Engineered Exosomes Based On Nanosecond Pulsed Electroporation For Glioblastoma Immunotherapy

Glioblastoma(GBM)is one of the most malignant primary central nervous system(CNS)tumours with an extremely poor prognosis.Over the past decade,immune checkpoint blockade(ICB)therapy has demonstrated significant potential in treating various tumours.However,the over-expressed programmed death-ligand(PD-L1)and the downregulation of the major histocompatibility complex-I(MHC-I)lead to the immunosuppressive tumour microenvironment(TME)and low immunogenicity of GBM,which impedes the ICB therapy.Interferon-gamma(IFN-γ)upregulates the expression of MHC-1 molecule in GBM cells,which exhibits prospects of enhancing the efficacy of the ICB therapy when combined with the anti-PD-L1 monoclonal antibody.However,solely IFN-γand anti-PD-L1 fail to reach GBM sites because of the blood-brain barrier(BBB).Hence,developing a novel therapeutically drug carrier that co-delivers IFN-γand anti-PD-L1 antibody throughout BBB is essential for GBM-targeting immunotherapy.Exosomes(Exo),as promising drug-delivery vehicles with satisfactory biocompatibility and biosafety,are able to deliver multiple biomacromolecules across BBB and protect them against degradation during circulation.However,the natural secretion of Exo from cells is inadequate.Hence,we developed the nanosecond pulse electroporation(ns EP)system that enabled high-throughput cell transfection meanwhile stimulated substantial cellular Exo release to circumvent limitations.In this study,we engineered a novel drug carrier called immune Exo(“im Exo”)that simultaneously encapsulated IFN-γm RNA and overexpressed CD64 on their surface(CD64-Exo),which immobilized anti-CD71 and anti-PD-L1 monoclonal antibodies.im Exo ensured GBM targeting and ICB therapeutical activity concurrently.They also mitigated the immunosuppressive TME,thereby enhancing the treatment effect of ICB therapy.The research content of the manuscript mainly includes the following parts:1.Optimization of Exo production using the ns EP system and demonstration of the release mechanismA novel ns EP system was proposed by coupling standard millisecond pulse electroporation(ms EP)with traditional ns EP,combined with microfluidic electroporation technology.Pulse amplitude,pulse width,and pulse frequency of the novel ns EP system were optimized to maximize the secretion of Exo without affecting cell viability.The pulse amplitude,width,and frequency were finalized at 180 V,600ns,and 100 k Hz for Gibco Mouse Embryonic Fibroblasts(MEFs)cells and 50 V,600ns,and 100 k Hz for Human embryonic kidney 293(HEK293T)cells,respectively.Our experimental results showed that 3.0×10~7 cells were processed in less than 5 min,demonstrating the high-throughput cell electroporation capacity of the ns EP system.Moreover,over 5-fold Exo production rates were observed in both MEF and HEK293T cell lines when applied optimized parameters in the ns EP system compared with the ms EP system.We also applied the proteomic analysis to illustrate the potential mechanism of Exo secretion based on the ns EP system.We discovered that mixed lineage kinase domain like pesudokinase plays a critical role in regulating Exo secretion.2.Production and characterization of the im ExoTo begin with,the MEF cell line was constructed and engineered to produce substantial CD64-enriched Exo consistently.In order to accomplish the specific loading of IFN-γm RNA in CD64-Exo,the RNA-binding polypeptide N pep was cloned onto the c-terminus of CD64.Meanwhile,a box B sequence was added at the 3′end of the IFN-γm RNA.The box B sequence is capable of self-assembly and has a neck-loop structure that binds readily to the N pep,causing a higher loading efficiency of IFN-γm RNA in CD64-Exo with an above 20%increment,which proved the targeted m RNA loading specificity of CD64-Exo.CD64 is an Fcγreceptor that binds to the Fc segment of immunoglobulin G(Ig G).Based on this specific property,we constructed a novel immunotherapy drug carrier called im Exo.In this study,two monoclonal antibodies,anti-CD71 and anti-PD-L1,were attached and immobilized onto CD64-Exo.We examined different CD64-Exo/anti-CD71/anti-PD-L1 ratios and achieved the highest loading capacity of two antibodies at the 1/1/3(w/w/w)ratio.3.Evaluation of anti-tumour efficacy of im Exo in vitroThe cellular uptake of im Exo was first evaluated since taken up by GBM cells is the crucial factor for im Exo to exert their therapeutic effects.Our results showed that the uptake of im Exo in GBM cells compared with non-orientated targeting Exos was increased by greater than two times.We also analyzed the uptake pathway and revealed that im Exo entered GBM cells through a CD71-mediated clathrin-dependent endocytosis pathway.Then we tested the anti-tumour activity of im Exo in vitro and found that IFN-γconcentration in the cell culture medium was increased as well as the MHC-1 expression was improved after taking up.In addition,no apparent liver and kidney toxicity and hemolysis were found in mice administrated with double doses,confirming the biosafety of im Exo for follow-up in vivo studies.4.Evaluation of anti-tumour efficacy of im Exo in vivoThe anti-tumour effect of im Exo was evaluated using two different GBM orthotopic models.Both models confirmed the excellent GBM targeting ability of im Exo.In the GL261 model,tumour growth was depressed by 70.5%in the treated group compared with the untreated group and by 52.2%compared with the free monoclonal antibody treated group.In addition,the median survival time of mice treated with im Exo was 53.5 days,which was significantly longer than 28.5 days in the untreated group.Also,the immunological analysis showed that the im Exo treatment remarkably increased the proportion of IFN-γexpressing cell populations in tumor tissues,allowing the improvement of the infiltration of CD8~+T cells.The GL261 model has moderate immune cell infiltration,modest immunosuppressive TME,and is sensitive to ICB therapy.To further prove the efficacy of im Exo,we assessed their anti-tumour effects in the SB28 model with highly immunosuppressive TME,which is considered to be majorly similar to the human GBM immune environment.Results demonstrated that the im Exo therapy successfully inhibited tumour growth by 79.5%compared with the control group,and the survival time was strikingly prolonged to 52days(27 days in the control group).Immunological analysis of tumour tissue showed that the im Exo treatment recovered the MHC-I expression in SB28 cells and increased infiltration of CD8~+T cells in tumour tissue simultaneously.Moreover,tumor-associated macrophages are massively infiltrated in the SB28 model,so we also evaluated the phenotype of macrophages in the tumor tissues after treatments.We noticed that the quantity of pro-inflammatory M1-polarized macrophages in tumour tissues was predominately increased after the im Exo treatment,which affirmed that im Exo could reprogram the GBM TME and enhance the immunogenicity of GBM,thus improving the immune response to the ICB treatment.In summary,we developed the ns EP system capable of generating substantial Exo via high-throughput cell stimulation.We also engineered a universal drug delivery system(CD64-Exo)that was able to transport biomacromolecular to the brain across BBB.In addition,the introduction of N pep/box B achieved the specific loading of targeted m RNA.As a proof of concept,we engineered im Exo via loading the IFN-γm RNA into CD64-Exo and conjugating anti-CD71/anti-PD-L1 antibody on their surface for GBM immunotherapy.im Exo exhibited extraordinary GBM tumour targeting ability and sufficient ICB therapy treatment effects.Moreover,they regulated TME,which significantly improved the effectiveness of ICB therapy for GBM.Importantly,our CD64-Exo platform is a versatile platform that has broad application prospects.