Stefaan De Koker

Polymeric multilayer capsules for drug delivery

The advent of Layer-by-Layer (LbL) assembly to fabricate polymeric as well as hybrid multilayer thin films has opened exciting avenues for the design of multifunctional drug carriers with extreme control over their physico-chemical properties. These polymeric multilayer capsules (PMLC) are typically fabricated by sequential adsorption of polymers onto a spherical substrate with dimensions varying from 10 nm to several microns and larger. In this critical review, we give an overview of the recent advances in the field of PMLC with respect to drug delivery and point out how sophisticated capsule engineering can lead to well-defined drug carriers with unique properties (139 references).

Polyelectrolyte microcapsules for biomedical applications

In this paper we review the recent contributions of polyelectrolyte microcapsules in the biomedical field, comprising in vitro and in vivodrug delivery as well as their applications as biosensors.

Polyelectrolyte Microcapsules as Antigen Delivery Vehicles To Dendritic Cells: Uptake, Processing, and Cross‐Presentation of Encapsulated Antigens

Degradable polyelectrolyte microcapsules (PMs; see picture) as antigen delivery vehicles are taken up by dendritic cells (DCs) by macropinocytosis. Following uptake, the shell of the microcapsules ruptures, resulting in the invasion of the capsules by the cellular cytoplasm, thus allowing DCs to efficiently process encapsulated antigen.

Intracellular Processing of Proteins Mediated by Biodegradable Polyelectrolyte Capsules

Multilayer polyelectrolyte capsules made by layer-by-layer assembly of oppositely charged biodegradable polyelectrolytes were filled with a model of a nonactive prodrug, a self-quenched fluorescence-labeled protein. After capsule uptake by living cells, the walls of the capsules were actively degraded and digested by intracellular proteases. Upon capsule wall degradation, intracellular proteases could reach the protein cargo in the cavity of the capsules. Enzymatic fragmentation of the self-quenched fluorescence-labeled protein by proteases led to individual fluorescence-labeled peptides and thus revoked self-quenching of the dye. In this way nonactive (nonfluorescent) molecules were converted into active (fluorescent) molecules. The data demonstrates that biodegradable capsules are able to convert nonactive molecules (prodrugs) to active molecules (drugs) specifically only inside cells where appropriate enzymes are at hand. In this way only cargo inside the capsules reaching cells is activated, but not the cargo in capsules which remain extracellular. The peptide fragments undergo further processing inside the cells, leading ultimately to exocytosis.

Type I IFN Counteracts the Induction of Antigen-Specific Immune Responses by Lipid-Based Delivery of mRNA Vaccines

The use of DNA and viral vector-based vaccines for the induction of cellular immune responses is increasingly gaining interest. However, concerns have been raised regarding the safety of these immunization strategies. Due to the lack of their genome integration, mRNA-based vaccines have emerged as a promising alternative. In this study, we evaluated the potency of antigen-encoding mRNA complexed with the cationic lipid 1,2-dioleoyl-3trimethylammonium-propane/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOTAP/DOPE ) as a novel vaccination approach. We demonstrate that subcutaneous immunization of mice with mRNA encoding the HIV-1 antigen Gag complexed with DOTAP/DOPE elicits antigen-specific, functional T cell responses resulting in specific killing of Gag peptide-pulsed cells and the induction of humoral responses. In addition, we show that DOTAP/DOPE complexed antigen-encoding mRNA displays immune-activating properties characterized by secretion of type I interferon (IFN) and the recruitment of proinflammatory monocytes to the draining lymph nodes. Finally, we demonstrate that type I IFN inhibit the expression of DOTAP/DOPE complexed antigen-encoding mRNA and the subsequent induction of antigen-specific immune responses. These results are of high relevance as they will stimulate the design and development of improved mRNA-based vaccination approaches.

Polymeric multilayer capsule-mediated vaccination induces protective immunity against cancer and viral infection

Recombinant antigens hold high potential to develop vaccines against lethal intracellular pathogens and cancer. However, they are poorly immunogenic and fail to induce potent cellular immunity. In this paper, we demonstrate that polymeric multilayer capsules (PMLC) strongly increase antigen delivery toward professional antigen-presenting cells in vivo, including dendritic cells (DCs), macrophages, and B cells, thereby enforcing antigen presentation and stimulating T cell proliferation. A thorough analysis of the T cell response demonstrated their capacity to induce IFN-γ secreting CD4 and CD8 T cells, in addition to follicular T-helper cells, a recently identified CD4 T cell subset supporting antibody responses. On the B cell level, PMLC-mediated antigen delivery promoted the formation of germinal centers, resulting in increased numbers of antibody-secreting plasma cells and elevated antibody titers. The functional relevance of the induced immune responses was validated in murine models of influenza and melanoma. On a mechanistic level, we have demonstrated the capacity of PMLC to activate the NALP3 inflammasome and trigger the release of the potent pro-inflammatory cytokine IL-1β. Finally, using DC-depleted mice, we have identified DCs as the key mediators of the immunogenic properties of PMLC.

Biodegradable Polyelectrolyte Microcapsules: Antigen Delivery Tools with Th17 Skewing Activity after Pulmonary Delivery

Because of their large surface area, the lungs appear an attractive route for noninvasive vaccine delivery, harboring the potential to induce local mucosal immune responses in addition to systemic immunity. To evoke adaptive immunity, Ags require the addition of adjuvants that not only enhance the strength of the immune response but also determine the type of response elicited. In this study, we evaluate the adjuvant characteristics of polyelectrolyte microcapsules (PEMs) consisting of the biopolymers dextran-sulfate and poly-l-arginine. PEMs form an entirely new class of microcapsules that are generated by the sequential adsorption of oppositely charged polymers (polyelectrolytes) onto a sacrificial colloidal template, which is subsequently dissolved leaving a hollow microcapsule surrounded by a thin shell. Following intratracheal instillation, PEMs were not only efficiently taken up by APCs but also enhanced their activation status. Pulmonary adaptive immune responses were characterized by the induction of a strongly Th17-polarized response. When compared with a mixture of soluble Ag with empty microcapsules, Ag encapsulation significantly enhanced the strength of this local mucosal response. Given their unique property to selectively generate Th17-polarized immune responses, PEMs may become of significant interest in the development of effective vaccines against fungal and bacterial species.

Surface‐Engineered Polyelectrolyte Multilayer Capsules: Synthetic Vaccines Mimicking Microbial Structure and Function

Immunizing: to evoke highly potent immune responses against recombinant antigens, hollow capsules consisting of layers of dextran sulphate and poly-L-arginine that encapsulate the antigen ovalbumin (orange circles) were coated with immune-activating CpG-containing oligonucleotides (green). These capsules were readily internalized by dendritic cells and showed activity in further immunization experiments.

Engineering Polymer Hydrogel Nanoparticles for Lymph Node-Targeted Delivery.

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.

Facile Two-Step Synthesis of Porous Antigen-Loaded Degradable Polyelectrolyte Microspheres**

Without doubt, the development of vaccines constitutes one of themajor breakthroughs of humanmedicine, allowing us to prevent numerous infectious diseases. Nevertheless, for major killers such as HIV, malaria, and tuberculosis, no effective vaccine is currently available. The failure of current vaccination strategies to elicit cellular immune responses, especially CD8 cytotoxic T cells (CTLs) that can recognize and eliminate infected cells, is considered to be one of the major reasons for this failure. Consequently, there is an urgent need to develop new vaccine formulations that can induce such CTL responses. One of the most promising approaches to achieve this goal is the encapsulation of antigens in particulate carriers with dimensions between 0.1 and 10 mm. A plethora of studies has now demonstrated that such carriers can strongly enhance antigen presentation by dendritic cells (DCs), the most potent antigen presenting cells capable of priming effector T cell responses, not only quantitatively but also qualitatively. Antigen being presented by a DC as an MHC/peptide complex (MHC=major histocompatibility complex) to the T cell receptor indeed constitutes the first step in the initiation of T cell responses. Two different pathways occur for antigen presentation by MHC I and MHC II to CD8 and CD4 T-cells, respectively. MHC I presentation is responsible for the processing and presentation of cytosolic proteins, which are cleaved by the proteasome, transported to the endoplasmatic reticulum, and subsequently loaded onto MHC I molecules. By contrast, MHC II presentation occurs for endocytosed proteins, which are degraded in endolysosomal compartments, loaded onto MHC II molecules and subsequently presented at the cell surface. The way in which antigen is internalized by a DC, however, strongly affects how the antigen is processed and presented by a DC, and consequently also the type and strength of immune response induced. Although soluble antigens are almost exclusively presented by MHC II to CD4 T cells, particulate antigens not only are far more efficiently taken up by DCs, but are also presented by MHC I to CD8 T cells, thus enabling the induction of CTL responses. Although particulate carriers have the capacity to evoke potent cellular immunity, their clinical application has been impeded largely by practical problems involving their generation, including a low antigen encapsulation efficiency, the use of chemical solvents and physical stresses that negatively affect antigen stability, and the involvement of complex and labor-intensive multistep processes to generate them. Polymeric multilayer capsules have emerged as promising microscopic carriers for the delivery of antigens to DCs, overcoming some of the problems described above. These capsules are based on alternate deposition of polymers (so-called layer-by-layer technology), either through electrostatic interaction or hydrogen bonding, onto a sacrificial template, followed by decomposition of the template, resulting in hollow capsules and allowing efficient antigen encapsulation under non-denaturing conditions. Several papers have now demonstrated the potential of these capsules to target antigens to APCs both in vitro and in vivo, resulting in strongly enhanced antigen presentation to CD4 and CD8 T cells and the induction of broad and strong immune responses. The major advantage for their success is presumably threefold: 1) They protect the antigen from degradation before reaching DCs; 2) Because of their size (1–10 mm), they are preferably targeted to DCs; and 3) Because of their soft thin shell, which is prone to the reductive conditions or endosomal proteases depending on the nature of the capsule shell, they allow the antigen to be readily processed upon internalization by DCs. Moreover, these capsules have been shown to be biocompatible and degradable, both in vitro and in vivo. Notwithstanding their excellent performance, polymeric multilayer capsules are fabricated in multiple steps, which is a rather cost-inefficient fashion involving the use of a large excess of polymer and several centrifugation steps during deposition of each single layer. Therefore, a simple and versatile strategy involving a minimum of process steps that mimics polymeric multilayer capsules would be of uttermost importance to allow this type of antigen carriers to reach the clinical stage. Herein, we report on the synthesis of porous antigenloaded degradable polyelectrolyte microspheres using spray drying as a simple, yet efficient, and scalable production [*] M. Dierendonck, Dr. S. De Koker, Prof. Dr. C. Vervaet, Prof. Dr. J.-P. Remon, Dr. B. G. De Geest Laboratory of Pharmaceutical Technology Department of Pharmaceutics, Ghent University Harelbekestraat 72, 9000 Ghent (Belgium) E-mail: br.degeest@ugent.be