Use of lentinan in preparation of pH-responsive nanomedicine carrier

Abstract

The application belongs to the technical field of biology, and discloses application of lentinan in preparation of a pH responsive nano drug carrier, wherein the nano drug carrier takes poly(beta-amino ester)-guanidine-phenylboronic acid as a hydrophilic shell, so that the bioavailability of the nano drug carrier is increased, the PBAE-G can respond to high-level reactive oxygen species (ROS) and low pH value in an inflammatory environment, and is converted into a membrane-permeable hydrophilic cationic polymer; and the nano drug carrier of the application loads lentinan on the surface of the PBAE-G hydrophilic shell, so that the nano drug carrier has high stability and pH responsiveness.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biotechnology, and particularly relates to application of lentinan in preparation of a pH-responsive nanomedicine carrier. BACKGROUND

[0002] Vaccination remains a reliable method for effectively preventing or alleviating a series of viral and bacterial diseases. Compared with traditional vaccines, protein subunit vaccines are sometimes considered to be advantageous because they are composed of highly purified components and can be well defined. However, due to the fact that they do not possess many inherent characteristics of actual pathogens, these protein subunit vaccines often induce less strong immune responses, and therefore adjuvants must be used to achieve effective immunogenicity and regulate the resulting immune response. Aluminum-based adjuvants are the most commonly used adjuvants, which can establish effective humoral immunity, although they are not effective in enhancing cellular immunity. As a vaccine adjuvant, polysaccharides have great development space due to their low toxicity, high safety and rapid immune response.

[0003] One of the biological properties of nanoparticles is easy uptake by a variety of cells. Using nanoparticles as vaccine adjuvants, on the one hand, the carrier properties of nanoparticles can be used to improve the phagocytic ability of antigen-presenting cells to antigens, and on the other hand, the effects of nanoparticles on immune cells can be used to trigger the body's innate immune response and ultimately induce effective specific immune responses. Nanodiamonds (NDs) are a structure with very promising physicochemical properties, which make them very suitable for use in a range of settings, including excellent thermal and chemical stability, a range of functional groups and large surface area. Therefore, biomedical researchers have recently investigated the utility of ND-based systems in vaccine delivery or drug carriers. Due to the good biocompatibility, unique mechanical properties, hydrophilicity and strong absorbance in the near-infrared (NIR) range of NDs, they are widely used in cell imaging, as biosensing probes or photothermal agents. NDs have a high specific surface area and many different functional groups, and can be used as an ideal drug delivery platform. In addition, NDs can stimulate toll-like receptor (TLR) signal activity, thereby more effectively activating antigen-presenting cells (APCs) such as macrophages or DCs. Considering that high ND loading rates of antigens or other proteins of interest can be obtained through physical adsorption or chemical modification, these nanomaterials are also excellent candidates for the design of vaccine delivery systems.

[0004] Cationic polymers can have different physicochemical properties and thus different functions by changing their chemical composition, molecular mass and molecular structure. Poly(beta-amino ester) (PBAE) is a class of biodegradable cationic polymers synthesized from acrylate and various amines through Michael addition reaction, which has inherent properties of tertiary amine and ester, and was first discovered by Langer laboratory. It has the advantages of cheap synthetic raw materials, simple synthesis steps, low cytotoxicity and high transfection efficiency. The main chain of PBAE contains tertiary amine groups, which endows it with obvious 'proton sponge' effect. Within the pH range of 3.5 to 7.2, they show excellent pH sensitivity, which is particularly suitable for intracellular delivery of genes, proteins, peptides and low molecular weight drugs. In recent years, polymer nanoparticles designed based on PBAE have wide application prospects in the field of medicine.

[0005] Lentinan (LNT) is an important bioactive component in Lentinula edodes, which has attracted increasing research interest due to its strong immunomodulatory and antioxidant activities. Studies have shown that LNT can stimulate the activation and maturation of various immune cell types, such as lymphocytes, dendritic cells (DCs), natural killer cells (NKs), and macrophages. By promoting cytokine production, lymphocyte proliferation, macrophage and DC activation, and T cell responses, LNT can induce more effective immune responses. LNT is a beta-1,3 beta-glucan with a branched comb-like primary structure containing two beta-1,6 glucosyl groups. Importantly, there is evidence to support the ability of LNT to enhance immunity as a vaccine adjuvant. However, since LNT cannot specifically target immune cells, it can be rapidly cleared from the body, highlighting the need for targeted delivery using appropriate functional systems.

[0006] There is no nanomedicine carrier composed of Lentinan functionalized cationic polymers. SUMMARY

[0007] The purpose of the present application is to provide the application of Lentinan in the preparation of pH-responsive nanomedicine carriers, which load Lentinan on the surface of PBAE-G hydrophilic shell, have high stability, pH responsiveness and biological safety.

[0008] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0009] The present application provides the application of Lentinan in the preparation of pH-responsive nanomedicine carriers.

[0010] In one technical solution, the nanomedicine carrier is a core-shell structure assembled with nanodiamond particles after loading Lentinan on the surface of cationic polymers.

[0011] In one technical solution, the cationic polymer is poly(beta-amino ester)-guanidine-boronic acid.

[0012] In one technical solution, the pH response range of the nanodrug carrier is 3.5-7.0.

[0013] In one technical solution, the bioactive substance carried by the nanodrug carrier is ovalbumin.

[0014] In one technical solution, the nanodrug carrier activates macrophages to produce immune response by activating the cGAS-STING signaling pathway.

[0015] Compared with the prior art, the beneficial effects of the present application are:

[0016] The nanodrug carrier of the present application uses poly(beta-amino ester)-guanidine-boronic acid as a hydrophilic shell. PBAE is a biodegradable cationic polymer that exhibits excellent affinity to lectins on bacteria and macrophages, increasing the bioavailability of NPs. The encapsulated target antigen, such as ovalbumin (OVA), can respond to high levels of reactive oxygen species (ROS) and low pH in inflammatory environments, converting into a membrane-permeable hydrophilic cationic polymer.

[0017] The nanodrug carrier of the present application loads lentinan on the surface of the PBAE-G hydrophilic shell, has high stability, and is pH-responsive. The nanodrug carrier carries OVA to prepare a nanovaccine adjuvant, which can enhance the activation of macrophages in vitro and induce strong cellular and humoral immunity in vivo without obvious toxicity, indicating that LNT-PBAE-G-ND is a safe and effective mRNA vaccine delivery carrier. In addition, the molecular mechanism of macrophage activation induced by the nanodrug carrier is also explored, and it is found that LNT-PBAE-G-ND@OVA activates macrophages by activating the cGAS-STING signaling pathway. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 For the generation and characterization of LNT-PBAE-G-ND, wherein Figure 1 A is the preparation process of PBAE-G-ND and LNT-PBAE-G-ND; Figure 1 B and Figure 1 C is the characterization of LNT-PBAE-G-ND preparation by transmission electron microscopy (B) and dynamic light scattering (C), respectively; Figure 1 D and Figure 1 E is the Zeta potential and PDI value of LNT-PBAE-G-ND preparation; Figure 1 F is the normalized ND and fluorescence spectrum (FL).

[0019] Figure 2 Stability of LNT-PBAE-G-ND and LNT-PBAE-G-ND@OVA, wherein Figure 2 A~2C are the hydrodynamic size, Zeta potential and PDI values of LNT-PBAE-G-ND and LNT-PBAE-G-ND@OVA samples after freeze-drying and dissolution in H solution, respectively, evaluated and showed no differences between these groups.

[0020] Figure 3 Release analysis of LNT and OVA in NPs, wherein Figure 3 A and Figure 3 B are the release profiles of LNT and OVA, respectively, at the indicated pH levels; Figure 3 C and Figure 3 E are the release profiles of LNT of NPs after activation of mouse neutrophils with IL-6, Figure 3 D and Figure 3 F are the release profiles of OVA of NPs after activation of mouse neutrophils with IL-6.

[0021] Figure 4 Uptake and cytotoxicity when macrophages are treated with LNT-PBAE-G-ND@OVA, wherein Figure 4 A macrophage viability was detected after 48 h of treatment with LNT-PBAE-G-ND and LNT-PBAE-G-ND@OVA; Figure 4 B is the internalization of macrophages after treatment with LNT-PBAE-G-ND or LNT-PBAE-G-ND@OVA (OVA-FITC: green; DAPI: blue; ND: red).

[0022] Figure 5 Effect of LNT-PBAE-G-ND@OVA treatment on macrophage activation, wherein Figure 5 A~5C are the expression of CD80 + , CD86 + and MHCII + in macrophages; Figure 5 D is the representative flow cytometry of CD80 + , CD86 + and MHCII + in macrophages.

[0023] Figure 6 Macrophage cytokine secretion, wherein Figure 6 A~6D are the concentrations of IL-12, IL-1β, TNF-α and IL-6 released by macrophages, respectively, data are mean ± SEM, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001.

[0024] Figure 7 Results of the mouse immune response, where Figure 7 A is the determination of OVA-specific IgG levels on the indicated days; Figure 7 B is the determination of IgG1 and IgG2a levels on the indicated days, data are mean ± SEM, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001.

[0025] Figure 8 Results of the changes in the ratio of T lymphocyte subsets, where Figure 8 A and 8B are the ratio of CD4+ / CD8+ T cells and CD3+ / CD8+ T cells in the spleen of mice 42 days after immunization, detected by flow cytometry, data are mean ± SEM, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001.

[0026] Figure 9 Serum cytokine levels determined by ELISA 28 and 42 days after primary immunization, where Figure 9 A and 9B are the concentration levels of Th1 cytokines IFN-γ and TNF-α in the serum of mice; Figure 9 C and 9D are the concentration levels of Th2 cytokines IL-4 and IL-6 in the serum of mice, data are mean ± SEM, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001.

[0027] Figure 10 In vivo biological safety analysis of LNT-PBAE-G-ND@OVA, where Figure 10 A is the hematological index of mice on days 7 and 14 after immunization; Figure 10 B is the analysis of serum biochemical indicators (ALT, AST, ALP, LDH and BUN) of mice on days 7 and 14 after immunization, data are mean ± SEM, n = 3, ns: not significant; Figure 10 C is the analysis of heart, lung, liver, spleen and kidney tissue samples of mice on days 7 and 14 after immunization by H&E staining, scale bar: 40 μm.

[0028] Figure 11 Changes in mRNA expression of macrophages after 48 h of LNT-PBAE-G-ND@OVA treatment, where Figure 11 A is the comparison by hierarchical clustering heat map when comparing control and LNT-PBAE-G-ND cells, using red and green to identify up- and down-regulated genes, respectively; Figure 11 B is the KEGG pathway enrichment analysis of DEGs; Figure 11C is nod-like pathway gene significantly enriched, green indicates up-regulation, red indicates down-regulation; Figure 11 D is LNT-PBAE-G-ND@OVA promotes the activation of cGAS-STING-TBK1-IRF3 pathway in macrophages. The relative protein expression of cGAS, STING, TBK1 and IRF3 was detected by western blotting, data are mean ± SEM, n = 3, *P < 0.05.

[0029] Figure 12 D is the effect of LNT-PBAE-G-ND@OVA treatment for 48 h on the expression of macrophage costimulatory molecules after inhibiting cGAS-STING signaling pathway, wherein Figure 12 A is the determination of cGAS and STING levels by western blotting; Figure 12 B is the relative protein level of cGAS-STING pathway components; Figure 12 C- Figure 12 D is the expression level of macrophage CD40, CD80 and CD86 Figure 12 C and representative FACS Figure 12 D; Figure 12 E is the effect of cGAS-STING pathway inhibition on macrophage IL-6, IL-12, IL-1β and TNF-α secretion, data are mean ± SEM, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001. DETAILED DESCRIPTION

[0030] The following examples are used to illustrate the present application, but not to limit the scope of protection of the present application. If not specifically indicated, the technical means used in the examples are conventional means known to those skilled in the art. The test methods in the following examples are conventional methods, unless otherwise specified.

[0031] Example 1

[0032] 1. Materials and methods

[0033] 1.1 Reagents

[0034] Nanodiamonds (NDs) were from Jiaozuo Tianbao Material Technology Co., Ltd. Lentinan (LNT) was from Shanghai Yuanye Biological Technology Co., Ltd. Hydrazine hydrate was from National Medicine Chemical Reagent Co., Ltd. Poly(beta-amino ester)-guanidine-phenylboronic acid (PBAE-G) was purchased from Aladdin Reagent Co., Ltd. in Shanghai, China. TRIzol and fetal bovine serum (FBS) were from Biyun Tian Biological Technology. Ovalbumin (OVA) and CCK-8 kit were from Sigma-Aldrich (MO, USA). DAPI was from Solarbio Technology Co., Ltd. IgG, IgG2a, IgG2b were from abclon Biotech Co., Ltd. in Wuhan, China. APC anti-mouse CD86 and FITC anti-mouse MHCII were from eBioscience, Inc. (CA, USA). Rabbit monoclonal antibodies specific for cGAS (E5V3W) (#79978), STING (D2P2F) (#13647), TBK1 / NAK (E8I3G) (#38066), IRF-3 (D6I4C) (#11904), and NF-kB p65 (D14E12) (#8242) were from CST. RU320521 (CAS No. 2262452-06-0) was from Sigma.

[0035] 1.2 LNT-PBAE-G-ND and LNT-PBAE-G-ND@OVA synthesis

[0036] The synthesis route of LNT-PBAE-G-ND and LNT-PBAE-G-ND@OVA is shown in Figure 1 A. LNT was reacted with PBAE-G polymer in DMSO for 8 h with molecular sieves as a dehydrating agent, then the mixture was filtered to obtain a solution containing LNT-PBAE-G. Then, a 50 mL mixture containing NDs (157 mg, 0.4 mmol) and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 77 mg, 0.4 mmol) in ethanol was prepared, then mixed at 60 °C for 30 min to facilitate the activation of the carboxyl group. The mixture was then added dropwise to the LNT-PBAE-G (149 mg) solution heated to 60 °C. After mixing for 24 h, it was added to a dialysis bag with a 4.5 kDa MWCO and exchanged with pure O2 for 48 h to eliminate unreacted EDC or LNT-PBAE-G-ND in the mixture. The sample was then lyophilized, and the LNT-PBAE-G-ND crude product was washed with ethanol three times to facilitate the removal of unreacted NDs.

[0037] Drug-loaded system NPs were prepared by suspending 300 mg of LNT-PBAE-G-ND in 3 mL of DMSO, followed by the addition of 10 mg of OVA. The synthesized mixture was then gradually mixed with 15 ml of PBS (pH = 8.0), followed by dialysis (MWCO: 4.5 kDA) against PBS (pH = 7.4) to remove all organic solvents, resulting in LNT-PBAE-G-ND@OVA. The same method was also used for the preparation of empty NPs.

[0038] 1.3 Properties and stability of LNT-PBAE-G-ND@OVA

[0039] The morphology of the LNT-PBAE-G-ND@OVA formulation was evaluated using a scanning electron microscope (SEM, model S-4800 II FESEM, Hitachi High Technologies, Japan). Dynamic light scattering (DLS) was used to evaluate NP size and Zeta potential values. The binding efficiency of LNT in the LNT-PBAE-G-ND formulation was evaluated using the phenol-sulfuric acid method, and the binding efficiency (EE%) was determined as follows:

[0040] EE% = (1 - Q1 / Q2) x 100%,

[0041] where Q2 corresponds to the amount of LNT and OVA, and Q1 corresponds to the amount of unbound LNT and OVA.

[0042] The stability of the samples was evaluated by dissolving the lyophilized LNT-PBAE-G-ND@OVA in ddH, followed by the evaluation of Zeta potential and particle size as described above.

[0043] 1.4 OVA and LNT release assay

[0044] The release of OVA and LNT under inflammatory microenvironment was investigated. Macrophages were incubated with LNT-PBAE-G-ND@OVA and IL-6 (10 pg / mL) concentrations for 4, 8, 12, 24, 48, 96, 192, and 384 h, and the release rates of OVA and LNT were observed.

[0045] 1.5 Cytotoxicity analysis

[0046] Mouse macrophages were treated with a range of concentrations of LNT-PBAE-G-ND@OVA or LNT-PBAE-G-ND, with DMEM as a control. At the appropriate time points, the cells were rinsed with PBS, and then cytotoxicity was evaluated using a CCK-8 kit, with absorbance analyzed at 450 nm using a microplate reader. The viability (%) relative to the control group was determined.

[0047] 1.6 Macrophage surface marker analysis

[0048] Flow cytometry was used to detect the expression of CD80, CD86 and MHCII on the surface of macrophages. Cells were treated with DMEM (negative control), LPS (500 ng / mL, positive control), LNT-PBAE-G-ND@OVA (10 pg / mL), OVA (10 pg / mL) or LNT (10 pg / mL). After 48 h, cells were stained with appropriate antibodies and analyzed by flow cytometry (C6, Becton, Dickinson and Company).

[0049] 1.7 Macrophage uptake assay

[0050] Macrophages were added to 6-well plates and treated with LNT-PBAE-G-ND@OVA (10 pg / mL) or LNT-PBAE-G-ND (10 pg / mL), respectively, to determine the uptake efficiency of LNT-PBAE-G-ND@OVA. After washing with PBS three times, cells were fixed in 4% paraformaldehyde for 15 min and stained with DAPI (50 pL) for 5 min. Uptake efficiency of LNT-PBAE-G-ND@OVA was then determined by confocal laser scanning microscopy.

[0051] 1.8 Immunization of mice

[0052] BALB / c mice (female, 6 weeks old) were obtained from the Comparative Medicine Center of Zhengzhou University. Experimental mice were housed in accordance with the Guide for the Care and Use of Laboratory Animals issued by the Henan Institute of Animal Husbandry and Economy IACUC, which was approved by IACUC (No: 017731, 2011bad34b02). Mice were injected subcutaneously with saline, OVA, LNT, LNT / OVA, LNT-PBAE-G-ND or LNT-PBAE-G-ND@OVA, LNT and OVA were used at the same concentration (500 pg / mL), while LNT-PBAE-G-ND and LNT-PBAE-G-ND@OVA were dosed according to the binding rate (%EE; 674.6 pg / mL, 697.2 pg / mL). After 14 days, mice were given a muscle-enhancing injection of 0.2 mL of the same amount of treatment at the corresponding dose. Blood samples were collected on days 14, 28 and 42 after the second immunization, and the mice were euthanized and the major organs were collected on day 42.

[0053] 1.9 Elisa assay

[0054] Elisa was used to analyze the serum samples of mice on days 14, 28 and 42 after the second immunization to detect OVA-specific IgG, IgG2a and IgG2b concentrations, as previously described. Elisa was used to detect inflammatory cytokine levels. The absorbance at 450 nm was measured by a microplate reader. Samples were divided into four parts.

[0055] 1.10 Lymphocyte immunophenotyping

[0056] On day 28 post the second immunization, T cell subsets were analyzed by collecting mouse splenocytes. These cells were then added to 24-well plates (1 x 10 6 / well) and 48h later, collected and stained with anti-CD3e-FITC, anti-CD4-APC and anti-CD8a-PE at 4°C in the dark for 30min. Cells were then washed with PBS 3 times, fixed with 4% paraformaldehyde and analyzed by flow cytometry. Analysis was done in triplicate.

[0057] 1.11 RNA-seq assay

[0058] Macrophages were added to 6-well plates (1 x 10 6 / well) and treated with LNT-PBAE-G-ND@OVA (10 pg / mL) in triplicate as described above with un-supplemented DMEM as control. RNA was extracted from these cells using TRIzol (Ambion) 48h later. RNA-seq analysis (PE 150) was then performed using Illumina Hiseq4000 platform (Biomake Scientific Co., Ltd, Beijing, China).

[0059] 1.12 qPCR-based validation

[0060] Nine different genes were randomly selected for RNA-Seq validation and then qPCR (+gDNA wiper) and ChamQ II Q RT SuperMix were performed according to the provided guidelines. TM qPCR was confirmed by qPCR using ChamQ -ΔΔCt Master Mix (Vazyme Biotech Co., China, Nanjing, China) according to the manufacturer’s instructions. Data were analyzed by the 2 6

[0061] 1.13 Western blotting

[0062] Macrophages were added to 6-well plates (1 x 10 6 / well) with LNT-PBAE-G-ND@OVA (10 pg / mL) in triplicate, with non-supplemented DMEM as control. Protein was extracted from samples using RIPA buffer (Bi Yun Tian Biotechnology) after 48 h treatment, and diluted to the same protein concentration with loading buffer and heated at 95 °C for 10 min. Protein was separated by SDS-PAGE and transferred to PVDF membrane, which was blocked with 5% skim milk for 2 h and then incubated with appropriate primary antibody at 4 °C overnight. After washing with PBST for 3 times, incubated with goat anti-rabbit IgG HRP for 2 h, washed for 3 times, and protein was detected using enhanced chemiluminescence (ECL) kit (Biosharp life sciences, Beijing, China).

[0063] 1.14 cGAS inhibition assay

[0064] Macrophages were added to 6-well plates (1 x 10 6 / well) with RU320521 (37.5 mM) pre-treatment for 4 h to inhibit cGAS. Then cells were treated with LNT-PBAE-G-ND@OVA (10 pg / mL) for 48 h. Cells without AG490 inhibitor treatment were treated with LNT-PBAE-G-ND@OVA (10 pg / mL) for 48 h. The above method was used to detect IL-12, IL-1 b, TNF-a levels in supernatant of CD11c positive cells and CD40, CD80, CD86 expression.

[0065] 1.15 Statistical analysis

[0066] Data are mean ± SEM, and comparisons were made by Duncan's multiple range test. The threshold of significance was P < 0.05.

[0067] 2 Results and discussion

[0068] 2.1 Preparation and characterization of LNT-PBAE-G-ND

[0069] Structural characteristics of PBAE-G polymer 1 H NMR ((400 MHz, DMSO-d6 δ): 6.5-8.0 (3H), 7.8 (1.21 hour), 7.4 (1.21 hour), 5.2 (1.21 hour), 4.3 (4 hour), 3.4 (8 hour), 3.0 (8 hour)).

[0070] The original ND NPs were produced by blasting method, and the nanoscale NDs produced by ultrasonic action were getting smaller and smaller, and the final NPs size was about 25 nm. The core-shell structure of LNT-PBAE-G-ND@OVA NPs was confirmed by transmission electron microscopy (TEM)Figure 1 B, 1C and Table 1), the final NPs size was ~145.1 nm.

[0071] Zeta potential of LNT-PBAE-G-ND and LNT-PBAE-G-ND@OVA were negative, -14.1 ± 0.98 mV and -17.78 ± 1.05 mV, respectively Figure 1 D), all of these NPs had low PDI values, which were consistent with low level of polydispersion Figure 1 E).

[0072] ND FL intensity values of the solutions containing LNT-PBAE-G-ND and LNT-PBAE-G-ND@OVA Figure 1 F). The solutions of LNT-PBAE-G-ND@OVA containing unbound LNT were evaluated for the synthesis of LNT-PBAE-G-ND@OVA using phenol-sulfuric acid method to calculate the LNT binding efficiency. In summary, 65.43% and 63.21% of LNT were successfully bound to LNT-PBAE-G-ND and LNT-PBAE-G-ND@OVA, respectively (Table 1), and this high LNT loading capacity might be due to the high surface area exhibited by the NDs.

[0073] Table 1 Size, Zeta potential and LNT and OVA grafting efficiency of LNF-PBAE-G-ND or LNF-PBAE-G-ND@OVAa

[0074]

[0075] 2.2 Nanoparticle stability

[0076] The stability of LNT-PBAE-G-ND and LNT-PBAE-G-ND@OVA NP samples was evaluated by measuring the PDI, particle size and Zeta potential values of these particles over a 4-week incubation period at 4°C Figure 2 ). LNT-PBAE-G-ND@OVA particles maintained a size of ~145-170 nm from day 1-35 (P > 0.05), while LNT-PBAE-G-ND lamellae maintained a size of 120-140 nm, with the smaller size corresponding to successful LNT modification, leading to a larger hydrated particle size Figure 2 A). The zeta potential and PDI values of LNT-PBAE-G-ND and LNT-PBAE-G-ND@OVA formulations remained negative and unchanged from day 1-35 Figure 2 B-2C, P > 0.05). Thus, both LNT-PBAE-G-ND and LNT-PBAE-G-ND@OVA were highly stable after repeated dissolution and lyophilization.

[0077] 2.3 Release of LNT and OVA from NPs

[0078] PBAE-G polymers can respond to high ROS levels and low pH, both of which are common under inflammatory microenvironment conditions, and drive the conversion of these polymers to a hydrophilic, cationic form. Vaccine adjuvants modulate inflammatory signaling pathways through immune stimulation, making LNT-PBAE-G-ND potentially highly sensitive to inflammatory conditions in a way that is favorable for immunity. LNT-PBAE-G-ND@OVA formulations released LNT and OVA more rapidly at pH 3.5 than at pH 7.0 Figure 3 A-3B). When a neutrophil inflammation model was established to mimic inflammatory microenvironments, under these conditions, LNT-PBAE-G-ND@OVA rapidly released high levels of LNT and OVA Figure 3 C-3F).

[0079] 2.4 Analysis of cytotoxicity and uptake of LNT-PBAE-G-ND@OVA treated macrophages

[0080] CCK-8 assays were used to detect the cytotoxic effects of LNT-PBAE-G-ND@OVA or LNT-PBAE-G-ND treated macrophages after 48 hours of exposure to different concentrations of these formulations. At the 40 pg / mL dose level, both formulations were non-toxic. At the 10 pg / mL concentration, both LNT-PBAE-G-ND and LNT-PBAE-G-ND@OVA promoted macrophage proliferation Figure 4 A). Therefore, all subsequent experiments were performed at the 10 pg / mL dose level.

[0081] The ability of macrophages to readily uptake antigens is necessary for their ability to stimulate subsequent immunity, both macrophages and neutrophils are early responders of the innate immune system, acting to rapidly destroy invading pathogens. Confocal microscopy was used to examine the internalization of LNT-PBAE-G-ND and LNT-PBAE-G-ND@OVA by macrophages, and it was found that OVA-FITC and NDs were readily internalized Figure 4 B), confirming the ability of LNT-PBAE-G-ND@OVA to be efficiently taken up by these apcs.

[0082] 2.5 Macrophage activation marker expression

[0083] Upon internalization of pathogen-derived proteins, macrophages are activated, allowing them to present these antigens to T cells, activating adaptive immunity. Therefore, macrophages are essential for both cellular and humoral immune response coordination, and upon activation, high levels of costimulatory markers such as CD80 + , CD86 +and MHCII + expression, enabling these cells to promote T cell activation. The expression of CD80 + , CD86 + , and MHCII + was detected by flow cytometry after LNT-PBAE-G-ND@OVA-treated macrophages. A significant upregulation of these three costimulatory markers was observed after LNT-PBAE-G-ND@OVA treatment compared to the control group (P < 0.05), reaching levels comparable to the LPS positive control group (P > 0.05, Figure 5 A-5D).

[0084] 2.6 Macrophage cytokine secretion

[0085] IL-12 is a pro-inflammatory cytokine that is important for T cell proliferation and IFN-γ production. When macrophages phagocytose antigens and are activated, they release a series of inflammatory cytokines, including IL-1β and IL-12. Therefore, by analyzing the levels of IL-12, IL-1β, TNF-α, and IL-6 in the supernatant after LNT-PBAE-G-ND@OVA treatment, the activation of macrophages was further evaluated. Consistent with the above data, higher levels of these four cytokines were released after LNT-PBAE-G-ND@OVA stimulation (P < 0.05 or P < 0.01) Figure 6 A-6D); these data suggest that LNT-PBAE-G-ND@OVA NPs can easily activate macrophages.

[0086] 2.7 Serum antibody production analysis in response to LNT-PBAE-G-ND@OVA

[0087] Antibody responses can be used to measure the impact of LNT-PBAE-G-ND@OVA treatment on the adaptive immune response, so the concentrations of IgG, IgG2a, and IgG2b were determined by ELISA. Mice were immunized once on day 0 and boosted on day 14. Animals were sacrificed on days 14, 28, and 42 to collect serum samples for antibody level determination, and cytokine levels were determined on days 28 and 42. The IgG levels were significantly higher after LNT-PBAE-G-ND@OVA treatment 28 and 42 days after immunization compared to LNT, OVA, LNT-PBAE-G-ND, or LNT / OVA treatment alone Figure 7 ). LNT-PBAE-G-ND@OVA treatment also resulted in higher IgG1 levels compared to other treatment groups, and the IgG2a response was stronger with LNT-PBAE-G-ND@OVA treatment compared to OVA treatment (P < 0.01). Immunoadjuvants can alter Th1 and Th2 CD4 +The relative induction of T cell responses can be further understood by measuring different serum antibody titers. Specifically, IgG1 is the major antibody subtype associated with Th2 immunity, while IgG2A and IgG2b are the major antibody subtypes associated with Th1 cellular immune responses. These results are consistent with the ability of LNT-PBAE-G-ND@OVA treatment to induce more effective humoral and cellular immunity.

[0088] 2.8 Effects on T lymphocyte subsets

[0089] Forty-two days after immunization, spleen cells were collected from mice, and the proportions of different T cell subsets (CD4+) were analyzed by flow cytometry. + / CD8 + and CD3 + / CD8 + T cells). Overall, LNT-PBAE-G-ND@OVA treatment is associated with higher CD4 counts. + Related + and CD3 + With CD8 + Compared with other experimental treatments, the T cell ratio ( Figure 8 This is consistent with the platform's ability to induce strong in vivo cellular immunity.

[0090] 2.9 Serum cytokine levels

[0091] Serum cytokine levels were measured by ELISA at 28 and 42 days post-primary immunization. Compared with other treatments, LNT-PBAE-G-ND@OVA treatment resulted in higher levels of Th1 cytokines (IFN-γ and TNF-α) (P<0.05). Figure 9 (A-9D). TNF-α is important for inducing Th1-based cellular immunity. Similarly, LNT-PBAE-G-ND@OVA treatment resulted in higher levels of IL-4 and IL-6 than other treatments (P<0.05). These data suggest that LNT-PBAE-G-ND@OVA immunization can induce strong Th1 and Th2 immune responses, consistent with the antibody data above.

[0092] 2.10 In vivo biocompatibility analysis of LNT-PBAE-G-ND@OVA

[0093] An effective vaccine adjuvant must possess good biocompatibility. Therefore, the ability of LNT-PBAE-G-ND@OVA to induce major organ toxicity was investigated. No changes in blood biochemical parameters were observed after treatment with this adjuvant candidate drug. Figure 10A-10B), and no difference in serum biochemical indicators of kidney or liver function was detected after treatment. Likewise, H&E staining of major organs (lungs, liver, spleen, kidneys, heart) after LNT-PBAE-G-ND@OVA treatment did not show any significant histological changes, with no evidence of inflammation or associated tissue damage Figure 10 C).

[0094] 2.11 LNT-PBAE-G-ND@OVA promotes immune-stimulatory cGAS-STING-TBK1-IRF3 pathway activation

[0095] To better understand the interaction ability between LNT-PBAE-G-ND@OVA and macrophage activation, the present application performed RNA-Seq analysis on these macrophages.

[0096] Figure 11 A shows a heatmap of the hierarchically clustered DEGs when comparing cells treated with saline or LNT-PBAE-G-ND@OVA. KEGG pathway analysis of these DEGs showed that multiple enriched pathways, including TLR and nod-like receptor signaling pathways, were upregulated after LNT-PBAE-G-ND@OVA treatment Figure 11 B and Figure 11 C), with results given according to q value and enrichment degree. The nod-like receptor and cGAS-STING signaling pathways were most upregulated. To further confirm these results and elucidate the potential molecular mechanism by which LNT-PBAE-G-ND@OVA can induce macrophage activation, the present application analyzed the key proteins of the cGAS-STING pathway and found that LNT-PBAE-G-ND@OVA upregulated cGAS, STING, TBK1, and IRF3 Figure 11 D). These data confirmed the ability of LNT-PBAE-G-ND@OVA to activate the cGAS-STING-TBK1-IRF3 signal.

[0097] 2.12 cGAS inhibitor treatment inhibits the ability of LNT-PBAE-G-ND@OVA to induce macrophage activation

[0098] Activation of the cGAS-STING pathway is essential for innate immunity, which can be activated in response to dsDNA released by bacteria and viruses that invade host cells. This dsDNA binding causes a change in the cGAS structure, which catalyzes the production of cGAMP, which in turn binds to STING, activating it and promoting the activation of TBK1. This further drives the expression of type I interferons (IFNs) and the upregulation of a series of inflammatory cytokines that can stimulate the induction of a stronger immune response. The cGAS inhibitor RU320521 was thus used to illustrate the interaction between the cGAS-STING-TBK1-IRF3 signal and the LNT-PBAE-G-ND@OVA-mediated activation and maturation of macrophages. Treatment with this inhibitor was sufficient to reduce the expression of cGAS and STING at the protein level Figure 12 A- Figure 12 B). Compared with macrophages treated with LNT-PBAE-G-ND@OVA alone, macrophages treated with RU320521 had significantly reduced surface CD80, CD86 and MHCII expression (P < 0.01, Figure 12 C- Figure 12 D), while the secretion of IL-6, IL-12, IL-1β and TNF-α by these cells was reduced (P < 0.01, Figure 12 E). In summary, these data suggest that the cGAS-STING pathway is required for the ability of LNT-PBAE-G-ND@OVA to easily activate macrophages.

[0099] In summary, the present application uses PBAE-G to design a nano-vaccine adjuvant: lentinan functionalized PBAE-G nanodiamond carrying ovalbumin (LNT-PBAE-G-ND@OVA). In vitro, LNT-PBAE-G-ND@OVA can transfect mRNA into multiple cell lines, increasing the immune response; in vivo, LNT-PBAE-G-ND@OVA / mRNA intramuscular injection can effectively deliver vaccines, inducing strong humoral and cellular immune responses, and without obvious toxicity, which shows that LNT-PBAE-G-ND is a safe and effective mRNA vaccine delivery carrier. In addition, the present application also explores the molecular mechanism of nano-vaccine adjuvant-induced macrophage activation, and finds that LNT-PBAE-G-ND@OVA activates macrophages by activating the cGAS-STING signaling pathway.

[0100] The above-described embodiments are merely preferred embodiments of the present application, merely used to explain the present application, and are not intended to limit the scope of the present application. For those skilled in the art, other embodiments can be easily obtained by substitution or change based on the technical content disclosed in the present specification. Therefore, any changes and improvements made on the principles of the present application shall be included in the scope of the present application.

Claims

1. Use of lentinan in the preparation of a pH-responsive nanomedicine carrier, characterized in that, The nano-drug carrier is a core-shell structure assembled by loading lentinan on the surface of a cationic polymer, poly(beta-amino ester)-guanidine-phenylboronic acid, and then together with nanodiamond particles, the nano-drug carrier activates macrophages to produce an immune response by activating the cGAS-STING signal pathway; the pH response range of the nano-drug carrier is 3.5-7.0, and the bioactive substance carried by the nano-drug carrier is ovalbumin.

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