A peptide immunomodulator-resistant nano-vaccine, a preparation method and application thereof

Abstract

The application discloses a tolerogenic peptide immunomodulator nanovaccine and a preparation method and application thereof. The tolerogenic peptide immunomodulator nanovaccine comprises an immunomodulator encapsulated polymer vesicle and a chemically bonded tolerogenic peptide. In order to reestablish antigen-specific immune tolerance in RA, the application develops a tolerogenic nanovaccine, i.e. a heat shock protein antigen peptide surface modified rapamycin encapsulated polymer vesicle (HRV). A PEG-P(TMC-DTC) based reduction-sensitive disulfide cross-linked polymer vesicle is used as a platform for displaying these antigens and presenting to DCs. The platform not only has flexibility in surface modification and loading of various anti-inflammatory therapeutic drugs (such as rapamycin), but also serves as an adjuvant to promote the establishment of immune tolerance. Hyaluronic acid (HA) hydrogel provides a reservoir for the tolerogenic nanovaccine, ensuring its sustained release to lymphoid organs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a tolerance vaccine, specifically a tolerance peptide immunomodulator nanovaccine and its preparation method and application. Background Technology

[0002] Rheumatoid arthritis (RA) is a systemic autoimmune disease that remains incurable. Patients manage the disease through long-term use of three classes of medications: nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and disease-modifying antirheumatic drugs (DMARDs). Essentially, rheumatoid arthritis is an autoimmune disease caused by the immune system recognizing its own antigens as allergens, leading to the production of autoantibodies that attack its own tissues. Therefore, inducing durable, antigen-specific immune tolerance is crucial for treating autoimmune diseases like RA. Immune tolerance therapy for rheumatoid arthritis has attracted widespread attention. Researchers have proposed various strategies, including adoptive immunotherapy that eliminates autoreactive immune cells through hematopoietic stem cell transplantation [see: Treatment of severe autoimmune disease by stem-cell transplantation], and the use of various immunomodulatory cells to rebuild autoimmune tolerance [see: Cell therapy for autoimmune diseases]. Although cell therapy has shown promising therapeutic effects, its complex treatment modalities, lengthy treatment cycles, and high costs limit its widespread clinical application. Tolerance peptide-based vaccines are a promising therapy for autoimmune diseases, which inhibit antigen-specific effector T cells and promote the proliferation of regulatory T cells (Tregs).

[0003] A significant challenge in inducing immune tolerance is that using autoantigens alone for immunization can exacerbate pro-inflammatory immune responses [see: Induction of immune tolerance in the treatment of rheumatoid arthritis]. Recent studies suggest that co-delivering autoantigens with immunomodulators may solve this problem. Existing techniques utilize lysine-proline-valine as small-molecule anti-inflammatory peptides, linking these peptides with the immunomodulator / autophagy inducer rapamycin via intermolecular forces such as hydrophobic interactions and hydrogen bonds, to prepare KPV-RAPA nanomedicines through nanoprecipitation. Existing techniques also disclose peptide nanomaterials for loading rapamycin. These peptide nanomaterials possess a specific structure and can self-assemble into nanoparticles, enabling the encapsulation of rapamycin. However, existing methods for loading immunomodulators with peptides suffer from problems such as leakage during delivery and deviations in therapeutic efficacy. Current technology involves dissolving biocompatible polymer matrix materials and autoantigens in pure water, adding immunomodulators, and stirring to obtain a suspension. This suspension is then added to a mold to form needle tips, dried, and a backing solution is poured in. Finally, the microneedle patch is demolded. Quality control of microneedles has always been a challenge in this field and a major reason limiting their mass production. Currently, mainstream testing methods for the mechanical strength of microneedles can only provide a rough assessment of the entire microneedle patch and cannot perform quality control on each individual needle tip unit. Existing technology uses oil emulsions (ILEs) as a core to encapsulate hydrophobic rapamycin in an oil phase. Polyethylene glycol is then modified onto the surface of the ILEs, and trace amounts of ferric hydroxide are added to the PEGylated ILEs to improve the encapsulation efficiency of the formulation for CitP (an important autoantigen in RA). This method is complex and difficult to control in terms of quality, especially given the large size of the nanoparticles (reaching 130 nm). Therefore, there is a need to develop new nanovaccines that can easily and effectively co-deliver autoantigens and immunomodulators to significantly alleviate inflammation and protect cartilage tissue. Summary of the Invention

[0004] This invention employs a novel method to prepare nanovaccines loaded with tolerance peptides and immunomodulators, with a particle size of approximately 60 nm. It successfully reduces the maturity and antigen-presenting capacity of BMDC cells, decreases the expression of pro-inflammatory factor IL-6, increases the expression of inhibitory factor IL-10, and increases the proportion of Treg cells in spleen cells.

[0005] The present invention adopts the following technical solution.

[0006] A tolerance peptide immunomodulator nanovaccine comprises an immunomodulator encapsulated polymer vesicle and a tolerance peptide.

[0007] Preferably, the tolerance peptide is linked to the immunomodulator encapsulated polymer vesicles via chemical bonds.

[0008] Further preferred, the surface of the immunomodulator encapsulated polymer vesicles is modified with a tolerance peptide.

[0009] In this invention, the tolerance peptides include one or more peptides derived from type II collagen, filaggrin, fibrinogen, vimentin, rheumatoid factor, citrullinated proteins, and heat shock proteins (HSPs). The heat shock proteins include large molecular weight heat shock proteins Hsp110, Hsp90, Hsp70, and Hsp60, as well as small molecular weight heat shock proteins. The large molecular weight heat shock proteins are ATP-dependent molecular chaperones, while the small molecular weight heat shock proteins function in an ATP-independent manner.

[0010] In this invention, the immunomodulators include one or more of dexamethasone, cyclosporine, tacrolimus, rapamycin, and their derivatives.

[0011] In this invention, the tolerance peptide immunomodulator nanovaccine can induce mature resistance in antigen-presenting cells and endow them with tolerance characteristics. By inhibiting the mTOR pathway of antigen-presenting cells, it downregulates the expression of surface maturation markers such as CD40, CD80, and CD86. At the same time, it allows the treated antigen-presenting cells to retain the ability to stimulate and activate regulatory T cells (Tregs). The co-delivery of self-antigens and immunomodulators significantly alleviates inflammation in mice and protects cartilage tissue.

[0012] In this invention, the polymer vesicles include hydrophilic segments and hydrophobic segments, and are assembled from polymers to form vesicles. The hydrophilic segments are attached to tolerance peptides, thereby enabling the surface modification of the immunomodulatory polymer vesicles with tolerance peptides. In other words, the tolerance peptides and the immunomodulatory polymer vesicles are connected by chemical bonds.

[0013] In this invention, the polymer assembled into polymer vesicles includes an amphiphilic block polymer, which includes hydrophilic segments and hydrophobic segments. Furthermore, the hydrophilic segments are attached to tolerance peptides, thereby modifying the surface of the assembled polymer vesicles with tolerance peptides.

[0014] Preferably, the hydrophobic segment includes a DTC (dithiopentane trimethylene carbonate) monomer repeating unit; more preferably, the hydrophobic segment includes a DTC monomer repeating unit and an ester monomer repeating unit, denoted as P (ester monomer unit-DTC).

[0015] Preferably, the polymer composed of hydrophilic and hydrophobic segments is hydrophilic segment-P (lipid monomer unit-DTC), which is an existing polymer.

[0016] Wherein: P (ester monomer unit - DTC) is a hydrophobic segment, which is a polymer segment of ester monomer and DTC monomer. Preferably, the hydrophilic segment is a polyethylene glycol segment (PEG); the molecular weight of the hydrophilic segment is 1000-15000 Da, and the molecular weight of the hydrophobic segment is 1-20 times that of the hydrophilic segment; the molecular weight of the PDTC segment is 5%-50% of the total molecular weight of the hydrophobic segment.

[0017] Preferably, the molecular weight of PEG is 2000-12000 Da; more preferably, the molecular weight of PEG is 3000-10000 Da; and even more preferably, the molecular weight of PEG is 4000-9000 Da.

[0018] Preferably, the molecular weight of the hydrophobic segment is 2 to 15 times that of the PEG molecular weight; more preferably, the molecular weight of the hydrophobic segment is 2 to 10 times that of the PEG molecular weight; even more preferably, the molecular weight of the hydrophobic segment is 2 to 8 times that of the PEG molecular weight, such as 3 times, 4 times, 5 times, or any multiple within the range; the molecular weight of PDTC is 8% to 40% of the molecular weight of the hydrophobic segment; preferably, the molecular weight of PDTC is 10% to 30% of the molecular weight of the hydrophobic segment, such as 11%, 12%, 15%, 20%, 25%, or any proportion within the range.

[0019] This invention discloses a method for preparing the above-mentioned tolerance peptide immunomodulator nanovaccine, which includes the following steps: mixing an amphiphilic block polymer containing the tolerance peptide and an immunomodulator, assembling them, and preparing the tolerance peptide immunomodulator nanovaccine.

[0020] Furthermore, the amphiphilic block polymer of the tolerance peptide and the immunomodulator were mixed in solution and assembled to prepare the tolerance peptide immunomodulator nanovaccine.

[0021] Furthermore, the amphiphilic block polymer solution containing the tolerance peptide and the immunomodulator solution are mixed and assembled to prepare a tolerance peptide immunomodulator nanovaccine.

[0022] Furthermore, the amphiphilic block polymer solution of the tolerance peptide and the immunomodulator solution were mixed and assembled in a buffer solution to prepare a tolerance peptide immunomodulator nanovaccine.

[0023] In this invention, assembly includes self-assembly.

[0024] In this invention, an amphiphilic block polymer containing a tolerance peptide is prepared by reacting a tolerance peptide with a functionalized amphiphilic block polymer.

[0025] In this invention, the solvent in the amphiphilic block polymer solution of the tolerance peptide includes dimethyl sulfoxide (DMSO); the concentration of the amphiphilic block polymer of the tolerance peptide is 1–1000 mg / mL, preferably 10–800 mg / mL, more preferably 30–600 mg / mL, even more preferably 50–500 mg / mL, more preferably 70–400 mg / mL, and most preferably 80–300 mg / mL, such as 90 mg / mL, 100 mg / mL, 150 mg / mL, 200 mg / mL, 250 mg / mL, or any value within the range.

[0026] In this invention, the solvent in the immunomodulator solution includes DMSO; the concentration of the immunomodulator is 10–1000 mg / mL, preferably 20–800 mg / mL, more preferably 30–500 mg / mL, even more preferably 40–300 mg / mL, and most preferably 50–100 mg / mL, such as 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, or any value within the range.

[0027] In this invention, the buffer solution is an existing product.

[0028] In this invention, the volume ratio of the amphiphilic block polymer solution containing the tolerance peptide to the immunomodulator solution is (1-100):1. Preferably, the volume ratio is (3-50):1. More preferably, the volume ratio is (5-30):1. Even more preferably, the volume ratio is (7-20):1, such as 8:1, 9:1, 10:1, or any ratio within the range.

[0029] This invention discloses an injectable tolerable peptide nanovesicle hydrogel system, comprising the aforementioned tolerable peptide immunomodulator nanovaccine and hydrogel.

[0030] In the injectable tolerable peptide nanovesicle hydrogel system of this invention, HRV achieved positive therapeutic effects in the ZIA mouse model, alleviating joint swelling, inducing joint tolDCs and lymph node Tregs, and successfully establishing local and peripheral tolerance in the joint. Furthermore, the HA-based dibenzocyclooctylene (-DBCO) and azide (-N3) functionalized injectable tolerable peptide nanovesicle hydrogel system (HRV@gel) can continuously release HRV, enabling long-term enrichment of HRV in LNs, establishing local and peripheral immune tolerance, alleviating joint swelling in CIA mice, and protecting the integrity of cartilage and bone.

[0031] In this invention, the hydrogel includes hyaluronic acid hydrogel.

[0032] Preferably, the hydrogel comprises a hyaluronic acid-based dibenzocyclooctylene (-DBCO) and azide (-N3) functionalized hydrogel.

[0033] This invention discloses a method for preparing an injectable tolerable peptide nanovesicle hydrogel system, comprising the following steps: mixing a hydrogel precursor and the above-mentioned tolerable peptide immunomodulatory nanovaccine to form an injectable tolerable peptide nanovesicle hydrogel system.

[0034] Furthermore, the hydrogel precursor solution and the above-mentioned tolerance peptide immunomodulator nanovaccine solution are mixed to form an injectable tolerance peptide nanovesicle hydrogel system.

[0035] In this invention, the hydrogel precursor includes functionalized hyaluronic acid, and further, the hydrogel precursor includes dibenzocyclooctylene-functionalized hyaluronic acid (HA-DBCO) and azide-functionalized hyaluronic acid (HA-N3).

[0036] Preferably, in the dibenzocyclooctylene-functionalized hyaluronic acid, the degree of substitution of dibenzocyclooctylene is 5-50%, more preferably 10-30%; the degree of substitution of DBCO refers to the proportion of carboxyl groups in hyaluronic acid that are replaced by DBCO, which is the theoretical degree of substitution.

[0037] Preferably, in the azide-functionalized hyaluronic acid, the degree of substitution of azide is 5-50%, more preferably 10-30%; the N3 substitution degree refers to the proportion of carboxyl groups in hyaluronic acid that are replaced by N3, which is the theoretical substitution degree.

[0038] In this invention, the concentration of the hydrogel precursor solution is 1 to 50 mg / mL, preferably 5 to 30 mg / mL, such as 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL or any concentration within the range.

[0039] Preferably, the weight ratio of dibenzocyclooctylene-functionalized hyaluronic acid (HA-DBCO) to azide-functionalized hyaluronic acid (HA-N3) is 1:(0.5-2), for example, 1:1.

[0040] This invention discloses the application of the above-mentioned tolerance peptide immunomodulator nanovaccine or injectable tolerance peptide nanovesicle hydrogel system in the preparation of anti-inflammatory drugs.

[0041] This invention discloses the application of the above-mentioned tolerance peptide immunomodulator nanovaccine or injectable tolerance peptide nanovesicle hydrogel system in the preparation of in vivo and / or in vitro inflammatory therapeutic drugs.

[0042] This invention discloses the application of the above-mentioned tolerance peptide immunomodulator nanovaccine or injectable tolerance peptide nanovesicle hydrogel system in the preparation of drugs for the prevention and treatment of arthritis.

[0043] This invention discloses the application of the above-mentioned tolerance peptide immunomodulator nanovaccine or injectable tolerance peptide nanovesicle hydrogel system in the preparation of drugs to relieve joint swelling.

[0044] Rheumatoid arthritis (RA) is an autoimmune disease characterized primarily by chronic synovitis, with pathological changes mainly concentrated in the joints: abnormal proliferation of the synovium to 10-20 layers, joint swelling, and deformity. This invention develops a tolerable nanovaccine, namely, rapamycin-encapsulated polymer vesicles (HRVs) with surface modification of heat shock protein antigen peptides. PEG-P (TMC-DTC)-based reductively sensitive disulfide-linked polymer vesicles are used as a platform to display these antigens and deliver them to dendritic cells (DCs). This platform not only possesses the flexibility of surface modification and loading various anti-inflammatory therapeutics (such as rapamycin), but also acts as an adjuvant to promote the establishment of immune tolerance. Targeting the pathological characteristics of RA, this invention's local targeted therapy strategy based on hydrogel delivery shows unique advantages. The establishment of immune tolerance requires maintaining long-term low concentrations of antigen peptides; the local drug delivery system can continuously release the drug, maintaining an effective concentration at the administration site for a long period, thereby modulating the immune response and inhibiting disease progression. In this invention, hydrogels, as a novel drug delivery system, possess excellent biocompatibility and controllable degradation characteristics. The three-dimensional network structure of hydrogels offers several advantages: efficient drug loading; direct injection into the vicinity of diseased tissue, minimizing off-target effects; prolonged drug residence time in vivo; and control of drug release rate through adjustment of cross-linking degree, thereby achieving continuous and stable drug release. Hyaluronic acid (HA) hydrogels are biocompatible and injectable. In-situ cross-linked HA hydrogels obtained through rapid chemical reactions such as Michael addition and click chemistry can continuously release drugs under physiological conditions. Attached Figure Description

[0045] Figure 1Synthesis and characterization of HSP-PEG-P(TMC-DTC). (a) Synthetic steps. (b) Mal-PEG-P(TMC-DTC) and (c) HSP-PEG-P(TMC-DTC). 1 H-NMR spectrum (400 MHz, DMSO- d 6 ).

[0046] Figure 2 Preparation and characterization of tolerance-derived peptide nanovesicles. (a) Schematic diagram of HRV preparation. (b) Particle size distribution of RV, HV, and HRV. (c) Particle size distribution of HRV after incubation for 24 hours in 50-fold dilution or in PB (pH 7.4) containing 10% FBS. (d) Changes in particle size and zeta potential of HRV during 60 days of storage. (e) Cumulative release curve of HSP peptides in HRV under enzymatic action (n = 3).

[0047] Figure 3 The process of BMDC uptake of HRV and presentation of HSP peptide by BMDC. (a) Flow cytometry analysis of BMDC internalization of HRV-cy5 (cy5: 3.3 μM) after 4 hours of incubation (n = 4), (b) Pearson coefficient between HRV and lysosomes and (c) cell membrane and MHCII (n = 3).

[0048] Figure 4 TolDCs were induced in vitro by HRV (n = 4). (a) Experimental procedure. CD11c + MFI of (b) CD86, (c) MHC I and (d) MHC II in DCs, and concentrations of (e) IL-6 and (f) IL-10 after BMDCs were incubated with HRV, HV or RV (HSP: 30 μg / mL, Rapa: 15 μg / mL) for 24 hours.

[0049] Figure 5 Treg cells were induced in vitro by HRV (n = 3). (a) CIA mouse spleen cell culture procedure. (b) Splenic CD3. + CD4 + The proportion of T cells. (c) Spleen CD25 + FoxP3 + The percentage of Treg cells.

[0050] Figure 6 The efficacy of RV, HV, and HRV in treating ZIA mice (n = 4). (a) Experimental setup (1.5, 3, 4.5 mg HSP / kg; 0.75 mg Rapa / kg). (b) Changes in knee circumference. (c) Changes in relative body weight.

[0051] Figure 7 HRV-induced tolDCs (n = 4) in ZIA mice. Splenic DCs CD86 (a), HRV-induced Tregs in ILNs and PLNs of ZIA mice (n = 4) (b), (c) HRV improved the Th17 phenotype of spleen T cells in ZIA mice (n = 4).

[0052] Figure 8 Preparation and infrared characterization of HA-DBCO and HA-N3. (a) Synthetic routes of HA-DBCO and HA-N3. (b) Infrared characterization of HA-DBCO, HA-N3, and HA.

[0053] Figure 9 1H-NMR spectra of HA with different degrees of DBCO substitution (400 MHz, D2O). (a) 10% HA-DBCO; (b) 20% HA-DBCO; (c) 30% HA-DBCO.

[0054] Figure 10 1H-NMR spectra of HA with different N3 substitution degrees (400 MHz, D2O). (a) 10% HA-N3; (b) 20% HA-N3; (c) 30% HA-N3.

[0055] Figure 11 (a) The gelation process of HA-DBCO and HA-N3. (b) The macroscopic gelation process of hydrogel.

[0056] Figure 12 The gelation time for injectable hydrogel formulations is as follows: (a) degree of DBCO- / N3- substitution, (b) concentration of HA-DBCO / N3, (c) gelation time at temperature, (d) concentration of HA-DBCO / N3, (f) storage modulus of DBCO- / N3- substitution, (e) concentration of HA-DBCO / N3, (g) loss modulus of DBCO- / N3- substitution. (h) hydrogel shear dilution.

[0057] Figure 13 The microstructure of the hydrogel is shown (scale bar: 25 μm). (a) Microstructure of hydrogels with different concentrations and degrees of substitution. (b) Schematic diagram of the preparation of HRV@gel and its microstructure.

[0058] Figure 14The in vitro release behavior of drugs in hydrogels (n = 3). (a) Effect of different concentrations of HAase on the release rate of BSA-cy5 in hydrogels. (b) Effect of different concentrations of hydrogel on the release rate of BSA-cy5. (c) Differences in release rates of BSA-cy5 and CC-cy5 with different molecular weights in hydrogels. (d) Release behavior of HRV-cy5 and its loaded Rapa in HRV-cy5@gel hydrogel.

[0059] Figure 15 To investigate the biocompatibility of the hydrogel in vitro. (a) Effect of DMTO on L929 cells and (b) Effect of hydrogel extract on 293T cell viability (n = 6). Effect of hydrogel on (c) 293T cells (n = 4) and (d) RAW264.7 cells (n = 3). (e) Viability staining of 293T cells in the gel. (Green: live cells. Red: dead cells. Scale bar: 100 μm).

[0060] Figure 16 Biodistribution of HRV-cy5 and HRV-cy5@gel in a CIA model (n = 3). (a) In vivo imaging and (b) Semi-quantitative analysis results in mice. (c) Ex vivo imaging of major organs and ILNs at 12 hours and 4 days post-injection and (d) Semi-quantitative analysis of ILNs. (e) DCs within ILNs and (f) HRV-cy5 uptake behavior of B cells. (g) Semi-quantitative analysis of HRV-cy5 distribution in major organs at 12 hours and 4 days post-injection.

[0061] Figure 17 Efficacy of HV, HRV, and HRV@gel treatment in CIA mice (n = 7). (a) Schematic diagram of the two dosing regimens (total dose: HSP peptide, 27.0 mg / kg; Rapa, 4.5 mg / kg). (b) Mean hind paw thickness, (c) arthritis score, and (d) changes in relative body weight (data are expressed as mean ± standard error). (e) Photograph of the right hind leg of the mouse on day 21.

[0062] Figure 18 To induce local joint tolerance in CIA mice using HRV@gel. Figure 3 On day 21 of treatment with regimen .10a, the concentrations of (a) IL-6, (b) TNF-α and (c) IL-10, and (d) the relative concentration of HSP antibody in the knee and ankle joint necrolysis fluid of CIA mice. (e) The proportion of CD86+ DCs and (f) the mean fluorescence intensity of MHC II.

[0063] Figure 19To induce peripheral lymph node tolerance in CIA mice using HRV@gel (n = 7). (a) Proportion of CD86+ DCs in ILNs and PLNs, and (b) MFI of MHC II. (c) Proportion of Tregs in the CD4+ cell population in lymph nodes. (d) Proportion of B cells in ILNs and PLNs.

[0064] Figure 20 To induce peripheral spleen tolerance in CIA mice using HRV@gel (n = 6). Splenic DCs: (a) proportion of CD86+ cell population and (b) mean MHC II fluorescence intensity. Percentages of (c) CD4+ T cells, (d) Treg cells, and (e) CD8+ T cells in the spleen, and the Treg to CD8+ T cell ratio (f). (g) Percentage of B cells in the spleen. (h) Percentage of memory B cells among B cells. Detailed Implementation

[0065] To re-establish antigen-specific immune tolerance in rheumatoid arthritis (RA), this invention develops a tolerance-enhancing nanovaccine: rapamycin-encapsulated polymer vesicles (HRVs) with surface-modified heat shock protein antigen peptides. PEG-P (TMC-DTC)-based reductively sensitive disulfide-linked polymer vesicles are used as a platform to display these antigens and deliver them to dendritic cells (DCs). This platform not only offers the flexibility of surface modification and loading various anti-inflammatory therapeutics (such as rapamycin) but also acts as an adjuvant to promote the establishment of immune tolerance. Hyaluronic acid (HA) hydrogels provide a reservoir for this tolerance-enhancing nanovaccine, ensuring its sustained release into lymphoid organs.

[0066] Establishing immune tolerance requires maintaining long-term low concentrations of antigenic peptides. Based on the positive therapeutic effects of HRV in the ZIA mouse model, which alleviated joint swelling, induced joint tolDCs and lymph node Tregs, and successfully established local and peripheral tolerance, this invention designs an injectable tolerance peptide nanovesicle hydrogel system (HRV@gel) functionalized with HA, dibenzocyclooctylene (-DBCO), and azide (-N3). Studies have shown that HRV@gel continuously releases HRV, leading to long-term enrichment of HRV in lymph nodes, establishing local and peripheral immune tolerance, alleviating joint swelling in CIA mice, and protecting the integrity of cartilage and bone.

[0067] The following specific experiments illustrate the technological advancements of this invention. All raw materials used are existing products, and the specific preparation operations and performance testing employ conventional techniques. Among them, PEG-P(TMC-DTC) (M n = 7.5-(15.0-2.0) kg / mol), PEG-P(TMC-DTC)-cy5(M n= 7.5-(15.0-2.0) kg / mol) and Mal-PEG-P(TMC-DTC) (M n = 7.5-(15.0-2.0) kg / mol) were prepared according to the previously reported method. HSP70 B29 peptide (CGPGPGVLRIINEPTAAAIAY), 5-(2-aminoethylamino)-1-naphthalenesulfonic acid-coupled HSP peptide (EDANs-HSP, CGPGPGVLRIINE(EDANs)PTAAAIAY) and 4-((4-(dimethylamino)phenyl)azo)benzyl-coupled HSP peptide (Dabcyl-HSP, C(Dabcyl-Ahx)VLRIINEPTAAAIAY) were commercially available products.

[0068] Cell Culture and Animal Experiments. RAW264.7 mouse macrophage cell lines, 8-week-old male C57BL / 6 mice, and 8-week-old male DAB / 1 mice were commercially available products. Bone marrow-derived dendritic cells (BMDCs) were directly extracted and cultured from mouse bone marrow using standard techniques. Specific procedures were described in previous reports, briefly as follows: To obtain spleen cells, the spleens of CIA mice were ground through a 70 μm cell sieve, followed by centrifugation at 350 g for 5 minutes to collect the cell pellet. The cells were resuspended in ACK lysis buffer and incubated for 5-10 minutes to lyse red blood cells. The resulting single-cell suspension was used for subsequent experiments. All animal experimental procedures were approved by the Experimental Animal Center of Soochow University and the Animal Care and Use Committee of Soochow University, and all animal research protocols complied with the "Guidelines for the Care and Use of Laboratory Animals."

[0069] All data presented in this invention are expressed as mean ± standard deviation (SD); other methods will be described separately. Statistical differences between groups were analyzed using one-way ANOVA.* p < 0.05 indicates a significant difference; highly significant differences are indicated by **. p < 0.01、*** p < 0.001 and **** p < 0.0001 indicates.

[0070] Synthesis Example 1 HSP peptide-functionalized copolymer HSP-PEG-P(TMC-DTC) was synthesized via a Michael addition reaction between the thiol group of the HSP peptide (CGPGPGVLRIINEPTAAAIAY) and Mal-PEG-P(TMC-DTC). Specifically, under nitrogen protection, an HSP peptide solution (37 mg / mL, DMSO) was added to a stirred Mal-PEG-P(TMC-DTC) copolymer (M n= 7.5-(15.0-2.0) kg / mol) DMSO solution (100 mg / mL), with a molar ratio of 2:1. The reaction was carried out at 37 °C for 24 hours, followed by dialysis with dichloromethane (molecular weight cutoff: 7 kDa) and concentration by rotary evaporation. HSP-PEG-P(TMC-DTC) was recovered by cold ether precipitation, filtration, and vacuum drying, and weighed. The HSP-PEG-P(TMC-DTC) was... 1 H-NMR characterization was used for end-group analysis, and the degree of functionalization of HSP peptides was quantified using the MicroBCA method.

[0071] like Figure 1 As shown in figure a, the -SH residue on the N-terminal cysteine ​​of the peptide chain undergoes a Michael addition reaction with the maleimide on Mal-PEG-P(TMC-DTC) to obtain the HSP-PEG-P(TMC-DTC) block polymer. Compared to Mal-PEG-P(TMC-DTC) ( Figure 1 b), HSP-PEG-P(TMC-DTC) 1 H-NMR nuclear magnetic spectrum ( Figure 1 The disappearance of the Mal characteristic peak δ 7.00 in c) indicates that HSP was successfully grafted to the PEG end of the polymer. 1 H-NMR end-group analysis showed that, based on the integral ratio of the methyl protons (δ 0.7-0.9 ppm, 19.6H) to the methylene protons (δ 3.1 ppm, 41.6H) of the HSP peptide, the functionalization degree of the peptide exceeded 93.6%. Figure 1 c).

[0072] EDANS-HSP-PEG-P(TMC-DTC) and Dabcyl-HSP-PEG-P(TMC-DTC) were synthesized according to the above method, wherein EDANS-HSP and Dabcyl-PEG-P(TMC-DTC) reacted in a molar ratio of 3:1.

[0073] Example 1: HSP70 B29 peptide-functionalized nanovesicles (HRV) loaded with rapamycin (Rapa) HRP was obtained by mixing HSP-PEG-P (TMC-DTC) (100 mg / mL, 90 μL) and rapamycin (50 mg / mL, 10 μL) in DMSO and adding them to 0.9 mL of HEPES buffer (5 mM, pH 6.8). The mixture was then dialyzed in PB buffer (10 mM, pH 7.4) for 4 hours (molecular weight cutoff: 7 kDa), with the medium changed every hour.

[0074] Following the above method, RV was prepared using PEG-P (TMC-DTC) and rapamycin (DMSO); HV was prepared using HSP-PEG-P (TMC-DTC); cy5-labeled HRV (HRV-cy5) was prepared by HSP-PEG-P (TMC-DTC) and PEG-P (TMC-DTC)-cy5 at a mass ratio of 99:1; EDANS-labeled HRV (HRV-EDANs) was prepared by HSP-PEG-P (TMC-DTC) and EDANS-PEG-P (TMC-DTC) at a mass ratio of 70:30; and EDANS and Dabcyl co-labeled HRV (HRV-FRET) was prepared by mixing four polymers, HSP-PEG-P (TMC-DTC), PEG-P (TMC-DTC), EDANS-HSP-PEG-P (TMC-DTC), and Dabcyl-HSP-P (TMC-DTC), at a mass ratio of 10 / 8 / 1 / 1.

[0075] Particle size, particle size distribution, zeta potential, and particle size changes of vesicles were determined using dynamic light scattering (DLS), and the results were tracked after 50-fold dilution in buffer containing 10% FBS and long-term storage. The Rapa concentration in HRV was determined by HPLC using a C18 reverse-phase column (4.6 × 150 mm, 5 µm). The mobile phase was a mixture of A / B = 70 / 30 (v / v), where phase A was acetonitrile and phase B was pure water. The flow rate was 1 mL / min, the detection wavelength was 278 nm, and the elution time was 16 min. The drug loading efficiency (DLE) and drug loading amount (DLC) of Rapa in HRV were determined by UV-Vis spectrophotometry at 278 nm and obtained based on the Rapa standard curve using conventional drug loading characterization methods. The concentration of Rapa in HRV was determined by self-assembly (…). Figure 2 a) Grafted HSP peptide nanovesicles (HRV), Rapa-loaded nanovesicles (RV), and grafted HSP peptide nanovesicles (HV) were prepared. Their particle size was approximately 67 nm, and their PDI was less than 0.1, as shown in Table 1. The HSP concentrations of the various nanoparticles at a final concentration of 9 mg / mL were determined using a MicroBCA kit to be 0, 703.6, and 696.4 μg / mL, respectively (Table 2.1). This indicates that the grafting rate of HSP onto the polymer was 90.0%, which is consistent with... 1 The H-NMR spectra results were similar. Notably, the HRV exhibited stability at high dilution, in the presence of 10% FBS, and during 60 days of storage, with minimal size change. Figure 2 c, d).

[0076] Table 1 Characterization of RV, HV and HRV

[0077] a Measured by MicroBCA; b Measured by HPLC; c, d Measured by DLS (n = 3).

[0078] To assess the intracellular behavior of HSP peptides, the release kinetics of HSPs under simulated endosome / lysosome (containing CTSB, pH 4.5, and 10 mM GSH) and cytoplasmic (containing PK, pH 7.4, and 10 mM GSH) conditions were investigated. Cumulative release results indicated that while most HSP peptides remained on nanovesicles in the presence of GSH, they were efficiently cleaved and released under lysosomal and cytoplasmic conditions. Figure 2 e). These results validate that the HSP peptide is enzymatically released from HRV and subsequently presented to T cells by antigen-presenting cells, which is crucial for establishing RA immune tolerance. Example 2

[0079] To investigate the uptake and presentation of HRV by antigen-presenting cells, BMDCs were incubated with 1% Cy5-labeled HRV (or RV-Cy5). Confocal microscopy results showed that HSP peptides on the nanovesicle surface slightly inhibited BMDC uptake, and flow cytometry results further confirmed this. Figure 3 a). These results are attributed to the negative charge and large size of the HRV surface.

[0080] Co-localization behavior of HRV in BMDC lysosomes. BMDCs were seeded on PDL-pretreated 24-well plate coverslips (1 × 10⁻⁶). 6 / well), and after overnight culture, incubate with HRV-FRET (0.8 μg / mL) for 2, 4, or 8 hours. Stain cells with 594-WGA (10 μg / mL, 100 μL, 10 min), lysosomal green probe (10000-20000×, 400 μL HBSS, 15 min), and PE-cy5-MHC II (75×, 50 μL, 10 min), and fix with 4% paraformaldehyde. Wash three times with PBS between each two steps. Fix coverslips onto microscope slides and observe under a fluorescence microscope. After BMDC uptake of HRV-FRET, EDANS-HSP is digested. After the FRET effect disappears, the fluorescence intensity of EDANS-HSP increases. Fluorescence microscopy images show that the fluorescence intensity of EDANS gradually increases over time, and the Pearson correlation coefficient with lysosomal colocalization increases from 0.14 to 0.33 (**, Figure 3 b). Furthermore, anti-MHC II antibody staining and co-localization analysis with the cell membrane indicated that MHC II is mainly located on the cell surface ( Figure 3 c). Combined images of EDANs, lysosomes, and MHC II revealed that the HSP peptide was presented by MHC II molecules on the cell surface, and that antigen presentation increased over time. These results highlight that HRV releases the HSP peptide antigen upon endocytosis, which is subsequently presented to CD4 by MHC II. + T cells, thereby promoting a strong immune response. The tolerance peptide HSP on HRV is presented via MHC II of DCs and is essential for establishing immune tolerance in RA.

[0081] In vitro study of HRV-induced tolerance in BMDC cells. BMDCs were seeded in 12-well plates (1 × 10⁻⁶). 6 / well), treated with HRV, RV, HV (HSP peptide: 30 μg / mL; Rapa: 15 μg / mL) or PBS (n = 4). After 12 hours, LPS (100 ng / mL) was added and incubated for another 12 hours. The culture medium was collected, and the concentrations of IL-6 and IL-10 were determined using an ELISA kit. BMDCs were stained with FITC-CD11c antibody, PE-CD86, PE-cy5-MHC I, and PerCP / cy5.5-MHC II antibody, and analyzed by flow cytometry. The ability of HRV to induce tolDCs and Tregs and inhibit inflammatory responses was investigated by stimulating BMDCs with LPS. Figure 4 a). Flow cytometry analysis showed that CD86 was present in LPS-stimulated BMDCs. + CD11c + The proportion of DCs increased significantly, confirming their mature state under inflammatory conditions. Subsequent treatment of these mature DCs (mDCs) with HRV, RV, or HV resulted in a substantial reduction in mDCs (****, Figure 4 b). It is worth noting that MHC I (***, Figure 4 c) and MHC II molecules (*, Figure 4 d) The decreased expression indicates that HRV treatment also significantly reduced their antigen-presenting capacity; compared to the effects shown by HV and RV in reducing mDCs and MHC expression, HRV demonstrated superior performance in these aspects. Notably, HRV significantly inhibited the secretion of the pro-inflammatory cytokine IL-6 while promoting the secretion of the anti-inflammatory cytokine IL-10. Figure 4 e, f).

[0082] A study on the in vitro induction of Tregs in the spleen by HRV. The spleen of CIA mice was used for this study. The CIA mouse modeling method was as follows: A mixture of type II collagen (CII) and complete Freund's adjuvant (v / v = 1 / 1) was prepared. A single immunization was induced in male DBA / 1 mice by subcutaneous injection of this mixture (2.5 mL / kg) at multiple points in the base of the tail, marked as day 0. A second immunization was induced 21 days later by subcutaneous injection of a mixture of CII and incomplete Freund's adjuvant (v / v = 1 / 1) as described above. The spleen of the CIA mice was aseptically removed, immersed in PBS containing 1% penicillin-streptomycin solution, then homogenized, filtered, and lysed to obtain a single-cell suspension. Cells were counted and measured at 3 × 10⁻⁶ cells / mL. 6 Cells were seeded at a density of 100 μg / mL in 6-well plates. Cells were treated with HRV, RV, HV (HSP, 30 μg / mL; Rapa, 15 μg / mL) or PBS for 12 h, followed by incubation with LPS (100 ng / mL) for 3 days (n = 3). Cells were stained with FITC-CD3, PE-CD4, PE-Cy7-CD25, and APC-Foxp3 antibodies, and analyzed by flow cytometry. Spleen cells were isolated from a CIA mouse model, stimulated with LPS, and then co-cultured with RV, HV, or HRV for 3 days (…). Figure 5 a). Flow cytometry analysis showed that, compared with the LPS group, HRV reduced total CD4+. + The proportion of T cells decreased from 14.9% to 9.5%, while the total CD3 content decreased. + CD4 + Treg cells (CD25) + FoxP3 + The proportion of ) increased by 2.3 times (****, Figure 5 (b, c). This indicates that HRV establishes immune tolerance by inducing tolDC and Treg, which provides a potential therapeutic pathway for modulating autoimmune responses. Example 3

[0083] 10 mg / mL Zymosan A dissolved in sterile physiological saline was resuspended, boiled, and then emulsified by sonication for 10 minutes. Subsequently, 50 μL of the freshly emulsified yeast polysaccharide solution was injected into the knee joint cavity of C57BL / 6 mice to establish a ZIA mouse model. At predetermined time points, the thickness (a, anteroposterior diameter) and width (b, lateral diameter) of the mouse knee joint were measured using calipers, and the knee joint circumference (L) was calculated using the following formula: L = πb + 2(ab).

[0084] See Figure 6a. Twenty-four hours after ZIA modeling in mice (n = 4, designated as day 0), significant redness and swelling of the knee joints indicated successful establishment of the ZIA model. Subcutaneous injections of HV (1.5 mg HSP / kg), HV (3 mg HSP / kg), HV (4.5 mg HSP / kg), RV (0.75 mg Rapa / kg), HRV (1.5 mg HSP / kg, 0.75 mg Rapa / kg), HRV (3 mg HSP / kg, 0.75 mg Rapa / kg), and HRV (4.5 mg HSP / kg, 0.75 mg Rapa / kg) were administered near the ILNs and ALNs of ZIA mice. The drugs were administered every two days for a total of two administrations. Mice in the Health group and PBS group served as controls. The extent of leg swelling was observed (…). Figure 6 (b) It can be seen that, except for a transient increase in leg circumference in the RV group after administration on day 0, all doses of HRV and the gel treatment group showed excellent effects in relieving joint swelling. Among them, the HRV (4.5 mg HSP / kg, 0.75 mg Rapa / kg) group had the best treatment effect, showing a significant difference from the PBS group (****). In the middle and late stages of treatment, the HRV (3.0 mg HSP / kg, 0.75 mg Rapa / kg) group showed poor efficacy, and the rate of swelling reduction in mice slowed down. This indicates that the HSP dose is crucial for treatment; one day after modeling, the body weight of mice other than the healthy group was significantly reduced (5%-10%) due to acute inflammation (). Figure 6 c) As time went on, the mice's weight returned to the normal range.

[0085] DCs are common antigen-presenting cells and play an important role in the body's immune response. For example... Figure 7 As shown in Figure a, compared to the PBS group, the expression level of CD86 in DCs cells in the spleen was significantly reduced in HRV (4.5 mg HSP / kg, 0.75 mg Rapa / kg) (*); the expression level of CD80 also showed a similar trend. In the joint microenvironment, HRV drugs could inhibit the expression of CD80 in DCs cells. This indicates that HRV plays a crucial role in improving the immune microenvironment at the joints of rheumatoid arthritis. Tregs mainly exert their immunosuppressive function by metabolic destruction of target cells, inhibition of cytokine secretion, and direct cell-cell contact, playing an important role in inflammation suppression and the establishment of immune tolerance. Lymph nodes are the main site of peripheral tolerance, where DCs can induce T cells to migrate from peripheral tissues to the T lymph node region of secondary lymph nodes, inducing Tregs, a type of immunomodulatory cell. Compared to the PBS group, this invention effectively increases CD3 expression. + CD4 + The proportion of Treg cells in T cells ( Figure 7(b) The spleen is the largest lymphoid organ, with functions including blood storage, hematopoiesis, removal of senescent red blood cells, and immune response. Considering that the pathogenesis of RA is associated with CD4... + T cell subset imbalance; analysis of splenic CD4 + T cell differentiation. The Th17 / Treg cell pair plays an important role in the immune response. Compared with the PBS group, the proportion of Th17 cells was decreased in the RV, HV, and HRV groups. Figure 7 c) The proportion of Treg in the spleen of mice in the RV, HV, and HRV groups was higher than that in the PBS group. Example 4

[0086] Sodium hyaluronate (400 mg, 1 MDa) was dissolved in 17.3 mL of deionized water, followed by the addition of DMTMM (273 mg, 1 mM), and then DBCO- (…). CH 2 2-NH2 (0.2 mM, DMSO solvent) was reacted at 37 °C for 24 hours. A mixed solvent of deionized water / DMSO (4:1 volume ratio) was used as the dialysate, and dialyzing was performed for 24 hours (the molecular weight cutoff of the dialysis bag was 3500 Da). Dialysis was then performed with deionized water for one day, followed by lyophilization to obtain 20% HA-DBCO. 0.1 mM and 0.3 mM DBCO- ( ) were added to 10% HA-DBCO and 30% HA-DBCO, respectively. CH 2 The reaction is 2-NH2, and the other steps are the same as described above.

[0087] To synthesize 10% HA-N3, 20% HA-N3, and 30% HA-N3, (0.2 mM, 0.1 mM), (0.3 mM, 0.2 mM), and (0.4 mM, 0.3 mM) of (DMTMM, N3-) were added to 1 mM HA, respectively. CH 2 (3-NH2). Other reaction conditions and purification steps are the same as described above.

[0088] 10 mg HA-DBCO and 10 mg HA-N3 were added to 1 mL PB (pH 7.4) buffer solution to obtain two solutions. These solutions were then mixed with 1 mL HRV solution (obtained by dialysis in Example 1) and loaded into a syringe for injection and measurement. The volume ratio of the three solutions was 1:1:1.

[0089] For hydrogels without HRV, add an equal volume of PB buffer.

[0090] This invention designs an injectable hydrogel system based on two-component modified hyaluronic acid. The system consists of dibenzocyclooctylene-functionalized hyaluronic acid (HA-DBCO) and azide-functionalized hyaluronic acid (HA-N3), both synthesized via a one-pot two-step method. Figure 8 a). FTIR results further confirmed the structures of the two polymers: HA-DBCO at 750 cm⁻¹ -1 The peak at 2150 cm⁻¹ shows the characteristic CH bending vibration of ortho-substituted benzene, while HA-N3 shows a peak at 2150 cm⁻¹. -1 The characteristic peak of N3 tensile vibration is observed at this location. Figure 8 b).

[0091] By adjusting DMTMM and DBCO-( CH 2 The amount of 2-NH2 added can be used to prepare HA-DBCO with different degrees of DBCO substitution. The degree of DBCO substitution refers to the proportion of carboxyl groups in hyaluronic acid replaced by DBCO. This can be determined using 1H NMR spectroscopy (NMR spectroscopy). 1 The chemical structure and degree of substitution (DS) of HA-DBCO were characterized by H-NMR. Figure 9 From the area integral ratios of the characteristic peaks of the benzene ring (δ = 7.5 ppm) and N-acetylglucosamine hyaluronic acid (δ = 2.0 ppm) of DBCO, the actual substitution degrees of 10%, 20%, and 30% HA-DBCO are 9.4%, 15.2%, and 22.1%, respectively. Taking 10% HA-DBCO as an example, the substitution degree is calculated as follows: HA acetyl group has 3 H atoms, and the peak area is defined as 3. DBCO benzene ring has 8 H atoms, so theoretically, the peak area of ​​10% HA-DBCO at chemical shift δ 7.5 ppm is 0.8. The actual peak area is 0.75, therefore the grafting rate of 10% HA-DBCO is 93.8%, and the actual substitution degree is 9.4%. Similarly, the actual substitution degrees of 20% and 30% HA-DBCO are 15.2% and 22.1%, respectively.

[0092] By adjusting DMTMM and N3-( CH 2 The amount of 3-NH2 added can be used to prepare HA-N3 with different degrees of N3 substitution. HA-N3... 1 H-NMR spectral analysis showed that ( Figure 10From the area integral ratio of the characteristic peak of the hydrogen atom on carbon 2 of N3 (δ = 1.8 ppm) and the characteristic peak of the HA acetyl group (δ = 2.0 ppm), it can be seen that the actual substitution degrees of HA-N3 with different substitution degrees of 10%, 20%, and 30% are 10.0%, 17.0%, and 19.0%, respectively. Taking 10% HA-N3 as an example, the substitution degree is calculated as follows: the HA acetyl group has 3 H atoms, and the peak area is defined as 3. N3-( CH 2 Since there are two H atoms at the e-position of 3-NH2, the theoretical peak area of ​​10% HA-N3 at a chemical shift δ 1.8 ppm is 0.2. In reality, the actual peak area of ​​10% HA-N3 at a chemical shift δ 1.8 ppm is also 0.2. Therefore, the theoretical grafting rate of 10% HA-N3 is 100.0%, and the actual degree of substitution is 10.0%. Similarly, the actual degrees of substitution for 20% and 30% HA-N3 can be obtained.

[0093] For ease of representation, each degree of substitution HA-DBCO in this invention is named according to its theoretical degree of substitution.

[0094] This invention achieves rapid hydrogel formation by mixing HA-DBCO and HA-N3 in PBS, based on click chemistry. The reaction formula is as follows: Figure 11 a. Macroeconomic changes such as Figure 11 b.

[0095] Using equal volumes of PB buffer, 15 mg / mL 20% HA-DBCO and HA-N3 as the basic formulation, HA-DBCO / N3 with substitution degrees of 10%, 20%, and 30%, concentrations of 5, 10, 15, and 20 mg / mL, and gelation times at temperatures of 25 °C and 37 °C are as follows. Figure 12 As shown, (a) degree of DBCO- / N3- substitution, (b) concentration of HA-DBCO / N3, (c) gelation time at temperature, (d) concentration of HA-DBCO / N3, (f) storage modulus of degree of DBCO- / N3- substitution, (e) concentration of HA-DBCO / N3, (g) loss modulus of degree of DBCO- / N3- substitution, and (h) hydrogel shear dilution. The hydrogel accelerates gelation after injection into the body, which is beneficial for the drug to be encapsulated in the hydrogel. The hydrogel exhibits significant shear thinning properties, which enable it to be injected smoothly using a standard syringe.

[0096] SEM characterization revealed that the hydrogel possesses a loose, porous three-dimensional network structure, which is beneficial for the effective encapsulation and sustained release of the HRV nanovaccine. After hydrogel formation, it was lyophilized, and SEM images were obtained of the hydrogel's microstructure. The pore sizes of 5, 10, and 15 mg / mL HA-DBCO / N3 were approximately 80, 20, and 10 μm, respectively. Figure 13 a); After PB or HRV is encapsulated in the hydrogel, its internal pore size is about 20 μm ( Figure 13 b).

[0097] In this invention, 10 mg / mL was selected as the polymer concentration for subsequent studies. Example 5

[0098] HA-DBCO / N3 with a DBCO / N3 functionalization of 20% and a concentration of 10 mg / mL was selected as the hydrogel raw material. One mL of hydrogel was premixed overnight in a 7 mL centrifuge tube. The volume ratio of HA-DBCO:HA-N3:BSA-cy5 / CC-cy5 in the hydrogel was 1:1:1. The concentration of BSA-cy5 was 2 mg / mL. The next day, to simulate the release of substances from the hydrogel in vitro, 0.5 mL of PB buffer or PB buffer containing hyaluronidase (HAase, 50 U / mL) (n = 3) was added to a pipette. In the PB (pH = 7.4) release medium, the higher the concentration of HAase, the faster the release rate of BSA-cy5. The maximum cumulative release difference (15%) was reached in the different HAase concentration groups at approximately 8 days. Figure 14 a). By day 21, BSA-cy5 in the hydrogel was almost completely released. Different concentrations of hydrogel had little effect on the release rate of BSA-cy5. Figure 14 b), reaching the maximum cumulative release differential (7%) on day 5. The molecular weight of the protein drug slightly affects the release rate. Figure 3 .7c). The release rate of 16 kDa cytochrome CC is slightly faster than that of 66 kDa BSA.

[0099] The release behavior of vesicles and Rapa within vesicles in HRV-cy5@gel hydrogels was investigated in the presence and absence of HAase. Cumulative release curves showed that within 18 days, vesicles in the HAase-free group hardly escaped from the hydrogel, while only 13% of Rapa was released. Figure 14 d). In stark contrast, in the presence of HAase, vesicles follow a zero-order kinetic release pattern, with release corresponding to the degradation process of the hydrogel. The release rate of Rapa is significantly accelerated. The sustained-release nanovaccine properties are beneficial for continuously stimulating antigen-presenting cells and inducing tolerance.

[0100] The effect of the byproduct 4,6-dimethoxy-1,3,5-triazin-2(1H)-one (DMTO) during synthesis was investigated using L929 cells. L929 cells were seeded in 96-well plates (5,000 cells / well, n = 6) and cultured for 24 hours to allow adherence. 10 μL of DMTO solution (concentration range 1 μg / mL to 4 mg / mL) was added for 24 hours. Cell viability was assessed using a CCK-8 assay kit.

[0101] The biocompatibility of the hydrogel extract was investigated using 293T cells. 90 μL or 180 μL of hydrogel was added to 1 mL of complete culture medium. After 3 or 6 hours, this medium was added to 293T cells in 96-well plates (5000 cells / well, n = 4). Cell viability was assessed using a CCK-8 assay kit after 24 hours of incubation.

[0102] The biocompatibility of the hydrogel was investigated using Transwell assay. 50 μL of hydrogel was added to the upper chamber, and RAW264.7 (n = 3) or 293T cells (n = 4) were seeded in the lower chamber (5000 cells / well). After co-culturing for 48 hours, cell viability was assessed using a CCK-8 assay kit.

[0103] The proliferation of 293T cells in hydrogels was investigated. An equal volume of 293T cell suspension (5 × 10⁶ cells / well) was prepared in a 12-well plate. 6 The cells were mixed with HA-N3 (10 mg / mL) and then with an equal volume of HA-DBCO (10 mg / mL) to obtain a cell-loaded hydrogel (denoted as 293T@gel). After gelation, culture medium was added and the cells were incubated for 24, 48, and 72 hours. After staining with Calcein / PI reagent, the cells were imaged using an inverted fluorescence microscope.

[0104] HA exhibits good biocompatibility, as demonstrated by various testing methods. CCK-8 assay results showed that even when the concentration of the unpurified byproduct DMTO in the hydrogel reached eight times the total concentration of DMTO in the hydrogel-forming polymer, it did not show cytotoxicity to L929 cells after 24 hours of culture. Figure 15 a). More importantly, after three days of enzymatic degradation, neither the exudate nor the degradation products of the hydrogel caused cytotoxicity. Figure 15 b). Co-culturing the hydrogel with 293T and RAW264.7 cells not only did not produce toxicity, but also exhibited a cell proliferation-promoting effect. Figure 15 c, d). Live / dead cell staining experiments confirmed that 293T cells cultured in hydrogels maintained good cell viability and proliferation capacity. Figure 15e). These experimental results fully demonstrate the excellent biocompatibility and safety of the hydrogel, indicating its broad application prospects as an HRV reservoir in the preparation of tolerable vaccines.

[0105] Example 6: Treatment Experiment and Efficacy Evaluation of HRV in CIA Model Mice When the RA score was 2-3, CIA mice were divided into four groups and administered two different dosing regimens (n ​​= 7), with this day designated as day 0. In regimen 1, on days 0, 3, 6, 9, 12, and 15, 150 μL of HV, HRV (4.5 mg HSP / kg, 750 μg Rapa / kg), or PBS was subcutaneously injected at four sites near the ILNs and ALNs. In regimen 2, on days 0 and 9, 450 μL of HRV@gel (13.5 mg HSP / kg, 2.25 mg Rapa / kg) was subcutaneously injected at four sites near the ILNs and ALNs. Mice were monitored for symptoms every other day and scored according to the Chondrex protocol.

[0106] Mice were sacrificed on day 21 after treatment began. Blood samples were collected to determine the concentrations of IL-6 and HSP peptide-specific antibodies in plasma. ILNs, PLNs, and spleen were ground to obtain cell suspensions, and leg bones were harvested. Three intact hind leg bone sections from each group were used for H&E and SO-FG staining. Knee and ankle joints were photographed to analyze inflammatory infiltration and cartilage destruction. Other leg bones were preserved in PBS, homogenized, filtered, and centrifuged. The supernatant was used to determine the concentrations of IL-6, TNF-α, and IL-10, and the bottom cells were stained by flow cytometry. ILN and PLN cell suspensions were stained, and flow cytometry was used to detect the maturity of dendritic cells (DCs), the proportion of Tregs, and the proportion of B cells in lymph nodes. Spleen cell suspensions were stained, and flow cytometry was used to detect the maturity of DCs and CD4+ in lymph nodes. + T and CD8 + The proportions of T cells, Treg cells, B cells, and memory B cells were analyzed using FlowJo. Finally, one mouse from each group was dissected, and its heart, liver, spleen, lungs, and kidneys were fixed in 4% paraformaldehyde for H&E staining analysis to assess the toxic effects of the drug on the mouse organs.

[0107] Imaging results showed that the HRV-cy5 group rapidly diffused and accumulated in the ILNs region 12 hours after injection, and was then rapidly cleared on day 4 (***, Figure 16 a) The hydrogel effectively delayed the diffusion of HRV-cy5. The HRV-cy5@gel group maintained stable retention at the injection site for nearly four days. Although the average fluorescence intensity was slightly lower than that of the HRV-cy5 group at 6 and 12 hours, it was significantly higher than that of the HRV-cy5 group on day 4 (*). Figure 16(b) This result confirms that hydrogels are beneficial in prolonging the uptake of nanovaccines by antigen-presenting cells within ILNs. Lymph nodes, as key immune organs, are the primary site of interaction between DCs and tolerable nanovaccines, and this interaction is fundamental to the reconstruction of immune tolerance. To investigate the accumulation behavior of nanovaccines in ILNs, in vitro imaging of mouse ILNs was performed. The results showed that the HRV-cy5 group had a high accumulation of nanovaccines in ILNs 12 hours after injection, but decreased by about two orders of magnitude by day 4; conversely, the HRV-cy5@gel group showed a moderate but gradually increasing trend in nanovaccine deposition in ILNs from 12 hours to 4 days. Figure 16 c, d). Notably, the uptake of HRV-cy5 by DCs and B cells showed significant differences over time: the uptake level in the HRV-cy5 group gradually decreased from 12 hours to day 4 (**), while the uptake in the HRV-cy5@gel group increased by 2.7 to 6.7 times ( Figure 16 e, f). The gradual increase in the uptake capacity of antigen-presenting cells within lymph nodes further confirms that hydrogels are beneficial for maintaining contact between the nanovaccine and antigen-presenting cells. Furthermore, biodistribution analysis of the nanovaccine in major organs showed that the cumulative amount in the HRV-cy5@gel group remained at a low level at both time points. Figure 16 g). These demonstrate the potential of HRV-cy5@gel to reconstruct immune tolerance through sustained targeted delivery.

[0108] To evaluate the therapeutic effects of HRV and HRV@gel in inflammatory autoimmune diseases, an experiment was conducted using a CIA mouse model. Figure 17 a). When the arthritis score (AS) reached 2-3 (recorded as day 0), mice received two treatment regimens: subcutaneous injections at four sites near ILNs and ALNs. The HRV or HV group received the injections 6 times, while the HRV@gel group received the injections 2 times, but the total dose was the same for both groups (HSP peptide: 27 mg / kg, Rapa: 4.5 mg / kg). Figure 17 a). PBS group and healthy mice were used as controls. The results showed that, compared with the PBS group, HV treatment significantly reduced claw thickness (**, Figure 17 (b) However, the results did not reach the levels of healthy mice. Despite some improvement, claw thickness remained high, and the AS score rose to 7, indicating limited effectiveness in reducing toe joint swelling. In contrast, HRV treatment significantly suppressed the inflammatory response, with a marked reduction in AS score and claw thickness, effectively alleviating toe joint swelling. This effect is consistent with the known mechanism of action of Rapa, a potent antigen-induced T-cell proliferation inhibitor often used to prevent organ transplant rejection.

[54] Notably, HRV@gel treatment significantly reduced AS scores ( Figure 17 c) reached a level comparable to the healthy group, without causing significant changes in body weight. Figure 17 (d) highlights the efficacy and good safety of this delivery system. By day 21, HRV@gel treatment continued to suppress hind paw thickness and AS score (2.0), while the PBS group showed erythema and damage, significantly increased paw thickness, and an AS score of 10.0, as shown in the photograph. Figure 17 e). These findings strongly demonstrate the therapeutic efficacy of HRV@gel for CIA, highlighting the crucial role of sustained release of tolerable nanovaccines in achieving potent RA treatment.

[0109] Immunological analyses were performed on CIA mice on day 21. Compared to the PBS group, IL-6 levels were significantly reduced in the HRV and HRV@gel groups (****), while the HRV@gel group also significantly inhibited TNF-α secretion (**). These cytokine levels were regulated to levels comparable to or lower than those in healthy mice. Figure 18 a, b). Notably, this decrease was accompanied by a 3.6 to 4.4-fold increase in IL-10 production, highlighting the important role of IL-10 in promoting tissue homeostasis during the inflammatory response. Figure 18 c). Against this backdrop, analysis of IgG levels against HSP70 B29 showed significantly increased expression in the PBS group mice, while the concentrations in the HRV and HRV@gel groups were decreased in the RA knee joint (****, Figure 18 d).

[0110] Dendritic cells (DCs), as primary antigen-presenting cells, play a crucial role in antigen recognition, presentation, and subsequent activation of adaptive immune responses. In the inflammatory joint microenvironment, the activation level and antigen-presenting capacity of DCs reflect the in vivo inflammatory state and act as a bridge between local and peripheral tolerance. Flow cytometry analysis on day 21 showed that, compared with healthy mice, CD86 levels were significantly higher in the CIA model (PBS group). + The proportion of mDCs increased significantly (62.0%), confirming their important role in RA progression. Notably, HV, HRV, and HRV@gel treatments all significantly reduced the proportion of mDCs (****, Figure 18 e). Furthermore, the MFI of MHC II is reduced (*, Figure 18 f) This indicates that HRV@gel also significantly reduced the antigen-presenting capacity of DCs. These results suggest that HRV@gel effectively promoted the reconstruction of local immune tolerance in the diseased joints of CIA mice by alleviating two typical features of autoimmune diseases: cytokine imbalance and DC activation.

[0111] In a clinical context, subcutaneous administration often leads to lymphatic diffusion, which significantly impacts therapeutic efficacy. As a primary site of peripheral tolerance, lymph nodes mainly present antigens to T cells via antigen-presenting cells in the lymph. Both local and peripheral tolerance are crucial for effectively restoring immune homeostasis.

[16] Flow cytometry analysis of lymph node cell suspensions showed that the proportion of mDCs was significantly reduced in the HRV group and the HRV@gel group, and the expression of MHC II molecules was decreased. Figure 19 (a, b) The sustained antigen release from this hydrogel may facilitate continuous stimulation of lymph nodes, thereby promoting peripheral immune tolerance in RA. During the establishment of immune tolerance, dendritic cells (DCs) present antigens to T cells, prompting their migration from peripheral tissues to the T-cell regions of secondary lymphoid organs. This process promotes T-cell differentiation into Tregs, which is crucial for the establishment and maintenance of immune tolerance.

[101] Compared with the PBS group, the proportion of Tregs in lymph nodes was significantly increased in the HRV group (****), while it was further increased by 1.3 times in the HRV@gel group (**), indicating that continuous exposure to the tolerance antigen plays an important role in promoting the production of Tregs. Figure 19 c). Meanwhile, the superficial lymph node cortex contains a certain proportion of B cells in its lymph node nodule area. B cells are also antigen-presenting cells and can produce an immune response by secreting antibodies. For example... Figure 19 As shown in d, compared to the PBS group, the proportion of B cells in the HRV@gel group decreased by 50% (**). p Compared to the HRV group, the proportion of B cells in the HRV@gel group was significantly reduced (**). p This indicates that sustained-release nanomedicines can effectively reduce the proportion of B cells and inhibit the production of autoantibodies at the source.

[0112] As the largest lymphoid organ and a key site of peripheral immunity, the spleen plays a crucial role in antibody-mediated immune responses. HRV@gel's inhibitory effect on the proportion of mDCs and MHC II molecule expression in the spleen is comparable to its effect on LNs (…). Figure 20 (a, b). Furthermore, compared to the HRV group, HRV@gel treatment induced only 28.8% of total CD4. + T cells, but increased Tregs by 1.7 times ( Figure 20 c, d). Meanwhile, the HRV@gel group enables CD8... + The proportion of T cells decreased by 82.7% ( Figure 20 e). CD8 + T cells express granzyme B and selectively eliminate Foxp3. +Treg, thus weakening the inverse regulatory effect of Treg. Treg / CD8 ratio in the PBS group + The T / CD8 ratio was 0.32, indicating that the spleen of untreated CIA mice had very weak immunomodulatory capacity. After HRV@gel treatment, the Treg / CD8 ratio increased. + The ratio of T increased by 45 times (****, Figure 20 f). These results indicate that sustained antigen release plays a crucial role in promoting T cell tolerance. In addition to cellular immunity, B cell-mediated humoral immunity is also associated with the pathogenesis of rheumatoid arthritis (RA).

[103] Our analysis showed that, compared with the HRV group, HRV@gel treatment reduced the number of total B cells and memory B cells (CD27) in the spleen. + B220 + The proportion decreased by about half. Figure 20 (g, h), which is beneficial for the long-term maintenance of immune tolerance. Furthermore, antigen-specific IgG antibodies can activate B cells and promote the production of plasma cells and new IgG, potentially triggering a strong pro-inflammatory response, thus leading to the persistence of RA. In summary, these results demonstrate that HRV@gel exerts a systemic anti-inflammatory effect in CIA mice through sustained HRV release. This restorative approach helps modulate DCs, Tregs, and B cells, thereby effectively restoring immune homeostasis in RA mice.

[0113] Protecting bone and cartilage from damage during treatment is crucial for ensuring long-term joint function and health. On day 21, histopathological analysis of RA knee and ankle tissue sections was performed using H&E and SO-FG staining. H&E staining revealed extensive immune cell infiltration and angiogenesis in the synovial tissue of the PBS and HV groups. Neovascularization is a key factor in the formation and maintenance of pannus in RA, recruiting more immune cells to continuously attack joint tissue, further inducing joint lesions and cartilage destruction. Compared to the HRV group, the HRV@gel group showed significantly reduced immune cell infiltration and angiogenesis. Safranin O-Fix Green FCF (SO-FG) staining further revealed that the cartilage morphology in the HRV@gel group remained intact, very similar to that of healthy mice, indicating its protective effect on cartilage integrity. In contrast, the PBS, HV, and HRV groups showed significant cartilage damage and bone erosion. Particularly in the ankle joint, the PBS and HV groups exhibited significant loss of periarticular space, adhesion of bone tissue to adjacent connective tissue, and extensive inflammatory cell infiltration. HRV treatment effectively reduced inflammation and prevented bone damage, with HRV@gel exhibiting even stronger protective capabilities, maintaining a structure similar to healthy tissue. The enhanced osteoprotective effect was associated with increased IL-10 production in the joints of mice treated with HRV@gel. IL-10 is a key cytokine that maintains tissue homeostasis during infection and inflammation by limiting excessive inflammatory responses, upregulating innate immunity, and promoting tissue repair mechanisms. This treatment strategy may improve clinical efficacy by preventing joint destruction. Furthermore, H&E staining revealed no significant toxicity to major organs in treated mice.

[0114] This invention prepared a Rapa-encapsulated tolerance-derived peptide (HSP peptide) polymer nanovesicle vaccine (HRV). The HRV particles had a diameter of approximately 60 nm and a zeta potential of approximately -6 eV. It successfully reduced BMDC cell maturity and antigen-presenting capacity, decreased the expression of the pro-inflammatory cytokine IL-6, increased the expression of the inhibitory cytokine IL-10, and increased the proportion of Treg cells in spleen cells. The HSP peptide on the vesicle surface was cleaved via the endosome / lysosome pathway, and the MHC II on the cell surface presented the HSP peptide to CD4 cells. + T cells are activated, thereby promoting the establishment of immune tolerance. In animal ZIA models, HRV effectively alleviated joint swelling, induced Tregs in lymph nodes, induced tolDCs in joints, and promoted CD4+ in the spleen. + The balance of the Th17 / Treg immune cell pair in T cells has shifted to the right.

[0115] This invention prepared a hydrogel local delivery system (HA-DBCO / N3, 10 mg / mL 20% DS, HA-DBCO, HA-N3 and drug mixed in equal volumes). The biocompatibility of the hydrogel was demonstrated through various methods. The release rate of BSA-cy5 from the hydrogel increased with increasing HAase concentration in the release medium, with the maximum cumulative release difference (15%) reaching approximately 8 days between groups. Regarding the nanoparticles in the gel, within 18 days, vesicles in the HAase-free group hardly escaped from the hydrogel, while Rapa only released a small amount (13%). In in vivo studies, HRV@gel further improved the therapeutic effect on CIA mice, significantly reducing joint swelling, joint pro-inflammatory factor concentrations, HSP-specific antibody concentrations, and DC maturity. It also reduced DC maturity and B cell proportions in lymph nodes and spleen, increased Treg cell proportions, and significantly improved cartilage and bone damage in the knee and ankle joints of mice.

Claims

1. A tolerance peptide immunomodulator nanovaccine, comprising an immunomodulator encapsulated polymer vesicle and a tolerance peptide.

2. The tolerance peptide immunomodulator nanovaccine according to claim 1, characterized in that, The tolerance peptides and immunomodulators are encapsulated in polymer vesicles by chemical bonds; the tolerance peptides include one or more peptides derived from type II collagen, filaggrin, fibrinogen, vimentin, rheumatoid factor, citrullinated protein, and heat shock protein; the immunomodulators include one or more of dexamethasone, cyclosporine, tacrolimus, rapamycin, and their derivatives.

3. The tolerance peptide immunomodulator nanovaccine according to claim 2, characterized in that, Immunomodulators encapsulate polymer vesicles with surface-modified tolerance peptides.

4. The tolerance peptide immunomodulator nanovaccine according to claim 1, characterized in that, The polymers assembled into polymer vesicles include amphiphilic block polymers; the amphiphilic block polymers include hydrophilic segments and hydrophobic segments.

5. The tolerance peptide immunomodulator nanovaccine according to claim 4, characterized in that, The hydrophobic segment includes repeating units of dithiopentane trimethylene carbonate monomer; the hydrophilic segment includes polyethylene glycol segments.

6. The method for preparing the tolerance peptide immunomodulator nanovaccine according to claim 1 includes the following steps: mixing an amphiphilic block polymer containing the tolerance peptide and an immunomodulator, assembling them, and preparing the tolerance peptide immunomodulator nanovaccine.

7. An injectable tolerance peptide nanovesicle hydrogel system, comprising the tolerance peptide immunomodulator nanovaccine of claim 1 and the hydrogel.

8. The method for preparing the injectable tolerable peptide nanovesicle hydrogel system according to claim 7, comprising the following steps: mixing the hydrogel precursor and the tolerable peptide immunomodulator nanovaccine to form the injectable tolerable peptide nanovesicle hydrogel system.

9. The use of the tolerable peptide immunomodulatory nanovaccine of claim 1 or the injectable tolerable peptide nanovesicle hydrogel system of claim 7 in the preparation of anti-inflammatory drugs, or in the preparation of drugs for the treatment of in vivo and / or in vitro inflammation, or in the preparation of drugs for the prevention and treatment of arthritis, or in the preparation of drugs for the relief of joint swelling.

10. An anti-inflammatory drug, characterized in that, The active ingredient of the anti-inflammatory drug includes the tolerance peptide immunomodulator nanovaccine of claim 1 or the injectable tolerance peptide nanovesicle hydrogel system of claim 7.

Images