Autoantigen-Dexamethasone Conjugate-Loaded Liposomes Halt Arthritis Development in Mice.

There is no curative treatment for chronic auto-inflammatory diseases including rheumatoid arthritis, and current treatments can induce off-target side effects due to systemic immune suppression. This work has previously shown that dexamethasone-pulsed tolerogenic dendritic cells loaded with the arthritis-specific antigen human proteoglycan can suppress arthritis development in a proteoglycan-induced arthritis mouse model. To circumvent ex vivo dendritic cell culture, and enhance antigen-specific effects, drug delivery vehicles, such as liposomes, provide an interesting approach. Here, this work uses anionic 1,2-distearoyl-sn-glycero-3-phosphoglycerol liposomes with enhanced loading of human proteoglycan-dexamethasone conjugates by cationic lysine tetramer addition. Antigen-pulsed tolerogenic dendritic cells induced by liposomal dexamethasone in vitro enhanced antigen-specific regulatory T cells to a similar extent as dexamethasone-induced tolerogenic dendritic cells. In an inflammatory adoptive transfer model, mice injected with antigen-dexamethasone liposomes have significantly higher antigen-specific type 1 regulatory T cells than mice injected with antigen only. The liposomes significantly inhibit the progression of arthritis compared to controls in preventative and therapeutic proteoglycan-induced arthritis mouse models. This coincides with systemic tolerance induction and an increase in IL10 expression in the paws of mice. In conclusion, a single administration of autoantigen and dexamethasone-loaded liposomes seems to be a promising antigen-specific treatment strategy for arthritis in mice.

Live Cell Imaging by Förster Resonance Energy Transfer Fluorescence to Study Trafficking of PLGA Nanoparticles and the Release of a Loaded Peptide in Dendritic Cells.

Our previous study demonstrated that a selected ß-lactoglobulin-derived peptide (BLG-Pep) loaded in poly(lactic-co-glycolic acid) (PLGA) nanoparticles protected mice against cow's milk allergy development. However, the mechanism(s) responsible for the interaction of the peptide-loaded PLGA nanoparticles with dendritic cells (DCs) and their intracellular fate was/were elusive. Förster resonance energy transfer (FRET), a distance-dependent non-radioactive energy transfer process mediated from a donor to an acceptor fluorochrome, was used to investigate these processes. The ratio of the donor (Cyanine-3)-conjugated peptide and acceptor (Cyanine-5) labeled PLGA nanocarrier was fine-tuned for optimal (87%) FRET efficiency. The colloidal stability and FRET emission of prepared NPs were maintained upon 144 h incubation in PBS buffer and 6 h incubation in biorelevant simulated gastric fluid at 37 °C. A total of 73% of Pep-Cy3 NP was internalized by DCs as quantified using flow cytometry and confirmed using confocal fluorescence microscopy. By real-time monitoring of the change in the FRET signal of the internalized peptide-loaded nanoparticles, we observed prolonged retention (for 96 h) of the nanoparticles-encapsulated peptide as compared to 24 h retention of the free peptide in the DCs. The prolonged retention and intracellular antigen release of the BLG-Pep loaded in PLGA nanoparticles in murine DCs might facilitate antigen-specific tolerance induction.

Polyplex designs for improving the stability and safety of RNA therapeutics.

Nanoparticle-based delivery systems have contributed to the recent clinical success of RNA therapeutics, including siRNA and mRNA. RNA delivery using polymers has several distinct properties, such as enabling RNA delivery into extra-hepatic organs, modulation of immune responses to RNA, and regulation of intracellular RNA release. However, delivery systems should overcome safety and stability issues to achieve widespread therapeutic applications. Safety concerns include direct damage to cellular components, innate and adaptive immune responses, complement activation, and interaction with surrounding molecules and cells in the blood circulation. The stability of the delivery systems should balance extracellular RNA protection and controlled intracellular RNA release, which requires optimization for each RNA species. Further, polymer designs for improving safety and stability often conflict with each other. This review covers advances in polymer-based approaches to address these issues over several years, focusing on biological understanding and design concepts for delivery systems rather than material chemistry.

Modulating albumin-mediated transport of peptide-drug conjugates for antigen-specific Treg induction.

The therapeutic potential of antigen-specific regulatory T cells (Treg) has been extensively explored, leading to the development of several tolerogenic vaccines. Dexamethasone-antigen conjugates represent a prominent class of tolerogenic vaccines that enable coordinated delivery of antigen and dexamethasone to target immune cells. The importance of nonspecific albumin association towards the biodistribution of antigen-adjuvant conjugates has gained increasing attention, by which hydrophobic and electrostatic interactions govern the association capacity. Using an ensemble of computational and experimental techniques, we evaluate the impact of charged residues adjacent to the drug conjugation site in dexamethasone-antigen conjugates (Dex-K/E4-OVA323, K: lysine, E: glutamate) towards their albumin association capacity and induction of antigen-specific Treg. We find that Dex-K4-OVA323 possesses a higher albumin association capacity than Dex-E4-OVA323, leading to enhanced liver distribution and antigen-presenting cell uptake. Furthermore, using an OVA323-specific adoptive-transfer mouse model, we show that Dex-K4-OVA323 selectively upregulated OVA323-specific Treg cells, whereas Dex-E4-OVA323 exerted no significant effect on Treg cells. Our findings serve as a guide to optimize the functionality of dexamethasone-antigen conjugate amid switching vaccine epitope sequences. Moreover, our study demonstrates that moderating the residues adjacent to the conjugation sites can serve as an engineering approach for future peptide-drug conjugate development.

Tuning Surface Charges of Peptide Nanofibers for Induction of Antigen-Specific Immune Tolerance: An Introductory Study.

Induction of antigen-specific immune tolerance has emerged as the next frontier in treating autoimmune disorders, including atherosclerosis and graft-vs-host reactions during transplantation. Nanostructures are under investigation as a platform for the coordinated delivery of critical components, i.e., the antigen epitope combined with tolerogenic agents, to the target immune cells and subsequently induce tolerance. In the present study, the utility of supramolecular peptide nanofibers to induce antigen-specific immune tolerance was explored. To study the influence of surface charges of the nanofibers towards the extent of the induced immune response, the flanking charge residues at both ends of the amphipathic fibrillization peptide sequences were varied. Dexamethasone, an immunosuppressive glucocorticoid drug, and the ovalbumin-derived OVA323-339 peptide that binds to I-A(d) MHC Class II were covalently linked at either end of the peptide sequences. It was shown that the functional extensions did not alter the structural integrity of the supramolecular nanofibers. Furthermore, the surface charges of the nanofibers were modulated by the inclusion of charged residues. Dendritic cell culture assays suggested that nanofiber of less negative ζ-potential can augment the antigen-specific tolerogenic response. Our findings illustrate a molecular approach to calibrate the tolerogenic response induced by peptide nanofibers, which pave the way for better design of future tolerogenic immunotherapies.

Retinoic Acid-Containing Liposomes for the Induction of Antigen-Specific Regulatory T Cells as a Treatment for Autoimmune Diseases.

The current treatment of autoimmune and chronic inflammatory diseases entails systemic immune suppression, which is associated with increased susceptibility to infections. To restore immune tolerance and reduce systemic side effects, a targeted approach using tolerogenic dendritic cells (tolDCs) is being explored. tolDCs are characterized by the expression of CD11c, the major histocompatibility complex (MHC)II and low levels of co-stimulatory molecules CD40 and CD86. In this study, tolDCs were generated using a human-proteoglycan-derived peptide (hPG) and all-trans retinoic acid (RA). RA-tolDCs not only display a tolerogenic phenotype but also can induce an antigen-specific regulatory T cell (Treg) response in vitro. However, further analysis showed that RA-tolDCs make up a heterogeneous population of DCs, with only a small proportion being antigen-associated tolDCs. To increase the homogeneity of this population, 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG)-containing liposomes were used to encapsulate the relevant antigen together with RA. These liposomes greatly enhanced the proportion of antigen-associated tolDCs in culture. In addition, in mice, we showed that the liposomal co-delivery of antigen and RA can be a more targeted approach to induce antigen-specific tolerance compared to the injection of RA-tolDCs, and that these liposomes can stimulate the generation of antigen-specific Tregs. This work highlights the importance of the co-delivery of an antigen and immunomodulator to minimize off-target effects and systemic side effects and provides new insights in the use of RA for antigen-specific immunotherapy for autoimmune and chronic inflammatory diseases.

Preparation of mRNA Polyplexes with Post-conjugated Endosome-Disruptive Peptides.

Successful delivery of mRNA into the cytosol of professional antigen-presenting cells (APCs) poses one of the biggest challenges in developing effective mRNA vaccines to treat various cancers and viral infectious diseases. However, most polymeric mRNA delivery systems fail to transfect APCs. We have discovered that decoration of pH-sensitive endosome-disruptive GALA peptides on the surface of mRNA polyplexes leads to efficient targeting and transfection of APCs. GALA peptides not only enhance specific uptake in APCs through binding to sialic acid moieties, they also facilitate the endosomal escape of mRNA especially in dendritic cells (DCs). Here, we describe in detail the production of stabilized mRNA polyplexes post-conjugated with GALA peptides via copper-free click chemistry. Methods described here include the synthesis and purification of GALA peptides and its conjugation to mRNA polyplexes.

Optimization of Liposomes for Antigen Targeting to Splenic CD169+ Macrophages.

Despite promising progress in cancer vaccination, therapeutic effectiveness is often insufficient. Cancer vaccine effectiveness could be enhanced by targeting vaccine antigens to antigen-presenting cells, thereby increasing T-cell activation. CD169-expressing splenic macrophages efficiently capture particulate antigens from the blood and transfer these antigens to dendritic cells for the activation of CD8+ T cells. In this study, we incorporated a physiological ligand for CD169, the ganglioside GM3, into liposomes to enhance liposome uptake by CD169+ macrophages. We assessed how variation in the amount of GM3, surface-attached PEG and liposomal size affected the binding to, and uptake by, CD169+ macrophages in vitro and in vivo. As a proof of concept, we prepared GM3-targeted liposomes containing a long synthetic ovalbumin peptide and tested the capacity of these liposomes to induce CD8+ and CD4+ T-cell responses compared to control liposomes or soluble peptide. The data indicate that the delivery of liposomes to splenic CD169+ macrophages can be optimized by the selection of liposomal constituents and liposomal size. Moreover, optimized GM3-mediated liposomal targeting to CD169+ macrophages induces potent immune responses and therefore presents as an interesting delivery strategy for cancer vaccination.

Control over the fibrillization yield by varying the oligomeric nucleation propensities of self-assembling peptides.

Self-assembling peptides are an exemplary class of supramolecular biomaterials of broad biomedical utility. Mechanistic studies on the peptide self-assembly demonstrated the importance of the oligomeric intermediates towards the properties of the supramolecular biomaterials being formed. In this study, we demonstrate how the overall yield of the supramolecular assemblies are moderated through subtle molecular changes in the peptide monomers. This strategy is exemplified with a set of surfactant-like peptides (SLPs) with different ß-sheet propensities and charged residues flanking the aggregation domains. By integrating different techniques, we show that these molecular changes can alter both the nucleation propensity of the oligomeric intermediates and the thermodynamic stability of the fibril structures. We demonstrate that the amount of assembled nanofibers are critically defined by the oligomeric nucleation propensities. Our findings offer guidance on designing self-assembling peptides for different biomedical applications, as well as insights into the role of protein gatekeeper sequences in preventing amyloidosis.

Cationic synthetic long peptides-loaded nanogels: An efficient therapeutic vaccine formulation for induction of T-cell responses.

Recent studies have shown a high potency of protein-based vaccines for cell-mediated cancer immunotherapy. However, due to their poor cellular uptake, efficient immune responses with soluble protein antigens are often not observed. As a result of superior cellular uptake, nanogels loaded with antigenic peptides were investigated in this study as carrier systems for cancer immunotherapy. Different synthetic long peptides (SLPs) containing the CTL and CD4+ T-helper (Help) epitopes were synthesized and covalently conjugated via disulfide bonds to the polymeric network of cationic dextran nanogels. Cationic nanogels with a size of 210 nm, positive zeta potential (+24 mV) and high peptide loading content (15%) showed triggered release of the loaded peptides under reducing conditions. An in vitro study demonstrated the capability of cationic nanogels to maturate dendritic cells (DCs). Importantly, covalently SLP-loaded nanogels adjuvanted with poly(I:C) showed superior CD8+ T cell responses compared to soluble peptides and nanogel formulations with physically loaded peptides both in vitro and in vivo. In conclusion, covalently SLPs-loaded cationic nanogels are a promising system to provoke immune responses for therapeutic cancer vaccination.

mRNA Polyplexes with Post-Conjugated GALA Peptides Efficiently Target, Transfect, and Activate Antigen Presenting Cells.

Vaccines based on mRNA have emerged as potent systems to elicit CD8+ T cell responses against various cancers and viral infectious diseases. The efficient intracellular delivery of mRNA molecules encoding antigens into the cytosol of antigen-presenting cells (APCs) is still challenging, requiring cell attachment, active uptake, and subsequent endosomal escape. Here, we report a facile approach for the formulation of peptide-functionalized mRNA polyplexes using copper-free click chemistry to promote presentation of mRNA antigen by dendritic cells (DCs). After screening different membrane active peptides, GALA modified mRNA polyplexes (PPx-GALA) with a size around 350 nm and with a slightly negative surface charge (-7 mV), exhibited the highest EGFP-mRNA transfection in RAW 246.7 macrophages (∼36%) and D1 dendritic cells (∼50%) as compared to polyplexes decorated with melittin or LEDE peptides. Interestingly, we found that PPx-GALA enters DCs through sialic acid mediated endo/phagocytosis, which was not influenced by DC maturation. The PPx-GALA formulation exhibited 18-fold higher cellular uptake compared to a lipofectamine mRNA formulation without inducing cytotoxicity. Live cell imaging showed that PPx-GALA that were taken up by endocytosis induced calcein release from endosomes into the cytosol. DCs treated with PPx-GALA containing mRNA encoding for OVA displayed enhanced T cell responses and DC maturation. Collectively, these data provide a strong rationale for further study of this PPx-GALA formulation in vivo as a promising mRNA vaccine platform.