Design of protein-reactive polymers and core-crosslinked nanoparticles in view of vaccine delivery

Delivering tumour-associated antigens (TAAg) to dendritic cells (DCs), the most potent class of antigen presenting cells (APCs) of our immune system, has emerged as a promising anti-cancer therapy by harnessing patients’ own immune system at recognizing and eliminating metastatic growth. Currently, several clinical trials obtain promising results with adoptive transfer of DCs and T cells that were conditioned ex vivo with TAAg. An even more viable approach is direct in vivo delivery of peptide- or protein-based TAAg to DCs in combination with specific immune-activating stimuli. The major bottleneck of this approach is the lack of efficiency of TAAg to provide DCs with the correct stimuli to steer the immune response towards the induction of cytotoxic T cells (CTLs) that can recognize and kill cancer cells. With respect to the improvement of CTL responses, molecular adjuvants have been developed that bind to pathogen recognition receptors (PRR) – such as Toll-like receptors (TLRs) – which are expressed on the cell surface or endosomal membrane of DCs. However, a major hurdle that hampers successful clinical translation of these components is the systemic inflammation caused when they enter systemic circulation. The aim of this thesis is the exploration of a materials chemistry approach to engineer the immune system by developing well-defined polymeric materials that can accommodate vaccine antigens and immune-modulatory components, and delivery these to DCs. From the materials chemistry side, efforts will focus on elucidating optimal conjugation strategies to link polymers to proteins, assemble proteins and polymers into nanoparticles, and formulate molecular adjuvants in polymeric nanoparticles. From the immunological side, efforts will focus on elucidating the in vitro and in vivo behaviour of the developed systems

A generic polymer-protein ligation strategy for vaccine delivery

Although the field of cancer immunotherapy is intensively investigated, there is still a need for generic strategies that allow easy, mild and efficient formulation of vaccine antigens. Here we report on a generic polymer protein ligation strategy to formulate protein antigens into reversible polymeric conjugates for enhanced uptake by dendritic cells and presentation to CD8 T-cells. A N-hydroxypropylmethacrylamide (HPMA)-based copolymer was synthesized via RAFT polymerization followed by introduction of pyridyldisulfide moieties. To enhance ligation efficiency to ovalbumin, which is used as a model protein antigen, protected thiols were introduced onto lysine residues and deprotected in situ in the presence of the polymer. The ligation efficiency was compared for both the thiol-modified versus unmodified ovalbumin, and the reversibility was confirmed. Furthermore, the obtained nanoconjugates were tested in vitro for their interaction and association with dendritic cells, showing enhanced cellular uptake and antigen cross-presentation to CD8 T-cells

pH-Degradable mannosylated nanogels for dendritic cell targeting

We report on the design of glycosylated nanogels via core-cross linking of amphiphilic non-water-soluble block copolymers composed of an acetylated glycosylated block and a pentafluorophenyl (PFP) activated ester block prepared by reversible addition fragmentation (RAFT) polymerization. Self-assembly, pH-sensitive core-cross-linking, and removal of remaining PFP esters and protecting groups are achieved in one pot and yield fully hydrated sub-100 nm nanogels. Using cell subsets that exhibit high and low expression of the mannose receptor (MR) under conditions that suppress active endocytosis, we show that mannosylated but not galactosylated nanogels can efficiently target the 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

Transiently thermoresponsive polymers and their applications in biomedicine

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

Dual pH- and temperature-responsive RAFT-based block co-polymer micelles and polymer-protein conjugates with transient solubility

Via a smart combination of temperature-responsive and acid labile acetal monomers, copolymers are obtained with a 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

Core/shell protein-reactive nanogels via a combination of RAFT polymerization and vinyl sulfone postmodification

Aim: A promising nanogel vaccine platform was expanded toward antigen conjugation. Materials & methods: Block copolymers containing a reactive ester solvophobic block and a PEG-like solvophilic block were synthesized via reversible addition-fragmentation chain-transfer polymerization. Following self-assembly in DMSO, the esters allow for core-crosslinking and hydrophilization by amide bond formation with primary amines. Free thiols were accessed at the polymer chain ends through aminolysis of the reversible addition-fragmentation chain-transfer groups, and into the nanogel core by reactive ester conversion with cysteamine. Subsequently, free thiols were converted into vinyl sulfone moieties. Results: Despite sterical constraints, nanogel-associated vinyl sulfone moieties remained well accessible for cysteins to enforce protein conjugation successfully. Conclusion: Our present findings provide a next step toward well-defined vaccine nanoparticles that can co-deliver antigen and a molecular adjuvant

Super resolution imaging of nanoparticles cellular uptake and trafficking

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 tells 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

Degradable Ketal-based block copolymer nanoparticles for anticancer drug delivery: A systematic evaluation

Low solubility of potent (anticancer) drugs is a major driving force for the development of noncytotoxic, stimuli-responsive nanocarriers, including systems based on amphiphilic block copolymers. In this regard, we investigated the potential of block copolymers based on 2-hydroxyethyl acrylate (HEA) and the acidsensitive ketal-containing monomer (2,2-dimethyl-1,3-dioxolane-4-yl)methyl acrylate (DMDMA) to form responsive drug nanocarriers. Block copolymers were successfully synthesized by sequential reversible addition-fragmentation chain transfer (RAFT) polymerization, in which we combined a hydrophilic poly(HEA)x block with a (responsive) hydrophobic poly(HEAm-co-DMDMAn)y copolymer block. The DMDMA content of the hydrophobic block was systematically varied to investigate the influence of polymer design on physicochemical properties and in vitro biological performance. We found that a DMDMA content higher than 11 mol % is required for self-assembly behavior in aqueous medium. All particles showed colloidal stability in PBS at 37°C for at least 4 days, with sizes ranging from 23 to 338 nm, proportional to the block copolymer DMDMA content. Under acidic conditions, the nanoparticles decomposed into soluble unimers, of which the decomposition rate was inversely proportional to the block copolymer DMDMA content. Flow cytometry and confocal microscopy showed dose-dependent, active in vitro cellular uptake of the particles loaded with hydrophobic octadecyl rhodamine B chloride (R18). The block copolymers showed no intrinsic in vitro cytotoxicity, while loaded with paclitaxel (PTX), a significant decrease in cell viability was observed comparable or better than the two commercial PTX nanoformulations Abraxane and Genexol-PM at equal PTX dose. This systematic approach evaluated and showed the potential of these block copolymers as nanocarriers for hydrophobic drugs. (Chemical Equation Presented)

Transiently responsive protein–polymer conjugates via a 'grafting-from' RAFT approach for intracellular co-delivery of proteins and immune-modulators

We report on transiently responsive protein-polymer conjugates that temporarily change their protein conformation from the soluble to the particle-like state. 'Grafting-from' RAFT polymerization of a dioxolane-containing acrylamide with a protein macroCTA is used to design polymer-protein conjugates that self-assemble into nano-particles at physiological temperature and pH. Acid triggered hydrolysis of the dioxolane units into diolmoeities rendered the conjugates fully water soluble irrespective of temperature