Killer PLGA Nanoparticles Induce Immune Tolerance In A Murine Model Of Alloskin Transplantation

Background:The primary goal to control the rejection of transplanted cells/tissues is to mediate the immune system by inducing the antigen-specific tolerance which should not impair the host's overall immune system.The progress in the field of nanotechnology enabled the researchers to develop the immuno-tolerance system which specifically reacts with the alloreactive T cells,B cells and antigen presenting cells?APCs?produced in response to the allograft transplantation.Biomimetic nanoparticles have been reported as immune modulators in autoimmune diseases and allograft rejections by numerous researchers around the world.But most of the therapeutics underlay the mechanism of encapsulation and controlled release of antigens,toxins,or cytokines and the antigen-presenting by cellular uptake of nanoparticles?NPs?through phagocytosis and pinocytosis.Few researches focus on the antigen-specific contacts of NPs with T cells and the combined use of multiple regulatory molecules.Objectives:In present study,polylactic-co-glycolic acid nanoparticles?PLGA-NPs?were prepared and covalently co-coupled with targeting alloantigen?H-2K^b-Ig dimer?,multiple regulatory molecules?anti-Fas mAb,PD-L1-Fc,and TGF-??,and“self-marker”CD47-Fc.The biodegradable,targeting alloreactive T cell,and multipotent killer PLGA-NPs were administered i.v.into a single MHC-mismatched murine model of alloskin transplantation,followed by the investigation of therapeutic effects,precise mechanism,side effects,and organ toxicity issue.Methods and Results:1.Preparation and characterization of PLGA-NPs:Two sizes of PLGA-NPs were prepared,respectively,using double emulsion method.The shape and surface morphology of PLGA-NPs was observed by scanning electron microscopy?SEM? while as,the size distribution and zeta potential was measured using Zeta sizer.As shown,the PLGA-NPs in the diameters of 202.4 nm and 78.8 nm PLGA?termed 200 nm and 80 nm?were prepared with an average zeta potential of-3.09 mV and -8.63 mV,respectively.Spherical shape and smooth surface morphology of the NPs were confirmed under SEM.Then polyethyleneimine?PEI?was conjugated to the surface of NPs by using EDC/NHS chemistry to modify the surface of PLGA-NPs with adequate NH₂ cationic groups which can couple with protein. 2.Generation and phenotypic analysis of Killer NPs:Micro BCA protein assay was used to quantify the BSA protein immobilized on the surface of PLGA-NPs.The maximum amount of protein loaded onto the 1 mg of NPs was approximately 75.31g for 200-nm NPs and 77.51?g for 80-nm NPs.Based on these data,PLGA-NPs were fabricated as a scaffold to co-coupling H-2K^b-Ig dimer,anti-Fas mAb,PD-L1-Fc,TGF-?,and CD47-Fc onto their PEI-coated surface,termed Killer NPs.After staining with fluorescence mAbs,the images of confocal microscopy confirmed the adequate immobilization of H-2K^b-Ig dimer,anti-Fas mAb,PD-L1-Fc and CD47-Fc onto the Killer NPs.But TGF-?molecules coupled onto killer NPs were not detected due to the unavailable of fluorescence-labeled anti-TGF-?antibody.3.Skin transplantation and Killer NPs treatment:The alloskin transplantation modelwas established by using 7-8 weeks old male C57BL/6 mice?H-2K^b?and the K^b-mutant C57BL/6 mice(bm1 mice,H-2K^bm1)as donor and recipient,respectively.The generated killer NPs were injected intravenously into bm1 mice on day 9,11 and 13 after skin transplant and alloskin survival was inspected every day.Meanwhile,7 treatment groups with different control NPs were also carried out.On day 15,the infiltrations of H-2K^b-alloreactive T cells,CD4⁺and CD8⁺T cells were detected in alloskin graft using immunofluorescence staining.The 200-nm control treatment groups(PBS?Blank NPs?NP^aFas?NP^Kb?NP^CD47?NP^Kb/aFasand NP^{Kb/aFas/PD-L1/TGF?}groups)was 16,19,22,27,27,36 and 41 days,respectively.The H-2K^b-alloreactive T cells infiltrated into alloskin was decreased by 79.8% as detected by in-situ H-2K^b-Ig dimer staining.The infiltration of CD8⁺T cells and CE4+T cells was reduced by 48.9%and 36.4%,respectively,while as obvious decrease of inflammatory cells was also observed HE staining in alloskin graft.In addition,the treatment with 80-nm killer NPs only prolonged the alloskin survival for 22 days.The treatment with 200-nm killer NPs was found much more effective than the 80-nm killer NPs in the alloskin transplant model,thus all further experiments were performed by using the 200-nm killer NPs.4.In vivo mechanism of Killer NPs to inhibit allograft rejections:As detected by H-2K^b-Ig dimer staining and flow cytometry,the killer NPs treatment decreased the frequency of H-2K^b-alloreactive CD8⁺T cells in recipient spleen cells by 90.3%,while the CD47^-killer NPs(NP^{Kb/aFas/PD-L1/TGF?})and Anti-Fas^-killer NPs(NP^{Kb/PD-L1/TGF?/CD47})treatment decreased the frequency by 62.6%and 51.9%,respectively.However,the non-cognate H-2K^d killer NPs treatment only led to a decrease of 12.6%.Similarly,80.7%of H-2K^b-alloreactive CD8⁺T cells in recipient peripheral blood were depleted by killer NPs treatment.In parallel,nearly three-fold increased apoptosis of CD8⁺T cells in spleen in the killer NPs treatment group,and 113.5% increase in CD47^-killer NPs group and 48.2%increased in Anti-Fas^-killer NPs group were found while little higher apoptosis of CD8⁺T cells in the no-cognate H-2K^d killer NPs group than the blank NPs group,without significant difference.In peripheral blood,about two-fold increase of apoptotic CD8⁺T cells was also observed in Killer NPs group,while a 96.2%increase in CD47^-killer NPs group and a 58.6%increase in Anti-Fas^-killer NPs groups.Killer NPs treatment significantly inhibited the activation of CD8⁺T cells in spleen with a decrease of 67.5%,and inhibited the proliferation of recipient T cells in response to donor splenocytes was markedly inhibited by about 50%,but facilitated the proliferation of CD4⁺/CD25⁺/Foxp3⁺regulatory T cells,with an increase of 81.7%in spleen and 80.1%in lymph nodes.5.In vivo tracking,tissue distribution of killer NPs and their contacts with immune cells:Grafted bm1 mice were injected via the tail vein with ICG-encapsulated killer NPs,H-2K^b-killer NPs(NP^{aFas/PD-L1/TGF?/CD47}),CD47^-killer NPs(NP^{Kb/aFas/PD-L1/TGF?}),and blank NPs,respectively,and followed by in vivo and ex vivo near-infrared imaging.Whole-body imaging showed the rapid and selective accumulation of ICG-encapsulated killer NPs in spleen,liver,lungs,kidney,heart,lymph nodes and alloskin graft.The strongest fluorescent intensity was observed in mice from 30 min to 4 h after i.v.administration,with a retention time up to 30 h.As controls,H-2K^b-killer NPs?non-targeting killer NPs?,CD47^-killer NPs and Blank NPs showed the in vivo trafficking a little different from the killer NPs,and a shorter retention time?24 h,24 h,and 18 h,respectively?.Furthermore,the detection by flow cytometry also confirmed the presence of PE-labeled killer NPs in peripheral blood,spleen and lymph nodes.The frozen sections of spleen isolated at 4 h after injection were processed for immunofluorescence staining.The confocal fluorescence images demonstrated that PE-CD47^-killer NPs mainly distributed in the red pulp and marginal zone and presented many co-localizations with CD8⁺T cells,CD4⁺T cells,macrophage and dendritic cells as well as fewer contacts with B cells.However,more PE-CD47⁺killer NPs were observed in the red pulp and marginal zone and displayed much more co-localizations with CD8⁺ T cells,but much fewer contacts with other cells than PE-CD47^-killer NPs.These results indicate the direct contacts of killer NPs with CD8⁺T cells and very less engulfment by macrophage and dendritic cells in vivo since the targeting effects of H-2K^b-Ig dimers.6.Recipient overall immune function and organ toxicity:On day 15,killer NPs treatment did not decrease the frequencies of CD3⁺T cells,CD4⁺T cells,B cells and NK cells in recipient's spleen;did not inhibit the proliferation of recipient T cells in response to the third-party spleen cells in a third-party MLR assay;did not decrease the cytotoxicity of NK cells to tumor cells;and also did not impair the general anti-tumor effects of recipient mice in a tumor-bearing experiments.At the long time points?2,17 and 32 days after the final injection of killer NPs?,no obvious bystander killing to the eight cell populations in peripheral blood was found as detected by blood routine tests;the functions of liver and kidney of recipient mice were not impaired significantly as monitored by routine biochemical tests.Meanwhile,no visible damage was observed in spleen,kidney,liver,heart,and lungs in recipient mice after killer NPs treatment as analyzed by H&E staining.These data indicate that killer NPs treatment did not lead to the obvious bystander killing to nonpathogenic immune cells,the significant impairment of overall immune function and visible organ toxicity.Conclusion:The biodegradable and multipotent killer nanoparticles were successfully developed by co-coupling the targeting alloantigen,multiple modulators and phagocytosis-resistant molecule onto the 200-nm PLGA-NPs.The killer NPs circulate throughout vasculature into spleen and lymph nodes;make contacts with CD8⁺T cells to facilitate vigorous apoptosis;inhibit the activation and proliferation of alloreactive CD8⁺T cells,and induce regulatory T cells;finally deplete most of alloantigen-reactive CTLs in spleen,blood and alloskin graft,thus markedly inhibit the allograft rejections and prolong alloskin survival,without the obvious impairment of overall immune function and visible organ toxicity.The promising results highlight the therapeutic capability of the alloantigen-presenting and tolerance-inducing killer NPs as a novel antigen-specific immunotherapy for the treatment of allograft rejections.