Bionic nanodrug delivery system, preparation method and application in eye immunotherapy

Abstract

The application belongs to the technical field of biological medicine and nanomaterials, and particularly relates to a kind of bionic nano drug delivery system, preparation method and application in eye immunotherapy.The application is with mesoporous SiO2 Nanomicrosphere as core, after protonation treatment, load sCD83, and coat mature DC cell membrane, form DCM@MSN / sCD83 nano drug delivery system.The preparation method of the application has high biocompatibility and simple operation, and the system can actively target mature DC after vitreous injection, release sCD83 and induce mature DC to transform into resistant DC, secrete IL-10 and TGF-β and other anti-inflammatory factors, inhibit STING path activation, reduce retinal inflammatory damage;At the same time, it can penetrate blood-ocular barrier into peripheral circulation, induce mature DC differentiation in neck lymph nodes and spleen and other immune organs, realize local and systemic synergistic immunoregulation, and can be used for preparing medicaments for treating experimental autoimmune uveitis.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and nanomaterials technology, specifically relating to a biomimetic nanodrug delivery system, its preparation method, and its application in ocular immunotherapy. Background Technology

[0002] Autoimmune uveitis is a refractory autoimmune disease that can cause irreversible vision loss. Traditional treatments mainly rely on steroids such as dexamethasone (DEX), but these have significant side effects and limited efficacy. In recent years, soluble CD83 (sCD83) has shown therapeutic potential as a novel immunomodulatory molecule. It can exert anti-inflammatory effects by inducing the production of tolerant dendritic cells (tDCs), inhibiting pathogenic Th1 / Th17 cells, and promoting the differentiation of regulatory T cells (Tregs). However, sCD83 is easily degraded in vivo, lacks targeted delivery methods, and the presence of the blood-eye barrier makes it difficult to effectively reach ocular lesions, severely limiting its clinical application.

[0003] Mesoporous silica nanospheres (MSNs) are considered excellent drug carriers due to their large specific surface area, tunable pore size, good biocompatibility, and ease of functionalization. However, unmodified nanoparticles are easily recognized and cleared by the immune system, making it difficult to cross the blood-eye barrier and achieve precise delivery. Existing biomimetic modification technologies, such as erythrocyte membrane (RBCM) coating, can achieve immune evasion and long circulation, but this modification mainly relies on the EPR effect to passively accumulate at tumor or inflammatory sites, lacking the ability to actively recognize specific cell types, resulting in limited targeting efficiency and no immunomodulatory function.

[0004] Therefore, developing a nanomedicine delivery system that can protect sCD83 from degradation, actively target DC cells in ocular lesions, and possess immune escape and blood-eye barrier penetration capabilities is of great significance for the efficient and low-toxicity treatment of autoimmune uveitis. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to provide a biomimetic nanodrug delivery system, its preparation method, and its application in ocular immunotherapy. This invention uses mesoporous silica (MSN) as its core, which, after protonation, is loaded with soluble CD83 and coated with mature dendritic cell (DC) membranes to form a DCM@MSN / sCD83 nanocarrier. Specifically, it utilizes the homologous targeting of the mature dendritic cell membrane (DCM) to precisely recognize and act on DCs at inflamed sites, inducing their transformation into tolerant DCs and secreting anti-inflammatory factors such as IL-10 and TGF-β; simultaneously promoting CD4+... + T cells differentiate into regulatory T cells, avoiding the formation of pro-inflammatory T cells and inhibiting the secretion of pro-inflammatory factors such as Th1 / Th17, thereby achieving precise cell-level delivery and local-systemic synergistic immune regulation.

[0006] Specifically, the present invention provides the following technical solution: A first aspect of the present invention provides a biomimetic nanomedicine delivery system, comprising: a mesoporous silica nanosphere core; soluble CD83 loaded within the mesopores of the mesoporous silica nanosphere; and a mature dendritic cell membrane coating the surface of the mesoporous silica nanosphere.

[0007] Preferably, the mesoporous silica nanospheres have a uniform spherical structure, an average particle size of 200 nm, and a BET specific surface area of ​​68 m². 2 / g, with an average pore size of 5~10 nm.

[0008] A second aspect of the present invention provides a method for preparing the biomimetic nanomedicine delivery system described in the first aspect, comprising the following steps: Monodisperse silica nanospheres were prepared by microemulsion method using tetraethyl orthosilicate as the silicon source; mesoporous silica nanospheres were obtained by etching the surface of the nanospheres with alkaline solution to construct a mesoporous structure. The obtained mesoporous silica nanospheres were immersed in glacial acetic acid solution for protonation treatment, so that their surface was positively charged, thus obtaining protonated mesoporous silica nanospheres. The obtained protonated mesoporous silica nanospheres were immersed in a soluble CD83 saline solution, and sCD83 was loaded into the mesopores to obtain drug-loaded mesoporous silica nanospheres. The suspension containing the cell membrane of mature dendritic cells was mixed with the obtained drug-loaded mesoporous silica nanospheres, and after ultrasonic treatment, the uncoated particles were removed by centrifugation to obtain the DCM@MSN / sCD83 nanocarrier.

[0009] Preferably, the microemulsion method described herein specifically involves stirring and reacting the mixture in an ethanol-ammonia system at 60°C for 4 hours to obtain monodisperse SiO2 nanospheres.

[0010] Preferably, the alkaline etching specifically involves etching with a 0.1 mol / L NaOH aqueous solution for 4 hours to obtain mesoporous silica nanospheres.

[0011] Preferably, the protonation treatment specifically involves impregnation with 0.1 M glacial acetic acid for 2 hours to obtain protonated mesoporous silica nanospheres.

[0012] Preferably, the operation of loading sCD83 specifically involves: immersing protonated mesoporous silica nanospheres in a physiological saline solution containing sCD83, and continuously mixing for 2 hours to ensure that the drug fully fills the pore structure on the surface of the SiO2 microspheres, thereby achieving drug loading. The ratio of the amount of protonated mesoporous silica nanospheres to the physiological saline solution containing sCD83 is 5 mg: 1 mL, and the concentration of the physiological saline solution containing sCD83 is 100 ng / mL.

[0013] Preferably, the mature dendritic cell membrane is obtained by lysing and separating activated mature dendritic cells; the ratio of the suspension containing the mature dendritic cell membrane to the drug-loaded mesoporous silica nanospheres is 1 mL:5 mg.

[0014] The mature dendritic cell membrane is tightly bound to the surface of the protonated mesoporous silica nanospheres through electrostatic interaction. The surface of the mature dendritic cell membrane carries a negative charge, while the surface of the protonated mesoporous silica nanospheres carries a positive charge.

[0015] Preferably, the centrifugation process for removing uncoated particles is repeated 3 to 4 times.

[0016] A third aspect of the present invention provides the application of the biomimetic nanodelivery system described in the first aspect in the preparation of a drug for treating experimental autoimmune uveitis.

[0017] Preferably, the biomimetic nanodelivery system delivers drugs via intravitreal injection, targeting mature dendritic cells in the eye, spleen, and lymph nodes, inducing tolerant mature dendritic cells to produce IL-10 and TGF-β, downregulating STING expression, and reducing retinal inflammatory damage; the nanocarrier penetrates the blood-eye barrier and enters the peripheral circulation, distributing to the eye, cervical lymph nodes, and spleen, achieving synergistic local and systemic immune regulation.

[0018] One or more embodiments of the present invention have at least the following beneficial effects: (1) This invention uses mature DC cell membrane (DCM) to coat MSN nanocarriers. The "self" recognition signal on the surface of DC membrane reduces the immunogenicity of the carrier, enabling it to effectively evade recognition by the immune system and clearance by macrophages, and significantly prolonging the circulation time in vivo. At the same time, DCM endows the carrier with the ability to specifically recognize and adhere to homologous DCs, enabling it to actively target and be taken up by DCs at the site of inflammation, achieving precise delivery at the cell level. This overcomes the shortcomings of traditional drug carriers, such as lack of active targeting ability and passive enrichment due to diffusion effects.

[0019] (2) In this invention, sCD83 is encapsulated within a mesoporous MSN structure. The DCM encapsulation protects sCD83 from rapid degradation in vivo and enables sustained release to target cells. After the carrier is taken up by DCs, sCD83 activates the HIF-1α / STAT3 signaling pathway, inducing mature DCs to transform into tolerant DCs (tDCs), which then secrete large amounts of anti-inflammatory factors such as IL-10 and TGF-β. These anti-inflammatory factors can, on the one hand, inhibit the activation of the STING pathway and the expression of pro-inflammatory factors such as IL-6 in retinal Müller cells, directly alleviating optic nerve inflammation and damage; on the other hand, they can induce CD4+... + T cells differentiate into regulatory T cells and suppress pathogenic T cells such as Th1 / Th17, thus blocking the inflammatory cascade from the source of immune regulation.

[0020] (3) After the nanocarrier of the present invention is injected into the vitreous body, it can effectively penetrate the blood-eye barrier and enter the peripheral circulation. It is distributed to immune organs such as cervical lymph nodes and spleen with the blood circulation. While inhibiting inflammation in the eye, it induces systemic immune tolerance by targeting DCs in lymph nodes and spleen, thereby relieving eye tissue inflammation and systemic immune disorder at the same time.

[0021] (4) This invention induces DCs to secrete IL-10 and TGF-β through sCD83, thereby inhibiting the activation of the STING pathway in retinal Müller cells, blocking the transmission of inflammatory signals from the level of innate immunity, and reducing retinal inflammatory damage.

[0022] (5) Compared with traditional steroid drugs such as dexamethasone (DEX), the present invention significantly reduces the effective dose of sCD83 and prolongs the dosing interval through targeted delivery of DCM, reducing side effects such as metabolic disorders caused by systemic exposure. Experiments show that DCM@MSN / sCD83 is superior to the DEX loading group in inhibiting retinal inflammation, reducing STING expression and inducing Treg differentiation, providing a novel immunotherapy strategy that is highly effective and low in toxicity for autoimmune uveitis. Attached Figure Description

[0023] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0024] Figure 1 The figure shows the characterization results of the protonated MSN in Example 1 of the present invention; where A is a transmission electron microscope (TEM) image, B is the X-ray diffraction (XRD) pattern of the protonated MSN and the unetched SiO2 microspheres, C is the nitrogen adsorption-desorption isotherm, and D is the pore size distribution curve. Figure 2Characterization results of the DCM@MSN / sCD83 nanocarrier in Example 1 of the present invention; where A is a transmission electron microscopy photograph, B is an elemental distribution map, and C is the Western-blot detection result; Figure 3 In vitro cytotoxicity test (MTT) of the DCM@MSN / sCD83 nanocarrier in Experimental Example 1 of the present invention; Figure 4 Flow cytometry detection of the expression of DC maturation markers (CD80, CD86) and the inhibition of the expression of inflammatory cytokines IL-10 and TGF-β by aDC, aDCM@MSN / sCD83, aDCM@MSN / DEX, and aDCM@MSN in Experimental Example 2 of the present invention; ns indicates P>0.05, that is, the difference is not significant and there is no statistical significance; * indicates 0.01<P≤0.05, and the difference between the two groups is statistically significant; **** indicates P<0.0001, that is, the difference is extremely significant; Figure 5 In Experimental Example 3 of the present invention, aDC, aDCM@MSN / sCD83, aDCM@MSN / DEX, and aDCM@MSN were used for CD4 + CD25 - ​​​​​​​​​​​​​​​​​​Flow cytometry detection of different mouse parts in the EAU model group, DCM@MSN / sCD83 treatment group, DCM@MSN / DEX treatment group, and DCM@MSN treatment group in Experimental Example 5 of the present invention; wherein A is the flow cytometry detection of the expression of DC maturation markers (CD80, CD86) and the expression of anti-inflammatory cytokines IL-10 and TGF-β in the eye; B is the flow cytometry detection of the expression of DC maturation markers (CD80, CD86) and the expression of anti-inflammatory cytokines IL-10 and TGF-β in the lymph node; C is the flow cytometry detection of the expression of DC maturation markers (CD80, CD86) and the expression of anti-inflammatory cytokines IL-10 and TGF-β in the spleen; ns indicates P>0.05, that is, the difference is not significant and there is no statistical significance; * indicates 0.01<P≤0.05, and the difference between the two groups has statistical significance; ** indicates 0.001<P≤0.01, that is, the difference is relatively significant; *** indicates 0.0001<P≤0.001, that is, the difference is very significant; Figure 8 Cell uptake and blood-eye barrier penetration effect diagrams of DCM@MSN in Experimental Example 6 of the present invention; wherein, A is the co-localization photo of DCM@MSN in DC cells observed by laser confocal microscopy, B is the comparison diagram of the penetration efficiency of the simulated blood-eye barrier (Transwell model), ** indicates 0.001<P≤0.01, that is, the difference is relatively significant; Figure 9 In vivo circulation performance test result diagrams of DCM@MSN in Experimental Example 6 of the present invention, wherein, A is the curve of the density change of DCM@MSN in the blood detected by IVFC (in vivo flow cytometry) over time; B is the in vivo three-dimensional imaging diagram; C is the ex vivo organ three-dimensional imaging diagram; Figure 10 Fundus color photos and retinal H&E staining results of the EAU model group, Comparative Example 1-2 groups, and Example 1 group in Application Example 1 of the present invention; Figure 11 Flow cytometry detection result diagrams of the EAU model group and the normal control group in Application Example 1 of the present invention; wherein, A is the flow cytometry detection result diagram of the proportion of mature DCs in the eye, lymph node, and spleen; B is CD4 + CD25 - / CD4 + CD25 + Flow cytometry detection result diagram of the proportion of T cells; * indicates 0.01<P≤0.05, and the difference between the two groups has statistical significance; ** indicates 0.001<P≤0.01, that is, the difference is relatively significant; Figure 12Flow cytometry detection results of the control groups 3 - 5 in Application Example 1 of the present invention; wherein, A is the flow cytometry detection result of the mature marker CD80 in the eye, lymph nodes, and spleen; B is the flow cytometry detection result of the mature marker CD86 in the eye, lymph nodes, and spleen; C is the flow cytometry detection result of the inhibitory inflammatory cytokine IL - 10 expression in the eye, lymph nodes, and spleen; D is the flow cytometry detection result of the inhibitory inflammatory cytokine TGF - β expression in the eye, lymph nodes, and spleen; ns indicates P>0.05, that is, the difference is not significant and there is no statistical significance; * indicates 0.01<P≤0.05, and the difference between the two groups is statistically significant; **** indicates P<0.0001, that is, the difference is extremely significant. Figure 13 Retinal STING protein immunohistochemical staining results of the EAU model group, control groups 1 - 2, and Example 1 group in Application Example 1 of the present invention; Figure 14 Detection result graph of the immunomodulatory mechanism of the present invention; wherein, A is the heat map of RNA sequencing results; B is the detection result of the expression of P - HIF - 1α and P - STAT3 proteins by Western - blot; ns indicates P>0.05, that is, the difference is not significant and there is no statistical significance; * indicates 0.01<P≤0.05, and the difference between the two groups is statistically significant; *** indicates 0.0001<P≤0.001, that is, the difference is very significant; **** indicates P<0.0001, that is, the difference is extremely significant. Detailed implementation manners

[0025] It should be noted that the following detailed description is illustrative and aims to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0026] The protection scheme of the present invention will be described below through specific examples. It should be noted that these examples are only for the convenience of those skilled in the art to understand and should not be regarded as a limitation on the protection scope of the present invention. Unless otherwise stated, the reagents used in the examples can be obtained through commercial purchase.

[0027] Example 1: This example provides a biomimetic nano - drug delivery system and its preparation method Specifically, it includes the following steps: (1) Preparation of MSN: Take 25 mL of anhydrous ethanol, add a small amount of ammonia water, stir evenly and heat to 60 °C, add 1.5 mL of tetraethyl orthosilicate (TEOS), and stir and react for 4 h. Centrifuge to separate the white precipitate, wash it several times with deionized water and ethanol, and then dry it at 70 °C for 2 h to obtain silica nanospheres.

[0028] The prepared silica nanospheres were etched with 0.1 mol / L NaOH aqueous solution for 4 h to obtain mesoporous silica nanospheres (MSN). The prepared MSN was then immersed in 0.1 M glacial acetic acid and thoroughly mixed for 2 h, followed by repeated washing with deionized water to obtain protonated MSN.

[0029] Characterized, such as Figure 1 As shown in Figure A, the obtained protonated MSN is a uniform spherical structure with an average diameter of approximately 200 nm. like Figure 1 As shown in Figure B, the XRD pattern shows that the mesoporous SiO2 nanospheres and the unetched SiO2 nanospheres have diffraction peaks at the same positions, and the peaks are relatively wide with no other impurity peaks, indicating that both are amorphous SiO2 and the etching process does not affect the crystal structure of the material. like Figure 1 As shown in Figure C, the N2 adsorption-desorption isotherm indicates that the BET specific surface area of ​​the protonated MSN is 68 m² / g. like Figure 1 As shown in Figure D, the pore size distribution curve shows that the average pore size of the obtained protonated MSN is 6.78 nm.

[0030] (2) Preparation of DCM@MSN / sCD83: Take an appropriate amount of mature DC, wash once with PBS buffer, centrifuge to remove the supernatant, and add an appropriate amount of RIPA lysis buffer (1×10⁻⁶). 5 (100 μL), lyse for 15 min, centrifuge at 20000 r / min for 30 min, remove supernatant to obtain mature DC cell membrane (DCM), and uniformly disperse DCM in 1 mL of physiological saline solution to obtain DCM suspension.

[0031] Five mg of protonated MSN was immersed in 1 mL of sCD83 physiological saline solution (100 ng / mL), mixed continuously for 2 h, and the precipitate was collected by centrifugation to obtain sCD83-loaded MSN (MSN / sCD83).

[0032] 1 mL of DCM suspension (containing 1×10 cells) 5 The mixture was thoroughly mixed with 5 mg of MSN / sCD83, and after ultrasonic treatment, the uncoated particles were removed by centrifugation. This process was repeated 3 to 4 times to obtain DCM-coated mesoporous silica nanocarriers loaded with sCD83 (DCM@MSN / sCD83).

[0033] Characterized, such as Figure 2 As shown in Figure A, transmission electron microscopy reveals that the surface of the nanocarrier is coated with a clear cell membrane layer (indicated by the arrow), with a coating thickness of approximately 10 nm. like Figure 2As shown in Figure B, the elemental distribution diagram shows that silicon and membrane proteins (represented by nitrogen N) are co-localized on the surface of the nanocarrier, confirming successful cell membrane encapsulation. like Figure 2 As shown in Figure C, Western blot analysis results showed that CD45 and CD80 were both positive in DCM@MSN / sCD83, indicating that key proteins of the DC cell membrane were preserved after coating.

[0034] Comparative Example 1: The difference between this comparative example and Example 1 is that in step (2), sCD83 is replaced with dexamethasone acetate (DEX). Specifically, the dexamethasone acetate tablets are thoroughly ground and dissolved in physiological saline until the maximum solubility is achieved. The supernatant is centrifuged and used to impregnate protonated MSN for 2 hours. The precipitate is collected by centrifugation to obtain MSN loaded with DEX (MSN / DEX). Then, DCM is applied to obtain DCM@MSN / DEX nanocarriers. The content of other components and the preparation method are the same as in Example 1.

[0035] Comparative Example 2: The difference between this comparative example and Example 1 is that in step (2), the protonated MSN and DCM suspension are directly mixed thoroughly, and after ultrasonic treatment, the uncoated particles are removed by centrifugation. This process is repeated 3 to 4 times to obtain blank mesoporous silica nanocarriers (DCM@MSN) coated with DCM. The content of other components and the preparation method are the same as in Example 1.

[0036] Comparative Example 3: The difference between this comparative example and Example 1 is that the DCM coating step (2) is not performed, resulting in a naked drug-carrying MSN nanocarrier without DCM coating, denoted as MSN / sCD83. The content of other components and the preparation method are the same as in Example 1.

[0037] Comparative Example 4: The difference between this comparative example and Example 1 is that: the same amount of free sCD83 protein (0.1 μg / mouse) as in Example 1 was directly used and dissolved in physiological saline as comparative example 4.

[0038] Comparative Example 5: The difference between this comparative example and Example 1 is that: free sCD83 protein at a dose of 2 μg / mouse was directly used, dissolved in physiological saline, as comparative example 5.

[0039] Experimental Example 1: Evaluation of Cytotoxicity and Biocompatibility Bone marrow-derived dendritic cells (BMDCs) were extracted from the tibia and femur of C57BL / 6 mice. After euthanizing the C57BL / 6 mice, the bilateral tibia and femur were aseptically separated, the attached muscle tissue was removed, and the epiphyses at both ends of the bones were cut off to expose the bone marrow cavity. The bone marrow was repeatedly washed with pre-cooled complete culture medium to obtain a bone marrow cell suspension. After centrifugation and resuspending, red blood cells were lysed to remove red blood cell interference. After washing and purification, the cells were seeded in induction medium containing GM-CSF and IL-4 for in vitro culture. Cytokines were replenished by changing half the medium periodically. After 5-7 days of induction differentiation, the cells were collected in suspension to obtain immature dendritic cells (hereinafter referred to as DCs).

[0040] DCs were seeded into 96-well plates, approximately 1 × 10⁻⁶ per well. 4 Cells were stimulated with LPS (lipopolysaccharide of Gram-negative bacterial outer membrane) for 24 h to obtain mature DCs (hereinafter referred to as aDCs); they were then incubated with medium containing different concentrations (0, 10, 20, 50, 100, 200 μg / mL) of DCM@MSN / sCD83 in a CO2 environment (37℃) for 1 h, 2 h, 6 h, 12 h, 24 h, and 48 h, and cell viability was detected by MTT assay.

[0041] MTT assay for cell viability: 5 mg / mL MTT was dissolved in PBS, sterilized using a 0.22 μm filter membrane, and aliquoted at -20°C in the dark. Cells were seeded at an appropriate density in 96-well plates, with three to six replicates per group: experimental group, blank group (cells without culture medium), and negative control group (untreated cells). 20 μL of 5 mg / mL MTT (final concentration 0.5 mg / mL) was added to each well, and the plates were incubated at 37°C in a 5% CO2 incubator for 4 h in the dark. The culture medium was carefully aspirated from each well, and 150 μL of DMSO was added to each well. The plates were then shaken at low speed at room temperature in the dark for 10–15 min to fully dissolve the formazan. The absorbance (OD value) at 570 nm was measured using a microplate reader. Cell viability was calculated using the following formula: Cell viability (%) = (OD experimental group - OD blank group) / (OD control group - OD blank group) × 100%; The results are as follows Figure 3 As shown, when the concentration of DCM@MSN / sCD83 does not exceed 50 μg / mL, the cell survival rate is still above 90%, and when the concentration reaches 100 μg / mL, the cell survival rate is still >75%, indicating that DCM@MSN / sCD83 has good biocompatibility within the experimental dosage range.

[0042] Experimental Example 2: In vitro DC maturation inhibition experiment Add DC to LPS (1×10) 5Cells were incubated at 37°C for 24 h in a CO2 environment to obtain aDCs (mature DCs simulating inflammatory sites). The culture medium was then changed, and DCM@MSN / sCD83 (50 μg / mL), DCM@MSN / DEX (50 μg / mL) prepared in Comparative Example 1, and DCM@MSN (50 μg / mL) prepared in Comparative Example 2 were added respectively. Cells were incubated for a total of 24 h, and then collected for flow cytometry analysis. Results are as follows: Figure 4 As shown, flow cytometry analysis revealed that, compared with untreated aDCs stimulated by LPS, the expression of DC maturation markers (CD80 and CD86) in the DCM@MSN / sCD83 treatment group (aDCM@MSN / sCD83) was significantly downregulated; cytokine expression analysis showed that IL-10 (an anti-inflammatory factor) expression was significantly upregulated after DCM@MSN / sCD83 treatment.

[0043] Similarly, flow cytometry analysis of the DCM@MSN / DEX treatment group (aDCM@MSN / DEX) showed that CD80 and CD86 expression was downregulated, IL-10 expression was upregulated, but TGF-β expression was not significantly changed.

[0044] Flow cytometry analysis of the DCM@MSN treatment group (aDCM@MSN) showed a slight decrease in CD80 and CD86 expression, while IL-10 and TGF-β expression were not significantly upregulated.

[0045] The results above indicate that DCM@MSN / sCD83 can effectively inhibit the maturation of aDC and promote its conversion into tolerant DC.

[0046] Experiment Example 3: In vitro Treg-induced differentiation experiment Spleens were aseptically harvested from BALB / c mice, ground, sieved, and used to prepare a single-cell suspension. After lysing and washing red blood cells, purified CD4 cells were obtained by negative sorting with magnetic beads. + T cells; the T lymphocytes were mixed with aDCs induced by DCM@MSN / sCD83, DCM@MSN / DEX, and DCM@MSN at an appropriate ratio (mass ratio 1:1) and seeded into a culture plate. Complete culture medium was added, and the cells were co-cultured at 37°C and 5% CO2 for 24 h.

[0047] The results are as follows Figure 5 As shown, flow cytometry analysis reveals the CD4+ of the DCM@MSN / sCD83 processing group (aDC+DCM@MSN / sCD83). + CD25 - / CD4 + CD25 + The proportion of T cells is significantly reduced; under normal circumstances, CD4 + CD25 +T cells are typically regulatory T cells (Tregs), indicating that DCM@MSN / sCD83 can promote Treg production; The reduction in the proportion of Tregs in the DCM@MSN / DEX processing group (aDC+DCM@MSN / DEX) was weaker than that in the DCM@MSN / sCD83 group; The proportion of Tregs in the DCM@MSN treatment group (aDC+DCM@MSN) did not change significantly.

[0048] Experiment Example 4: In vivo flow cytometry detection of changes in the proportion of DCs and Tregs in various organs of EAU mice To further verify the immunomodulatory effects of DCM@MSN / sCD83 in vivo, flow cytometry was performed on EAU mice after drug administration. The specific procedures are as follows: First, an EAU model was established using C57BL / 6 mice. 350 μg of human photoreceptor intergenic retinoid peptide 1-20 (IRBP1-20, amino acid sequence: GPTHLFQPSLVLDMAKVLLD) was subcutaneously inoculated at six different sites (footpads, tail base, and flanks). This protein was emulsified in complete Freund adjuvant, and a single dose of 500 ng pertussis toxin (PTX) was administered intraperitoneally. The dosing cycle was as follows: starting on day 14 post-immunization, the drug was administered every other day for a total of four doses (days 14, 16, 18, and 20). Then, on day 21, single-cell suspensions were prepared from the eyes, cervical lymph nodes, and spleen for flow cytometry analysis to examine changes in the proportion of mature dendritic cells (DCs) and Tregs in each organ.

[0049] Results of the test on the changes in the proportion of mature DCs in various organs are as follows: Figure 6 As shown in Figure A, flow cytometry results revealed that, compared to the EAU model group, the proportion of dendritic cells (DCs) in the eye, cervical lymph nodes, and spleen was significantly decreased in the DCM@MSN / sCD83 treatment group (EAU+DCM@MSN / sCD83). This indicates that DCM@MSN / sCD83 reduces the role of DCs in the immune response by inhibiting their activation and maturation, thereby alleviating the inflammatory response. In the DCM@MSN / DEX treatment group (EAU+DCM@MSN / DEX), only the proportion of mature DCs decreased in the eye and spleen, while the proportion of DCs in the cervical lymph nodes was not significantly different from that in the EAU model group, indicating that DEX has a limited inhibitory effect on lymph node DCs. In the DCM@MSN treatment group (EAU+DCM@MSN), the proportion of mature DCs in all organs was not significantly different from that in the EAU model group, suggesting that the blank carrier itself does not possess immunomodulatory activity.

[0050] Changes in the proportion of Tregs in various organs, as follows Figure 6As shown in Figure B, compared with the EAU model group, the DCM@MSN / sCD83 treatment group (EAU+DCM@MSN / sCD83) showed lower levels of CD4+ in the eyes, cervical lymph nodes, and spleen of mice. + CD25 - / CD4 + CD25 + The proportions were significantly reduced, indicating that sCD83 can systematically promote Treg differentiation; CD4+ levels in the eyes and lymph nodes of the EAU@MSN / DEX treatment group (EAU+DCM@MSN / DEX) were significantly reduced. + CD25 - / CD4 + CD25 + The proportions were not significantly different from the EAU model group, except for CD4 in the spleen. + CD25 - / CD4 + CD25 + The proportion was significantly reduced, indicating that the induction effect of surface DEX on Tregs was mainly limited to peripheral immune organs, with limited effect on the local eye and draining lymph nodes; the proportion of Terg in each organ of the DCM@MSN treatment group (EAU+DCM@MSN) was not significantly different from that of the EAU model group.

[0051] The above results indicate that DCM@MSN / sCD83, after intravitreal injection, can penetrate the blood-eye barrier and enter the peripheral circulation, simultaneously inhibiting DC maturation and promoting Treg differentiation in the eye, neck lymph nodes, and spleen, thereby achieving synergistic local and systemic immune regulation.

[0052] Experiment Example 5: Flow Cytometry Detection of DC Maturation Markers and Cytokine Expression in Various Organs in Vivo To further investigate the mechanism of action of DCM@MSN / sCD83 nanocarriers on immunomodulation and their effects in different tissues, single-cell suspensions were prepared from the eyes, cervical lymph nodes, and spleen of EAU mice after drug administration (day 21). Flow cytometry was used to analyze the expression levels of mature DC markers CD80 and CD86, as well as anti-inflammatory cytokines IL-10 and TGF-β, in each organ.

[0053] Test results are as follows Figure 7 As shown in A~C: (1) Ocular DC markers and cytokine expression (e.g.) Figure 7 (As shown in A) Compared with the EAU model group, the expression levels of surface molecules CD80 and CD86 in ocular DCs of mice treated with DCM@MSN / sCD83 (EAU+DCM@MSN / sCD83) were downregulated, while the expression of TGF-β and IL-10 was upregulated, indicating that the nanocarrier can effectively inhibit DC maturation and promote its conversion to the tolerant type in the ocular region. The expression of surface molecules CD80 and CD86 in ocular DCs of the EAU@MSN / DEX treatment group (EAU+DCM@MSN / DEX) was also downregulated, while the expression of TGF-β and IL-10 was upregulated, but the upregulation of TGF-β was significantly weaker than that of the DCM@MSN / sCD83 treatment group. There were no significant differences in the indicators between the DCM@MSN treatment group (EAU+DCM@MSN) and the EAU model group.

[0054] (2) Cervical lymph node DC markers and cytokine expression (e.g.) Figure 7 (As shown in B) Compared with the EAU model group, the expression levels of surface molecules CD80 and CD86 in the DCs of cervical lymph nodes of mice treated with DCM@MSN / sCD83 (EAU+DCM@MSN / sCD83) were significantly downregulated, while the expression of TGF-β and IL-10 was significantly upregulated, indicating that the nanocarrier can reach the draining lymph nodes and exert an immunomodulatory effect after penetrating the blood-eye barrier. The expression of TGF-β and IL-10 in the DCs of cervical lymph nodes of mice treated with EAU@MSN / DEX (EAU+DCM@MSN / DEX) was upregulated, but the upregulation of TGF-β was weaker than that of the DCM@MSN / sCD83 treatment group, indicating that DEX has limited ability to regulate lymph node function. There were no significant differences in the indicators between the DCM@MSN treatment group (EAU+DCM@MSN) and the EAU model group.

[0055] (3) Splenic DC markers and cytokine expression (e.g.) Figure 7 (As shown in C) Compared with the EAU model group, the expression levels of surface molecules CD80 and CD86 in the spleen of mice treated with DCM@MSN / sCD83 (EAU+DCM@MSN / sCD83) showed no significant difference, but the expression of TGF-β and IL-10 was significantly upregulated, indicating that the nanocarrier mainly plays a functional immunomodulatory role rather than inhibiting DC maturation. In the EAU@MSN / DEX treatment group (EAU+DCM@MSN / DEX), the expression of IL-10 in the spleen DCs was upregulated, but the expression of TGF-β was not significantly different from that in the EAU model group, further indicating that sCD83 has a unique advantage in inducing TGF-β secretion. There were no significant differences in any indicators between the DCM@MSN treatment group (EAU+DCM@MSN) and the EAU model group.

[0056] In summary, the DCM@MSN / sCD83 synthesized in this invention can simultaneously downregulate DC maturation markers (CD80 / CD86) and upregulate anti-inflammatory factors (TGF-β / IL-10) in the eye; it also exhibits a dual function of inhibiting maturation and inducing tolerance in cervical lymph nodes; while in the spleen, it primarily promotes the secretion of anti-inflammatory factors without affecting the expression of maturation markers. This tissue-specific differential regulatory pattern demonstrates the unique advantage of DCM@MSN / sCD83 in achieving synergistic immunotherapy through "local targeting-peripheral regulation." In contrast, DCM@MSN / DEX and DCM@MSN have significantly insufficient induction of anti-inflammatory factors in lymph nodes and spleen, and are limited in their systemic immune regulation effects.

[0057] Experiment Example 6: Simulated Blood-Eye Barrier Penetration Test and In vivo Circulation Tracing (1) Simulated blood-eye barrier penetration test: To investigate whether nanoparticles can cross the blood-brain barrier, this experimental example uses Transwell to establish an in vitro blood-brain barrier model.

[0058] Thaw the matrix gel overnight at 4°C and mix well. Add 100 μL of matrix gel to the chamber, spread it evenly with the tip of a pipette, and incubate at 37°C for 30 minutes to allow the matrix gel to solidify. All culture dishes or media in contact with the matrix gel substrate should be pre-cooled below freezing. Throughout the procedure, the matrix gel substrate should be kept on ice, and excessive drying of the matrix gel substrate should be avoided during gelation.

[0059] Mouse retinal vascular endothelial cells (mREC cells) (5×10 5 Cells per well were seeded into the upper chamber of 24-well substrate-coated cells and cultured for 27 hours until 100% aggregation was achieved. The cells were then cultured in serum-free medium for another 12 hours. DiD-labeled DCM@MSN and DiD-labeled naked MSN (0.5 μg / μL) were seeded into the upper chamber, and the fluorescence intensity in the lower chamber was detected using a fluorescence microscope after 12 hours.

[0060] The co-localization effect of DCM@MSN was observed using laser confocal microscopy. DiD (red light) was used to label the cell membrane DCM encapsulating the nanocarrier; DiO (green light) was used to label the nanocarrier MSN / sCD83; and DAPI (blue light) was used to stain the cell nucleus. Figure 8Image A shows micrographs of DCs co-cultured with DCM@MSN labeled with DiD and DiO fluorescent probes under green (488 nm), red (570 nm), and blue (405 nm) excitation, as well as the superimposed images of each channel. By comparing the red and green light regions with the superimposed images of each channel, it was found that red light was uniformly distributed on the cell surface, while green light was mainly concentrated in the cell center and exhibited obvious granular bright spots. This indicates that during the endocytosis of DCM@MSN / sCD83, the DiD-labeled DCM fuses with the cell membrane of the DC itself, while the DiO-labeled MSN is concentrated inside the cell, outside the nucleus. Furthermore, the areas with high overlap between the red and green light regions are all located within the cell outline, demonstrating that the DCM@MSN nanocarrier can be effectively taken up by DCs, providing direct evidence for the subsequent intracellular release and immunomodulatory function of sCD83. Furthermore, according to Figure 8 The results showed that DiD-labeled DCM@MSNs were abundant in the lower chamber, while unmodified naked MSNs were scarce, suggesting that DCM@MSNs could more easily penetrate simulated retinal vascular endothelial cells and enter the lower chamber.

[0061] (2) Tracing within the body's circulation: In this study, in vivo flow cytometry (IVFC) was used to detect the concentration of DCM@MSN in the blood. DiD fluorescently labeled DCM@MSN was injected into the eye of C57BL / 6 mice via vitreous injection. Blood flow in the ear artery of the mice was measured at 6, 12, 24, 48, 72, and 96 hours, and the number of fluorescence peaks per unit time was recorded to calculate the concentration of DCM@MSN in the peripheral blood of the mice at the corresponding time points.

[0062] The distribution of DCM@MSN in various organs was observed using the DPM-IVFM-3D small animal three-dimensional imaging system. The administration method was consistent with the in vivo flow cytometry experiment, and the enrichment of DCM@MSN in different sites of the thoracic and peritoneal cavities of mice was detected at 6, 24, 48, and 72 hours. Mice were sacrificed after 72 hours, and cervical lymph nodes and spleen were removed for further analysis of the enrichment level of DCM@MSN in these two organs.

[0063] The results are as follows Figure 9 As shown in Figure A, DCM@MSN appeared in the blood 6 hours after ocular injection and gradually disappeared from the blood after 72 hours. In vivo three-dimensional imaging was used to study the aggregation location of DCM@MSN in mice. The results are as follows... Figure 9The results showed that 24 hours after ocular injection, mice exhibited significant fluorescence in the thoracic and peritoneal cavities, which disappeared after 72 hours. This indicates that DCM@MSN can enter the thoracic and digestive tracts through peripheral blood circulation, with a metabolic cycle of approximately 72 hours, further confirming its good penetration ability. To determine the accumulation of DCM@MSN in mouse organs, major organs of mice injected ocularly with DCM@MSN / DiD 24 hours prior were removed for three-dimensional imaging tests and compared with normal mice. Figure 9 As shown in Figure C, the cervical lymph nodes and spleen of the injected mice showed strong fluorescence, indicating that DCM@MSN mainly accumulated in these organs. After 72 h, almost no fluorescence signal was detected, which is consistent with the results of IVFC.

[0064] Application Example 1: An EAU model was established using C57BL / 6 mice. 350 μg of human photoreceptor intergenic retinoid peptide 1-20 (IRBP1-20, amino acid sequence: GPTHLFQPSLVLDMAKVLLD) was subcutaneously injected into six different sites (footpads, tail base, and lateral flanks). This protein was emulsified in complete Freund adjuvant, and a single dose of 500 ng pertussis toxin (PTX) was administered intraperitoneally. On day 14 post-immunization, mice were randomly divided into eight groups of 15 mice each. The dosing cycle was as follows: starting from day 14 post-immunization, the drug was administered every other day for a total of four doses (days 14, 16, 18, and 20). On day 21, eyes were harvested for H&E histological staining and clinical examination. Neck lymph nodes, spleen, and eyes were harvested for flow cytometry. Serum and aqueous humor were collected to detect cytokine concentrations. Specific groupings are as follows: Normal control group (control): Unimmunized healthy mice, injected intravitreal with an equal volume of physiological saline; EAU model group: After immunization, an equal volume of physiological saline was injected into the vitreous body; Example 1 group (EAU+DCM@MSN / sCD83): The nanocarrier suspension prepared in Example 1 was injected intravitreally, with a dose of 0.1 μg / animal of sCD83, 2 μL each time, once every other day; Comparative Example 1 (EAU+DCM@MSN / DEX): DCM@MSN / DEX was injected into the vitreous body, and the DEX content was equal to the molar amount of sCD83 in Example 1 (0.1 μg / animal). Comparative Example 2 (EAU+DCM@MSN): Intravitreal injection of blank DCM@MSN vector; Comparative Example 3 (EAU+MSN / sCD83): MSN / sCD83 without DCM encapsulation injected into the vitreous, containing 0.1 μg / animal of sCD83; Comparative Group 4 (EAU + low dose of free sCD83): Free sCD83 protein was injected intravitreally at a dose of 0.1 μg / animal; Comparative group 5 (EAU + high dose of free sCD83): Free sCD83 protein was injected intravitreally at a dose of 1 μg / animal.

[0065] The test results are as follows: (1) Fundus color photographs and H&E histological staining of the retina like Figure 10 As shown, fundus photography of the EAU model group revealed optic disc swelling and multiple vascular leaks, inflammatory infiltration, multifocal retinal damage, and disruption of the vascular barrier. HE staining showed disordered cell arrangement, inflammatory cell infiltration, and retinal folds. Fundus photography of the EAU+DCM@MSN / sCD83 and sCD83 groups showed normal retinal vessels and optic disc with no obvious lesions and no exudates on the retinal surface. HE staining showed clear retinal structure and regular cell arrangement. Fundus photography and HE staining of the comparative group 1 (EAU+DCM@MSN / DEX and DEX groups) showed some relief of inflammatory symptoms. Fundus photography and HE staining of the comparative group 2 (EAU+DCM@MSN and MSN groups) showed that ocular inflammation in the EAU+MSN group mice did not worsen, indicating that the nanocarrier itself does not have adverse effects on the mouse eyes.

[0066] (2) Streaming cytometry results validated by the EAU model like Figure 11 As shown in Figure A, compared with the normal control group, the proportion of mature dendritic cells (DCs) in the eye, cervical lymph nodes, and spleen of the EAU model group was significantly increased; Figure 11 As shown in Figure B, compared with the normal control group, the CD4 count in the EAU model group was significantly higher. + CD25 - / CD4 + CD25 + The proportion of Tregs was increased, indicating that the EAU model was successfully established and that inflammation continued to progress without treatment.

[0067] (3) To systematically evaluate the in vivo immunomodulatory effects of DCM@MSN / sCD83, flow cytometry was performed on the eyes, cervical lymph nodes, and spleen of mice after drug administration. Results showed: Compared with the EAU model group, the proportion of mature DCs and the expression levels of CD80 / CD86 in various organs of mice in Example 1 group (EAU+DCM@MSN / sCD83) were significantly decreased, while the expression of anti-inflammatory factors IL-10 and TGF-β was significantly upregulated, and CD4 was also significantly reduced. + CD25 + FOXP3+ The significantly increased proportion of Tregs indicates that this nanocarrier can achieve synergistic immunomodulation both locally and systemically. In contrast, Comparative Group 1 (EAU+DCM@MSN / DEX) mainly exerted immunomodulatory effects in the spleen, with significantly weaker induction of TGF-β and promotion of Tregs in the eye and lymph nodes compared to the sCD83 group; Comparative Group 2 (EAU+DCM@MSN) showed no significant changes in any of the indicators (e.g., Figure 6 , Figure 7 (As shown).

[0068] like Figure 12 As shown in Figures A-B, the expression of the ocular mature DC markers CD80 and CD86 was slightly downregulated in the three comparative groups (EAU+MSN / sCD83); Figure 12 As shown in C-D, the expression of anti-inflammatory factors IL-10 and TGF-β was slightly upregulated; there were no significant changes in any indicators of cervical lymph nodes and spleen, indicating that the naked carrier without DCM coating could not effectively penetrate the blood-eye barrier; in Comparative Group 4 (EAU + low dose of free sCD83), there were no significant changes in any indicators of the eye, cervical lymph nodes and spleen, indicating that low dose of free sCD83 had no therapeutic effect; in Comparative Group 5 (EAU + high dose of free sCD83), all indicators of the eye, cervical lymph nodes and spleen showed significant improvement, but the required dose was 10 times that of Group 1 in Example 1.

[0069] (4) Immunohistochemical staining of retinal STING protein like Figure 13 As shown, the area of ​​STING positive staining in Example 1 group (EAU+DCM@MSN / sCD83) shrank significantly, and the deeply stained area almost disappeared, leaving only a thin layer of light brown staining; the structure of each layer of the retina was arranged regularly, and the infiltration of inflammatory cells was significantly reduced; the retinal STING expression in Comparative Example 1 group (EAU+DCM@MSN / DEX) also decreased, but to a lesser extent than in Example 1 group; compared with the EAU model group, there was no significant difference in retinal STING expression in Comparative Example 2 group (EAU+DCM@MSN) and the EAU model group, indicating that blank DCM@MSN itself does not have an immunomodulatory effect in treating EAU, while sCD83 carried by MSN can regulate the immune inflammatory response by inhibiting the expression of retinal STING protein.

[0070] (5) Detection of immune regulation mechanisms like Figure 14 As shown, RNA sequencing results indicate that, compared to the DCM@MSN treatment group, DCM@MSN / sCD83 treatment upregulated intracellular RNA in dendritic cells (aDCs). mTORC1 and ANGPT4 Gene expression ( Figure 13 The two genes mentioned above are hypoxia-inducible factor-1α (A). HIF-1α In contrast, phosphorylated HIF-1α (P-HIF-1α) precisely reflects the cellular energy metabolism state and works synergistically with phosphorylated STAT3 (P-STAT3). Western blot experiments confirmed that after treatment with DCM@MSN / sCD83, the expression levels of intracellular P-HIF-1α and P-STAT3 were significantly increased. Figure 13 In the diagram, B and C represent the grayscale value calculated based on B, which indicates the intensity of the WB band. After treatment with DCM@MSN / DEX, P-STAT3 expression was upregulated, but P-HIF-1α expression was significantly reduced.

[0071] Therefore, DCM@MSN / sCD83 activates the HIF-1α / STAT3 signaling pathway, promotes the secretion of IL-10 and TGF-β, and induces dendritic cells (DCs) to form an immune-tolerant phenotype. These tolerant dendritic cells (tDCs) can further induce the generation of regulatory T cells and inhibit the STING signaling pathway in retinal cells, thereby alleviating experimental autoimmune uveitis.

[0072] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A biomimetic nanomedicine delivery system, characterized in that, include: Mesoporous silica nanosphere core; Soluble CD83 loaded within the mesoporous silica nanospheres; And the mature dendritic cell membranes that coat the surface of the mesoporous silica nanospheres.

2. The biomimetic nanomedicine delivery system according to claim 1, characterized in that, The mesoporous silica nanospheres exhibit a uniform spherical structure with an average particle size of 200 nm and a BET specific surface area of ​​68 m². 2 / g, with an average pore size of 5~10 nm.

3. A method for preparing the biomimetic nano-drug delivery system according to claim 1 or 2, characterized in that, Includes the following steps: Monodisperse SiO2 nanospheres were prepared by microemulsion method using tetraethyl orthosilicate as silicon source; mesoporous structures were constructed on the surface of the nanospheres by alkaline etching to obtain mesoporous silica nanospheres. The obtained mesoporous silica nanospheres were immersed in glacial acetic acid solution for protonation treatment, so that their surface was positively charged, thus obtaining protonated mesoporous silica nanospheres. The obtained protonated mesoporous silica nanospheres were immersed in a soluble CD83 saline solution, and sCD83 was loaded into the mesopores to obtain drug-loaded mesoporous silica nanospheres. The suspension containing the cell membrane of mature dendritic cells was mixed with the obtained drug-loaded mesoporous silica nanospheres, and after ultrasonic treatment, the uncoated particles were removed by centrifugation to obtain the DCM@MSN / sCD83 nanocarrier.

4. The preparation method according to claim 3, characterized in that, The microemulsion method described herein is as follows: stirring the reaction in an ethanol-ammonia system at 60°C for 4 hours to obtain monodisperse SiO2 nanospheres.

5. The preparation method according to claim 3, characterized in that, The alkaline etching process specifically involves etching with a 0.1 mol / L NaOH aqueous solution for 4 hours to obtain mesoporous silica nanospheres.

6. The preparation method according to claim 3, characterized in that, The protonation treatment specifically involves impregnation with 0.1 M glacial acetic acid for 2 hours to obtain protonated mesoporous silica nanospheres.

7. The preparation method according to claim 3, characterized in that, The specific operation of loading sCD83 is as follows: protonated mesoporous silica nanospheres are immersed in a physiological saline solution containing sCD83 and continuously mixed for 2 hours to ensure that the drug fully fills the pore structure on the surface of the SiO2 microspheres, thereby achieving drug loading. The ratio of the amount of protonated mesoporous silica nanospheres to the physiological saline solution containing sCD83 is 5 mg: 1 mL, and the concentration of the physiological saline solution containing sCD83 is 100 ng / mL.

8. The preparation method according to claim 3, characterized in that, The mature dendritic cell membrane is obtained by lysing and separating activated mature dendritic cells; the ratio of the suspension containing the mature dendritic cell membrane to the drug-loaded mesoporous silica nanospheres is 1 mL: 5 mg. The mature dendritic cell membrane is tightly bound to the surface of the protonated mesoporous silica nanospheres through electrostatic interaction. The surface of the mature dendritic cell membrane is negatively charged, while the surface of the protonated mesoporous silica nanospheres is positively charged. Preferably, the centrifugation process for removing uncoated particles is repeated 3 to 4 times.

9. The use of the biomimetic nanodelivery system according to claim 1 or 2 in the preparation of a drug for treating experimental autoimmune uveitis.

10. The application according to claim 9, characterized in that, The biomimetic nanodelivery system delivers drugs via intravitreal injection, targeting mature dendritic cells in the eye and lymph nodes, inducing tolerant mature dendritic cells to produce IL-10 and TGF-β, downregulating STING expression, and reducing retinal inflammatory damage; the nanocarrier penetrates the blood-eye barrier and enters the peripheral circulation, distributing to cervical lymph nodes and spleen, achieving synergistic local and systemic immune regulation.

Images