Systemically Administered TLR7/8 Agonist and Antigen-Conjugated Nanogels Govern Immune Responses against Tumors

[Image: see text] The generation of specific humoral and cellular immune responses plays a pivotal role in the development of effective vaccines against tumors. Especially the presence of antigen-specific, cytotoxic T cells influences the outcome of therapeutic cancer vaccinations. Different strategies, ranging from delivering antigen-encoding mRNAs to peptides or full antigens, are accessible but often suffer from insufficient immunogenicity and require immune-boosting adjuvants as well as carrier platforms to ensure stability and adequate retention. Here, we introduce a pH-responsive nanogel platform as a two-component antitumor vaccine that is safe for intravenous application and elicits robust immune responses in vitro and in vivo. The underlying chemical design allows for straightforward covalent attachment of a model antigen (ovalbumin) and an immune adjuvant (imidazoquinoline-type TLR7/8 agonist) onto the same nanocarrier system. In addition to eliciting antigen-specific T and B cell responses that outperform mixtures of individual components, our two-component nanovaccine leads in prophylactic and therapeutic studies to an antigen-specific growth reduction of different tumors expressing ovalbumin intracellularly or on their surface. Regarding the versatile opportunities for functionalization, our nanogels are promising for the development of highly customized and potent nanovaccines

Density of conjugated antibody determines the extent of Fc receptor dependent capture of nanoparticles by liver sinusoidal endothelial cells

Despite considerable progress in the design of multifunctionalized nanoparticles (NPs) that selectively target specific cell types, their systemic application often results in unwanted liver accumulation. The exact mechanisms for this general observation are still unclear. Here we asked whether the number of cell-targeting antibodies per NP determines the extent of NP liver accumulation and also addressed the mechanisms by which antibody-coated NPs are retained in the liver. We used polysarcosine-based peptobrushes (PBs), which in an unmodified form remain in the circulation for >24 h due to the absence of a protein corona formation and low unspecific cell binding, and conjugated them with specific average numbers (2, 6, and 12) of antibodies specific for the dendritic cell (DC) surface receptor, DEC205. We assessed the time-dependent biodistribution of PB-antibody conjugates by in vivo imaging and flow cytometry. We observed that PB-antibody conjugates were trapped in the liver and that the extent of liver accumulation strongly increased with the number of attached antibodies. PB-antibody conjugates were selectively captured in the liver via Fc receptors (FcR) on liver sinusoidal endothelial cells, since systemic administration of FcR-blocking agents or the use of F(ab')2 fragments prevented liver accumulation. Cumulatively, our study demonstrates that liver endothelial cells play a yet scarcely acknowledged role in liver entrapment of antibody-coated NPs and that low antibody numbers on NPs and the use of F(ab')2 antibody fragments are both sufficient for cell type-specific targeting of secondary lymphoid organs and necessary to minimize unwanted liver accumulation.Drug Delivery Technolog

The Influence of Block Ionomer Microstructure on Polyplex Properties: Can Simulations Help to Understand Differences in Transfection Efficiency?

Gene therapies enable therapeutic interventions at gene transcription and translation level, providing enormous potential to improve standards of care for multiple diseases. Nonviral transfection agents and in particular polyplexes based on block ionomers are-besides viral vectors and cationic lipid formulations-among the most promising systems for this purpose. Block ionomers combine a hydrophilic noncharged block, e.g., polyethylene glycol (PEG), with a hydrophilic cationic block. For efficient transfection, however, endosomolytic moieties, e.g., imidazoles, are additionally required to facilitate endosomal escape, which raises the general question how to distribute these functionalities within the block copolymer. Combining molecular dynamics simulation with physicochemical and biological characterization, this work aims to provide a first rational for the influence of block ionomer microstructure on polyplex properties, e.g., size, shape, and transfection efficiency. Our findings underline that a triblock microstructure is most efficient in compacting pDNA, which reduces polyplex size, enhances stability against degradation by DNase I, and thus provides better transfection performance.FWN – Publicaties zonder aanstelling Universiteit Leide

Combining reactive triblock copolymers with functional cross-linkers: A versatile pathway to disulfide stabilized-polyplex libraries and their application as pDNA vaccines

Therapeutic nucleic acids such as pDNA hold great promise for the treatment of multiple diseases. These therapeutic interventions are, however, compromised by the lack of efficient and safe non-viral delivery systems, which guarantee stability during blood circulation together with high transfection efficiency. To provide these desired properties within one system, we propose the use of reactive triblock copolypept(o)ides, which include a stealth-like block for efficient shielding, a hydrophobic block based on reactive disulfides for cross-linking and a cationic block for complexation of pDNA. After the complexation step, bifunctional cross-linkers can be employed to bio-reversibly stabilize derived polyplexes by disulfide bond formation and to introduce endosomolytic moieties at the same time. Cross-linked polyplexes show no aggregation in human blood serum. Upon cellular uptake and cleavage of disulfide bonds, the cross-linkers can interact with the endosomal membrane, leading to lysis and efficient endosomal translocation. In principal, the approach allows for the combination of one polymer with various different cross-linkers and thus enables the fast forward creation of a polyplex library. Here, we provide a first insight into the potential of this concept and use a screening strategy to identify a lead candidate, which is able to transfect dendritic cells with a model DNA vaccine.FWN – Publicaties zonder aanstelling Universiteit Leide