscess area of ​​the mice was observed by ultrasound imaging to evaluate the internal abscess area and angiogenesis. Then, the mouse skin tissue was dissected at the site of the infected abscess, and hematoxylin and eosin (H&E) staining and Giemsa staining sections were prepared. At the same time, in order to conduct in vivo antibacterial evaluation, the skin tissue at the infected abscess site was dissected from the mouse and cut into small pieces on the 15th day, and further preserved with sterile PBS. Subsequently, the antibacterial effect of each sample was evaluated in vivo by plating. Figure 22-23 As shown, the results showed that compared with other control groups, the skin of mice treated with RBCM@PPPB and PPPB nanoparticles after NIR recovered faster, with the smallest abscess area and the lowest bacterial residue, indicating that PIT based on RBCM@PPPB or PPPB nanoparticles treatment has a good therapeutic effect in resisting bacteria-induced abscesses.

[0109] Example 13

[0110] In vivo toxin response and bacterial targeting ability of RBCM@PPPB

[0111] A bilateral abscess model was established in mice to evaluate the in vivo retention and bacterial targeting of nanoparticles. Mice were anesthetized with 1% sodium pentobarbital, the hair on the back was cut, and 50 μL of the prepared MRSA suspension (1×10 7 CFUs / mL) and Escherichia coli (E.coli) / Δhla MRSA (1×10 7 CFUs / mL) were injected on both sides of the upper back, and then inoculated for 1 day to form an abscess. The next day, 50 μL of 1 mg / mL prepared nanoparticles (Ce6, Ce6-staining peptide, Ce6-staining PPPB, Ce6-staining RBCM@PPPB) were injected on both sides of the abscess area of ​​the mouse. Before injection, peptide (CARGG), PPPB and RBCM@PPPB nanoparticles were mixed with Ce6. The above mice were observed and recorded by live imaging at 0, 3, and 6 hours after injection. Fig.24 As shown, there were significant differences in the total radiation efficiency between the MRSA / E.coli and MRSA / Δhla MRSA groups after 6 h of RBCM@PPPB treatment, respectively, which proves that MRSA exotoxin secretion is a prerequisite for the exposure of targeting peptides and further retention of nanoparticles at the infection site after the nanoparticle toxin response.

[0112] Embodiment 14

[0113] RBCM@PPPB-mediated PIT induces long-term immune memory effect

[0114] In order to explore the long-term immune memory effect based on PIT induced by RBCM@PPPB nanoparticles, a secondary infection MRSA abscess model was used to observe whether these nanoparticles can prevent the recurrence of infection after the initial cure. Fig.25 The secondary infection model is that mice are first cured by PIT mediated by RBCM@PPPB nanoparticles after the initial infection, and then infected with the same dose of MRSA. Mice cured by RBCM@PPPB nanoparticles can be infected with MRSA again without showing obvious abscess symptoms, and the skin of the infected area is basically the same as that of healthy mice ( Fig.26 ), indicating that PIT mediated by RBCM@PPPB nanoparticles has an immune memory effect. Further investigation of its mechanism revealed that CD4 + Memory T cells (CD4 + CD44 + CD62L - ) and CD8+ memory T cells (CD8 + CD44 + CD62L - ) is significantly higher ( Fig. 27 ). Intralymphatic B220+ memory B cells (B220 + IgG + IgD - CD80 + ) was also significantly higher than that of the control group ( Fig.28 ). These results indicate that RBCM@PPPB nanoparticle-mediated PIT effectively induced systemic immune memory effects of both cellular and humoral immunity, effectively combating secondary bacterial infections and further reducing the risk of recurrence of bacterial infections.

[0115] In summary, the nanosystem prepared by the present invention has good biocompatibility and in vivo safety, which indicates that RBCM@PPPB nanoparticles do not induce exclusive adverse reactions, and the PIT mediated by them has great potential as a nano "in situ vaccine" for the treatment of bacterial infections and further clinical transformation.

[0116] Beneficial effects of the present invention: A PIT nanoparticle named RBCM@PPPB that integrates "exotoxin response" and "antigen enrichment" was developed, combining PTT and immunotherapy for the treatment of bacterial infections and recurrences. The nanoparticles under this strategy achieve precise bacterial killing based on PTT, and collect bacterial antigens as "in situ vaccines", enhance the antigen presentation process of DC cells, enhance long-term immune memory, and effectively avoid the recurrence of MRSA infection. This work provides a proof of concept for the antigen enrichment-enhanced photoimmunotherapy strategy, achieving the vaccination of "in situ vaccines" and enhancing immune memory to fight bacterial infections and recurrences.

[0117] The above description is a specific embodiment of the present invention and does not constitute a limitation on the protection scope of the present invention. Any modification and variation made to the technical concept of the present invention should be included in the protection scope of the present invention.

Claims

1. A bacterial vaccine, characterized in that: The invention comprises polydopamine and a specific bactericide, wherein the specific bactericide is used for killing bacteria, the polydopamine is used for in-situ enrichment of antigens to induce immune response, and the antigens are the killed bacteria.

2. The bacterial vaccine according to claim 1, characterized in that The specific bactericide is a photothermal bactericide Prussian blue nanoparticles.

3. The bacterial vaccine according to claim 2, characterized in that The polydopamine is wrapped on the outer surface of the Prussian blue nanoparticles to form a polydopamine coating on the outer surface of the Prussian blue nanoparticles. The polydopamine coating is grafted with a targeting peptide and is coated by a red blood cell membrane.

4. The bacterial vaccine according to claim 3, characterized in that The targeting peptide targets MRSA.

5. An antigen-enriched nanoparticle, characterized in that: The nanoparticles include a Prussian blue nanoparticle core, a polydopamine coating, and an erythrocyte membrane. Polydopamine is wrapped around the outer surface of the Prussian blue nanoparticle core to form the polydopamine coating on the outer surface of the Prussian blue nanoparticle. The erythrocyte membrane is coated with the polydopamine coating grafted with a targeting peptide.

6. Use of the bacterial vaccine according to any one of claims 1 to 4 or the antigen-enriched nanoparticles according to claim 5 in the preparation of a biomedical material for treating skin infection or in the preparation of a biomedical material for preventing recurrence of bacterial infection.

7. The use according to claim 6, characterized in that: The skin infection is MRSA skin infection, and the bacterial infection recurrence is MRSA infection recurrence.

8. A method for preparing the antigen-enriched nanoparticles according to claim 5, characterized in that: The following steps are involved: S1, dissolving potassium ferrocyanide and polyvinyl pyrrolidone in deionized water, stirring, then adding hydrochloric acid and stirring until the solution becomes transparent, dropping H2PtCl·6H2O and stirring, reacting in an oil bath, and collecting after centrifugation to obtain the core of the Prussian blue nanoparticles; S2, washing the core of the Prussian blue nanoparticles in S1, mixing with dopamine hydrochloride in Tris-HCl buffer and stirring, and collecting the polydopamine-coated nanoparticles PPB by centrifugation; S3, dissolving the PPB nanoparticles and MRSA targeting peptide in S2 in Tris-HCl buffer, stirring the mixture in the dark, and collecting the nanoparticles PPPB grafted with the targeting peptide by centrifugation; S4. The PPPB nanoparticles in S3 and the red blood cell membranes are mixed in a PBS buffer solution, and then coated with the aid of ultrasound. After centrifugation and washing, the antigen-enriched nanoparticles RBCM@PPPB nanoparticles are obtained.

9. The preparation method according to claim 8, characterized in that: In the step S1, the final concentration of H2PtCl·6H2O in the solution is 38.6 mM.

10. The preparation method according to claim 8, characterized in that: The step S2 is specifically as follows: 1 mg / mL Prussian blue nanoparticle core and 0.5 mg / mL dopamine hydrochloride are mixed in a pH=8.5, 10 mM Tris-HCl buffer and stirred overnight, then centrifuged at 12000 rpm for 30 min to collect, and washed three times with deionized water and ethanol respectively to obtain PPB nanoparticles; The step S3 is specifically as follows: dissolving the MRSA targeting peptide 5-TAMRA-CARGG-NH2 and 1 mg / mL PPB nanoparticles in a pH=8.5, 10 mM Tris-HCl buffer, placing the mixture in a dark place at 37°C and stirring overnight, and finally centrifuging at a speed of 12000 rpm for 30 min, washing three times with deionized water and ethanol respectively, to obtain PPPB nanoparticles, wherein the final concentration of the targeting peptide in the mixed solution is 50 μg / mL; The step S4 is specifically as follows: RBCM coating is performed under ultrasound-assisted conditions, 5 mL of 1 mg / mL PPPB nanoparticles and RBCM are coated under ultrasound-assisted conditions for 10 min, and then the product is collected by centrifugation at 12000 rpm for 30 min, and washed three times with phosphate buffer to obtain RBCM@PPPB nanoparticles.

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