Self-assembled PEG-b-PDPA-b-PGEM copolymer nanoparticles as protein antigen delivery vehicles to dendritic cells:preparation,characterization and cellular uptake

Antigen uptake by dendritic cells(DCs)is a key step for initiating antigen-specific T cell immunity.In the present study,novel synthetic polymeric nanoparticles were prepared as antigen delivery vehicles to improve the antigen uptake by DCs.Well-defined cationic and acid-responsive copolymers,monomethoxy poly(ethylene glycol)-block-poly(2-(diisopropyl amino)ethyl methacrylate)-block-poly(2-(guanidyl)ethyl methacrylate)(mPEG-b-PDPA-b-PGEM,PEDG)were synthesized by reversible addition-fragmentation chain transfer polymerization of 2-(diisopropylamino)ethyl methacrylate and N-(tert-butoxycarbonyl)amino ethyl methacrylate monomers,followed by deprotection of tert-butyl protective groups and guanidinylation of obtained primary amines.1H NMR,13C NMR and GPC results indicated the successful synthesis of well-defined PEDG copolymers.PEDG copolymers could self-assemble into nanoparticles in aqueous solution,which were of cationic surface charges and showed acid-triggered disassembly contributed by PGEM and PDPA moieties,respectively.Significantly,PEDG nanoparticles could effectively condense with negatively charged model antigen ovalbumin(OVA)to form OVA/PEDG nanoparticle formulations with no influence on its secondary and tertiary structures demonstrating by far-UV circular dichroism and UV-vis spectra.In vitro antigen cellular uptake by bone marrow DCs(BMDCs)indicated using PEDG nanoparticles as antigen delivery vehicles could significantly improve the antigen uptake efficiency of OVA compared with free OVA or the commercialized Alum adjuvant.Moreover,as the surface cationic charges of OVA/PEDG nanoparticle formulations reduced,the uptake efficiency decreased correspondingly.Collectively,our work suggests that guanidinylated,cationic and acid-responsive PEDG nanoparticles represent a new kind of promising antigen delivery vehicle to DCs and hold great potential to serve as immunoadjuvants in the development of vaccines.

Programmed nanoparticle-loaded microparticles for effective antigen/adjuvant delivery

A microscale vaccine containing SiO_(2)nanoparticles loaded in CaC〇3 microparticles was constructed using the co-precipitation method.The antigen ovalbumin(OVA)was covalently conjugated with SiO_(2)nanoparticles,and these nanoparticles and CpG were co-encapsulated into CaCO_(3)microparticles,generating a vaccine with a size of approximately 5.2μm.Scanning electron microscopy(SEM),energy-dispersive X-ray(EDX),elemental mapping,and Fourier transform infrared(FTIR)analyses confirmed the successful preparation of the microscale vaccine;the vaccine had good storage stability without sustained antigen release,and negligible cytotoxicity to dendritic cells(DCs)and macrophages.Compared to SiO_(2)nanoparticles,the microscale vaccine can significantly improve antigen/adjuvant uptake.DCs internalized the entire microscale vaccine into lysosomes via macropinocytosis,and an increase in antigen endo/lysosomal escape was observed by confocal User scanning microscopy(CLSM).Specifically,DCs pulsed with the vaccine were fully mature,expressing high levels of costimulatory molecules(CD40,CD80,and CD86),MHCⅡ,and MHCⅠand secreting high levels of proinflammatory cytokines(IL-12,TNF-α,IL-1β,and IL-6).In addition,the vaccine had good in vivo biocompatibility,could protect the antigen from rapid degradation,and increased the retention time in lymph nodes.SiO_(2)nanoparticles-in-CaCO_(3)microparticles were an excellent carrier for antigen and adjuvant delivery.Hopefully,this study can provide some information on the design of microscale carriers for vaccine delivery systems.

Radial porous SiO_(2) nanoflowers potentiate the effect of antigen/adjuvant in antitumor immunotherapy

Here,we reported a cancer nanovaccine based on SiO_(2)nanoflowers with a special radial pore structure,which greatly enhanced cross-presentation and induced the production of cytotoxic T lymphocyte cells secreting granzymes B and interferon-γ.The antigen ovalbumin was covalently conjugated onto the as-synthesized hierarchical SiO_(2)nanoflowers,and the adjuvant cytosine-phosphate-guanine was electrostatically adsorbed into their radial pore by simple mixing before use.The nanovaccine exhibited excellent storage stability without antigen release after 27 days of incubation,negligible cytotoxicity to dendritic cells,and a high antigen loading capacity of 430±66 mg·g^(−1)support.Besides,the nanovaccine could be internalized by dendritic cells via multiple pathways.And the enhancement of antigen/adjuvant uptake and lysosome escape of antigen were observed.Noteworthy,in vitro culture of bone marrow-derived dendritic cells in the presence of nanovaccine proved the activation of dendritic cells and antigen cross-presentation as well as secretion of proinflammatory cytokines.Besides,in vivo study verified the targeting of nanovaccine to draining lymph nodes,the complete suppression of tumor in six out of ten mice,and the triggering of notable tumor growth delay.Overall,the present results indicated that the nanovaccine can be served as a potential therapeutic vaccine to treat cancer.

Mature dendritic cell-derived dendrosomes swallow oxaliplatin-loaded nanoparticles to boost immunogenic chemotherapy and tumor antigen-specific immunotherapy

The cytomembrane-derived delivery platform represents a promising biomimetic strategy in oncotherapy.To achieve durable and reliable tumor inhibition,mature dendrosomes(mDs),which were isolated from bone marrow-derived dendritic cells undergoing CT26 tumor antigen(TA)stimulation,were fused with redox-responsive nanoparticles(NPs)that were composed of poly(disulfide ester amide)polymers with an intensified disulfide density and hydrophobic oxaliplatin(OXA)prodrugs with the ability to potentiate immunogenicity.In vitro and in vivo results revealed that NP/mDs could induce tumor cell death through mitochondrial pathway and thus created immunogenic microenvironments,but also elicited immunocyte differentiation by TA cross-dressing and infiltration by direct presentation.By further neutralizing immune-regulatory interaction,the administration of PD-L1 antibody(αPD-L1)greatly improved antitumor efficiency of NP/mDs.Furthermore,the effectors of host immune systems effectively inhibited the growth and metastasis of distal tumors,likely because the autologous TA evoked by OXA and allogeneic TA delivered by mDs acted as additional stimuli to reinforce the immune response of tumor-specific T cells and immunosurveillance toward oncogenesis.These results demonstrated that NP/mDs could simultaneously realize immunogenic chemotherapeutics and specific TA delivery.In combination withαPD-L1,the antitumor effect was further enhanced.Therefore,NP/mDs provide a promising strategy for the comprehensive treatment of malignancy.

Ovalbumin-loaded paramagnetic nano-triangles for enhanced dendritic cell stimulation,T_(1)-MR imaging,and antitumor immunity

Vaccine-based cancer immunotherapy has demonstrated a significant potential for cancer treatment in clinics.Although the efficiencies of vaccines are limited,they can be enhanced by a well-designed antigen delivery system that promotes sufficient antigen presentation of dendritic cells(DCs)for initiating high T cell immunity.Herein,antigen-loaded manganese oxide(Mn_(3)O_(4))triangular-shaped ultrasmall nanoparti-cles were prepared to stimulate DC-based immunotherapy under the guidance of T_(1)magnetic resonance imaging.The FDA-approved triblock copolymer Pluronic^(■)F-68 wasused not onlyto transferthe phase from hydrophobic to hydrophilic but also to enrich antigen loading and improve the biocompatibility of the prepared nanoparticles.Ovalbumin(OVA),a model antigen,was adsorbed on the surface of polymer-coated nanoparticles through electrostatic interaction to form Mn_(3)O_(4)@PF68-OVA nanoparticle-antigen complexes to stimulate DC-based immunization and antigen-specific T cell immunity.The Mn_(3)O_(4)@PF68-OVA nanovaccine(NV)induces negligible toxicity effects against 4T1 and bone marrow-derived dendritic cells(BMDCs)by conventional methods supports the proliferation of intestine organoids,which are an innovative three-dimensional cytotoxicity evaluation system,thereby indicating their potential safety for in vivo cancer therapies.The designed paramagnetic nanovaccine possessed excellent OVA delivery to dendritic-regulated antigen-specific T cells in vitro by stimulating the maturation level of BMDCs.In ad-dition,Mn_(3)O_(4)@PF68-OVA NVs enhance immunity in vivo by increasing the T-cells and M1 macrophages,which suggests improved immunity.Excitingly,vaccination with Mn_(3)O_(4)@PF68-OVA offer complete pro-tection in the prophylactic group and significant tumor inhibition in the therapeutic group against B16-OVA tumor.In addition,the designed nanovaccine demonstrated high T_(1)-MR imaging in the tumor,fur-ther justifying enhanced tumor accumulation and capability to real-time monitor the treatment proce-dure.This study presents a promising nanosystem to design an effective nanovaccine for T_(1)-MR imaging-guided tumor immunotherapy.