Agonists of Toll-like receptors (TLRs) are potent activators of the innate immune system and hold promise as vaccine adjuvant and for anticancer immunotherapy. Unfortunately, in soluble form they readily enter systemic circulation and cause systemic inflammatory toxicity. Here we demonstrate that by covalent ligation of a smallmolecule imidazoquinoline-based TLR7/8 agonist to 50-nm-sized degradable polymeric nanogels the potency of the agonist to activate TLR7/8 in in vitro cultured dendritic cells is largely retained. Importantly, imidazoquinoline-ligated nanogels focused the in vivo immune activation on the draining lymph nodes while dramatically reducing systemic inflammation. Mechanistic studies revealed a prevalent passive diffusion of the nanogels to the draining lymph node. Moreover, immunization studies in mice have shown that relative to soluble TLR7/8 agonist, imidazoquinoline-ligated nanogels induce superior antibody and T-cell responses against a tuberculosis antigen. This approach opens possibilities to enhance the therapeutic benefit of small-molecule TLR agonist for a variety of applications.nanotechnology | Toll-like receptor | dendritic cells | lymph node | vaccine A ctivation of the innate immune system crucially depends on the recognition of evolutionary conserved microbe-associated molecular patterns by host pattern recognition receptors (PRRs). Triggering of PRRs not only elicits a direct antimicrobial and inflammatory cascade but also activates the necessary antigen-presenting cells to subsequently prime antigen-specific T-and B-cell immune responses (1). Agonists of Toll-like receptors (TLRs) are among the most potent activators of the innate immune response identified to date and, thus, are under intensive investigation as adjuvants for vaccination (2) or as agents for anticancer immunotherapy (3). Especially promising in this context are agonists of the endosomal TLR7 and TLR8, which typically recognize singlestranded RNAs generated during viral infection (4) but can also be activated by synthetic small-molecule agonists (5). Molecular adjuvants (6, 7) that are agonists of TLR7/8 indeed activate a broad spectrum of antigen-presenting cells, both in mice (only TLR7) and humans, and typically induce high levels of type I IFN and IL-12, the most vital cytokines to drive the Th1 and cytotoxic T-cell responses required to combat intracellular infections and cancer (7,8).Due to their pharmacokinetic profile, most molecular adjuvants rapidly diffuse after administration and evoke systemic inflammatory responses that cause dose-limiting toxicity (9, 10). In the context of intratumoral administration, these dose-limiting toxicities prevent these compounds from reaching the necessary intratumoral concentration to yield real therapeutic benefit. In the context of vaccination, the rapid diffusion of molecular adjuvants dramatically lowers the ability of antigen and TLR agonist to reach the same antigen-presenting cells in the draining lymph node (DLN), which results in suboptimal immunity to the del...

The induction of antigen-specific adaptive immunity exclusively occurs in lymphoid organs. As a consequence, the efficacy by which vaccines reach these tissues strongly affects the efficacy of the vaccine. Here, we report the design of polymer hydrogel nanoparticles that efficiently target multiple immune cell subsets in the draining lymph nodes. Nanoparticles are fabricated by infiltrating mesoporous silica particles (ca. 200 nm) with poly(methacrylic acid) followed by disulfide-based crosslinking and template removal. PEGylation of these nanoparticles does not affect their cellular association in vitro, but dramatically improves their lymphatic drainage in vivo. The functional relevance of these observations is further illustrated by the increased priming of antigen-specific T cells. Our findings highlight the potential of engineered hydrogel nanoparticles for the lymphatic delivery of antigens and immune-modulating compounds.

The focus of this review is on the class of transiently thermoresponsive polymers. These polymers are thermoresponsive, but gradually lose this property upon chemical transformation - often a hydrolysis reaction - in the polymer side chain or backbone. An overview of the different approaches used for the design of these polymers along with their physicochemical properties is given. Their amphiphilic properties and degradability into fully soluble compounds make this class of responsive polymers attractive for drug delivery and tissue engineering applications. Examples of these are also provided in this review.

Understanding the interaction between synthetic nanostructures and living cells is of crucial importance for the development of nanotechnology-based intracellular delivery systems. Fluorescence microscopy is one of the most widespread tools owing to its ability to image multiple colors in native conditions. However, due to the limited resolution, it is unsuitable to address individual diffraction-limited objects. Here we introduce a combination of super-resolution microscopy and single-molecule data analysis to unveil the behavior of nanoparticles during their entry into mammalian cells. Two-color Stochastic Optical Reconstruction Microscopy (STORM) addresses the size and positioning of nanoparticles inside cells and probes their interaction with the cellular machineries at nanoscale resolution. Moreover, we develop image analysis tools to extract quantitative information about internalized particles from STORM images. To demonstrate the potential of our methodology, we extract previously inaccessible information by the direct visualization of the nanoparticle uptake mechanism and the intracellular tracking of nanoparticulate model antigens by dendritic cells. Finally, a direct comparison between STORM, confocal microscopy, and electron microscopy is presented, showing that STORM can provide novel and complementary information on nanoparticle cellular uptake.

Nanomaterials hold potential of altering the interaction between therapeutic molecules and target cells or tissues. High aspect ratio nanomaterials in particular have been reported to possess unprecedented properties and are intensively investigated for their interaction with biological systems. Graphene oxide (GOx) is a water-soluble graphene derivative that combines high aspect ratio dimension with functional groups that can be exploited for bioconjugation. Here, we demonstrate that GOx nanosheets can spontaneously adsorb proteins by a combination of interactions. This property is then explored for intracellular protein vaccine delivery, in view of the potential of GOx nanosheets to destabilize lipid membranes such as those of intracellular vesicles. Using a series of in vitro experiments, we show that GOx nanosheet adsorbed proteins are efficiently internalized by dendritic cells (DCs: the most potent class of antigen presenting cells of the immune system) and promote antigen cross-presentation to CD8 T cells. The latter is a hallmark in the induction of potent cellular antigen-specific immune responses against intracellular pathogens and cancer.

Via a smart combination of temperature-responsive and acid labile acetal monomers, copolymers are obtained with à la carte lower critical solution temperature behavior. RAFT copolymerization of these monomers using, respectively, a PEG-functionalized or amine-reactive NHS-functionalized chain transfer agent allows designing of micelles and polymer-protein conjugates with transient solubility properties within a physiologically relevant window.

a Efficient polymer-protein conjugation is a crucial step in the design of many therapeutic protein formulations including nanoscopic vaccine formulations, antibody-drug conjugates and to enhance the in vivo behaviour of proteins. Here we aimed at preparing well-defined polymers for conjugation to proteins by reversible addition-fragmentation chain transfer (RAFT) polymerization of both acrylates and methacrylamides with protein-reactive chain transfer agents (CTAs). These RAFT agents contain either a N-hydroxysuccinimide (NHS) or pentafluorophenyl (PFP) ester moiety that can be conjugated to lysine residues, and alternatively a maleimide (MAL) or pyridyl disulfide (PDS) moiety that can be conjugated to cysteine residues. Efficiency of the bioconjugation of these polymers to bovine and avian serum albumin was investigated as a function of stoichiometry, polymer molecular weight and the presence of reducing agents. A large molar excess of polymer was required to obtain an acceptable degree of protein conjugation.However, protein modification with N-succinimidyl-S-acetylthiopropionate (SATP) to introduce sulfhydryl groups onto primary amines, significantly increased conjugation efficiency with MAL-and PDS-containing polymers.

We report on the design of glycosylated nanogels via core-cross-linking of amphiphilic non-watersoluble block copolymers composed of an acetylated glycosylated block and a pentafluorophenyl (PFP) activated ester block prepared by RAFT polymerization. Self-assembly, pH-sensitive corecross-linking and removal of remaining PFP esters and protecting groups is achieved in one-pot yielding fully hydrated sub-100 nm nanogels. Using cell subsets that exhibit high and low expression of the mannose receptor under conditions that suppress active endocytosis, we show that mannosylated but not galactosylated nanogels can efficiently target the mannose receptor (MR) that is expressed on the cell surface of primary dendritic cells (DCs). These nanogels hold promise for immunological applications involving DCs and macrophage subsets.