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New and improved nanomaterials are constantly being developed for biomedical purposes. Nanomaterials based on elastin-like polypeptides (ELPs) have increasingly shown potential over the past two decades. These polymers are artificial proteins of which the design is based on human tropoelastin. Due to this similarity, ELP-based nanomaterials are biodegradable and therefore well suited to drug delivery. The assembly of ELP molecules into nanoparticles spontaneously occurs at temperatures above a transition temperature (T t ). The ELP sequence influences both the T t and the physicochemical properties of the assembled nanomaterial. Nanoparticles with desired properties can hence be designed by choosing the appropriate sequence. A promising class of ELP nanoparticles are micelles assembled from amphiphilic ELP diblock copolymers. Such micelles are generally uniform and well defined. Furthermore, site-specific attachment of cargo to the hydrophobic block results in micelles with the cargo shielded inside their core, while conjugation to the hydrophilic block causes the cargo to reside in the corona where it is available for interactions. Such control over particle design is one of the main contributing factors for the potential of ELP-based micelles as a drug delivery system. Additionally, the micelles are easily loaded with protein or peptide-based cargo by expressing it as a fusion protein. Small molecule drugs and other cargo types can be either covalently conjugated to ELP domains or physically entrapped inside the micelle core. This review aims to give an overview of ELP-based micelles and their applications in nanomedicine., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Regulatory T cells (Tregs) are vital for maintaining a balanced immune response and their dysfunction is often associated with auto-immune disorders. We have previously shown that antigen-loaded anionic liposomes composed of phosphatidylcholine (PC) and phosphatidylglycerol (PG) and cholesterol can induce strong antigen-specific Treg responses. We hypothesized that altering the rigidity of these liposomes while maintaining their size and surface charge would affect their capability of inducing Treg responses. The rigidity of liposomes is affected in part by the length and saturation of carbon chains of the phospholipids in the bilayer, and in part by the presence of cholesterol. We used atomic force microscopy (AFM) to measure the rigidity of anionic OVA 323 -containing liposomes composed of different types of PC and PG, with or without cholesterol, in a molar ratio of 4:1(:2) distearoyl (DS)PC:DSPG (Young's modulus (YM) 3611 ± 1271 kPa), DSPC:DSPG:CHOL (1498 ± 531 kPa), DSPC:dipalmitoyl (DP)PG:CHOL (1208 ± 538), DPPC:DPPG:CHOL (1195 ± 348 kPa), DSPC:dioleoyl (DO)PG:CHOL (825 ± 307 kPa), DOPC:DOPG:CHOL (911 ± 447 kPa), and DOPC:DOPG (494 ± 365 kPa). Next, we assessed if rigidity affects the association of liposomes to bone marrow-derived dendritic cells (BMDCs) in vitro. Aside from DOPC:DOPG liposomes, we observed a positive correlation between liposomal rigidity and cellular association. Finally, we show that rigidity positively correlates with Treg responses in vitro in murine DCs and in vivo in mice. Our findings underline the suitability of AFM to measure liposome rigidity and the importance of this parameter when designing liposomes as a vaccine delivery system., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)

Atherosclerosis is the predominant underlying pathology of many types of cardiovascular disease and is one of the leading causes of death worldwide. It is characterized by the retention of oxidized low-density lipoprotein (ox-LDL) in lipid-rich macrophages (foam cells) in the intima of arteries. Autoantigens derived from oxLDL can be used to vaccinate against atherosclerosis. However, a major challenge is the induction of antigen-specific Tregs in a safe and effective way. Here we report that liposomes containing the anionic phospholipid 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG) induce Tregs that are specific for the liposomes' cargo. Mechanistically, we show a crucial role for the protein corona that forms on the liposomes in the circulation, as uptake of DSPG-liposomes by antigen-presenting cells is mediated via complement component 1q (C1q) and scavenger receptors (SRs). Vaccination of atherosclerotic mice on a western-type diet with DSPG-liposomes encapsulating an LDL-derived peptide antigen significantly reduced plaque formation by 50% and stabilized the plaques, and reduced serum cholesterol concentrations. These results indicate that DSPG-liposomes have potential as a delivery system in vaccination against atherosclerosis., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

In this study, we investigated the potential of intradermal delivery of nanoparticulate vaccines to modulate the immune response of protein antigen using hollow microneedles. Four types of nanoparticles covering a broad range of physiochemical parameters, namely poly (lactic-co-glycolic) (PLGA) nanoparticles, liposomes, mesoporous silica nanoparticles (MSNs) and gelatin nanoparticles (GNPs) were compared. The developed nanoparticles were loaded with a model antigen (ovalbumin (OVA)) with and without an adjuvant (poly(I:C)), followed by the characterization of size, zeta potential, morphology, and loading and release of antigen and adjuvant. An in-house developed hollow-microneedle applicator was used to inject nanoparticle suspensions precisely into murine skin at a depth of about 120μm. OVA/poly(I:C)-loaded nanoparticles and OVA/poly(I:C) solution elicited similarly strong total IgG and IgG1 responses. However, the co-encapsulation of OVA and poly(I:C) in nanoparticles significantly increased the IgG2a response compared to OVA/poly(I:C) solution. PLGA nanoparticles and liposomes induced stronger IgG2a responses than MSNs and GNPs, correlating with sustained release of the antigen and adjuvant and a smaller nanoparticle size. When examining cellular responses, the highest CD8 + and CD4 + T cell responses were induced by OVA/poly(I:C)-loaded liposomes. In conclusion, the applicator controlled hollow microneedle delivery is an excellent method for intradermal injection of nanoparticle vaccines, allowing selection of optimal nanoparticle formulations for humoral and cellular immune responses., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Particulate carrier systems are promising drug delivery vehicles for subunit vaccination as they can enhance and direct the type of T cell response. In order to develop vaccines with optimal immunogenicity, a thorough understanding of parameters that could affect the strength and quality of immune responses is required. Pathogens have different dimensions and stimulate the immune system in a specific way. It is therefore not surprising that physicochemical characteristics of particulate vaccines, such as particle size, shape, and rigidity, affect multiple processes that impact their immunogenicity. Among these processes are the uptake of the particles from the site of administration, passage through lymphoid tissue and the uptake, antigen processing and activation of antigen-presenting cells. Herein, we systematically review the role of the size, shape and rigidity of particulate vaccines in enhancing and skewing T cell response and attempted to provide a "roadmap" for rational vaccine design., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The ideal vaccine is a simple and stable formulation which can be conveniently administered and provides life-long immunity against a given pathogen. The development of such a vaccine, which should trigger broad and strong B-cell and T-cell responses against antigens of the pathogen in question, is highly dependent on tailored vaccine delivery approaches. This review addresses vaccine delivery in its broadest scope. We discuss the needs and challenges in the area of vaccine delivery, including restrictions posed by specific target populations, potentials of dedicated stable formulations and devices, and the use of adjuvants. Moreover, we address the current status and perspectives of vaccine delivery via several routes of administration, including non- or minimally invasive routes. Finally we suggest possible directions for future vaccine delivery research and development., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The physical stability of polyelectrolyte nanocomplexes composed of trimethyl chitosan (TMC) and hyaluronic acid (HA) is limited in physiological conditions. This may minimize the favorable adjuvant effects associated with particulate systems for nasal and intradermal immunization. Therefore, covalently stabilized nanoparticles loaded with ovalbumin (OVA) were prepared with thiolated TMC and thiolated HA via ionic gelation followed by spontaneous disulfide formation after incubation at pH 7.4 and 37°C. Also, maleimide PEG was coupled to the remaining thiol-moieties on the particles to shield their surface charge. OVA-loaded TMC/HA nanoparticles had a size of around 250-350nm, a positive zeta potential and OVA loading efficiencies up to 60%. Reacting the thiolated particles with maleimide PEG resulted in a slight reduction of zeta potential (from +7 to +4mV) and a minor increase in particle size. Stabilized TMC-S-S-HA particles (PEGylated or not) showed superior stability in saline solutions compared to non-stabilized particles (composed of nonthiolated polymers) but readily disintegrated upon incubation in a saline buffer containing 10mM dithiothreitol. In both the nasal and intradermal immunization study, OVA loaded stabilized TMC-S-S-HA particles demonstrated superior immunogenicity compared to non-stabilized particles (indicated by higher IgG titers). Intranasal, PEGylation completely abolished the beneficial effects of stabilization and it induced no enhanced immune responses against OVA after intradermal administration. In conclusion, stabilization of the TMC/HA particulate system greatly enhances the immunogenicity of OVA in nasal and intradermal vaccination., (Copyright © 2011. Published by Elsevier B.V.)

In this study we explored the immunization route-dependent adjuvanticity of cationic liposomes loaded with an antigen (ovalbumin; OVA) and an immune potentiator (CpG). Mice were immunized intranodally, intradermally, transcutaneously (with microneedle pre-treatment) and nasally with liposomal OVA/CpG or OVA/CpG solution. In vitro, OVA/CpG liposomes showed enhanced uptake by DCs of both OVA and CpG compared to OVA+CpG solution. A similar enhanced uptake by DCs was observed in vivo when fluorescent OVA/CpG liposomes were administered intranodally. However, after transcutaneous and nasal application a lower uptake of OVA/CpG liposomes compared to an OVA+CpG solution was observed. Moreover, the IgG titers after nasal and transcutaneous administration of OVA/CpG liposomes were reduced compared to administration of an OVA+CpG solution. Although serum IgG titers may suggest limited added value of liposomes to the immunogenicity, for all routes, OVA/CpG liposomes resulted in elevated IgG2a levels, whereas administration of OVA+CpG solutions did not. These data show that encapsulation of antigen and adjuvant into a cationic liposome has a beneficial effect on the quality of the antibody response in mice after intranodal or intradermal immunization, but impairs proper delivery of antigen and adjuvant to the lymph nodes when the formulations are administered transcutaneously or nasally., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Nasal vaccination is a promising, but challenging vaccination strategy. Poor absorption by the nasal epithelium and failure to break nasal tolerance are regarded as important reasons for poor efficacy of nasally applied vaccines. Formulation of the antigen into mucoadhesive nanoparticles, made of N-trimethyl chitosan (TMC) crosslinked with tripolyphosphate (TPP), has been shown to overcome these obstacles. However, although nasally administered antigen loaded TMC/TPP nanoparticles induce a strong humoral response, antibody subtyping indicates a Th2 bias. To design a nasal antigen delivery system capable of inducing stronger Th1 type responses, TPP as a crosslinking agent was replaced by unmethylated CpG DNA, a TLR-9 ligand and a potent inducer of Th1 responses, to prepare ovalbumin (OVA) loaded TMC nanoparticles (TMC/CpG/OVA). Several physicochemical characteristics of TMC/CpG/OVA (size, zetapotential, loading efficiency and antigen release profile) were assessed and compared to TMC nanoparticles prepared by crosslinking with TPP (TMC/TPP/OVA). Mice were nasally administered TMC/TPP/OVA and TMC/CpG/OVA after which antibody responses in serum and nasal washes were assessed and T-cell activation in the spleens determined. TMC/CpG/OVA showed similar physical properties as TMC/TPP/OVA in terms of particle size (380 nm), zetapotential (+21 mV) and antigen release characteristics. Nasal administration of TMC/CpG/OVA and TMC/TPP/OVA to mice resulted in comparable serum IgG levels (ca. 1000 fold higher than those induced by unadjuvanted OVA) and local secretory IgA levels. Moreover, TMC/CpG/OVA induced a 10 fold higher IgG2a response than TMC/TPP/OVA and enhanced the number of OVA specific IFN-gamma-producing T-cells in the spleen. In conclusion, OVA loaded TMC nanoparticles, containing CpG as adjuvant and crosslinker, are capable of provoking strong humoral as well as Th1 type cellular immune responses after nasal vaccination., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Particulate delivery systems such as liposomes and polymeric nano- and microparticles are attracting great interest for developing new vaccines. Materials and formulation properties essential for this purpose have been extensively studied, but relatively little is known about the influence of the administration route of such delivery systems on the type and strength of immune response elicited. Thus, the present study aimed at elucidating the influence on the immune response when of immunising mice by different routes, such as the subcutaneous, intradermal, intramuscular, and intralymphatic routes with ovalbumin-loaded liposomes, N-trimethyl chitosan (TMC) nanoparticles, and poly(lactide-co-glycolide) (PLGA) microparticles, all with and without specifically selected immune-response modifiers. The results showed that the route of administration caused only minor differences in inducing an antibody response of the IgG1 subclass, and any such differences were abolished upon booster immunisation with the various adjuvanted and non-adjuvanted delivery systems. In contrast, the administration route strongly affected both the kinetics and magnitude of the IgG2a response. A single intralymphatic administration of all evaluated delivery systems induced a robust IgG2a response, whereas subcutaneous administration failed to elicit a substantial IgG2a response even after boosting, except with the adjuvanted nanoparticles. The intradermal and intramuscular routes generated intermediate IgG2a titers. The benefit of the intralymphatic administration route for eliciting a Th1-type response was confirmed in terms of IFN-gamma production of isolated and re-stimulated splenocytes from animals previously immunised with adjuvanted and non-adjuvanted liposomes as well as with adjuvanted microparticles. Altogether the results show that the IgG2a associated with Th1-type immune responses are sensitive to the route of administration, whereas IgG1 response associated with Th2-type immune responses were relatively insensitive to the administration route of the particulate delivery systems. The route of administration should therefore be considered when planning and interpreting pre-clinical research or development on vaccine delivery systems., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The nose is a promising immunization site and intranasal (i.n.) vaccination studies with whole inactivated influenza virus (WIV) adjuvanted with N,N,N-trimethylchitosan (TMC-WIV) have shown promising results. In this study, the influence of TMC on the i.n. delivery of WIV was studied in mice by comparing the nasal residence time and the specific location in the nasal cavity of WIV and TMC-WIV. Additionally, the local toxicity profile of the WIV formulations was assessed. In vivo fluorescence imaging was used to study the nasal residence time and the fate of the bulk vaccine in mice that received vaccines fluorescently labeled with IRDye800CW. An immunohistochemical (IHC) staining method for nasal cross-sections was developed to visualize the antigen in the nasal cavity. Therefore, mice were sacrificed at different time points after vaccination with various vaccine formulations and nasal cross-sections were made. The local toxicity was assessed using hematoxylin and eosin staining for the nasal cross-sections. No significant differences in the nasal residence time between WIV and TMC-WIV were observed. However, IHC revealed a striking difference in the location and distribution of WIV in the nasal cavity. When formulated as plain WIV, positive staining was mainly found in the nasal cavity, presumably in mucus blobs. TMC-coated WIV, on the other hand, was mostly present as a thin layer on the epithelial surfaces of the naso- and maxilloturbinates. This difference in staining pattern correlates with the observed differences in immunogenicity of these two vaccines and indicates that TMC-WIV results in a much closer interaction of WIV with the epithelial surfaces than WIV alone, potentially leading to enhanced uptake and induction of immune responses. This study further shows that both WIV and TMC-WIV formulations induce minimal local toxicity. Taken altogether, these results provide more insight in the mode of action and safety of TMC and justify further research to develop TMC-adjuvanted nasal vaccines., (Copyright 2010 Elsevier B.V. All rights reserved.)

Subunit vaccines are generally safer, but often less effective than live attenuated vaccines as they lack the necessary co-stimulatory factors. The formulation of an adjuvant like N-trimethyl chitosan (TMC) with an antigen can overcome its poor immunogenicity. Recent data suggest the importance of incorporating the antigen and the adjuvant into one entity for maximum immunostimulatory effect, e.g. by using (nano)particles. In the present paper we introduce the conjugation of an antigen, ovalbumin (OVA), to TMC as an alternative to nanoparticles for subunit vaccination. OVA was covalently linked to TMC using thiol chemistry (SPDP method). The uptake of the resulting TMC-OVA conjugate by dendritic cells (DC) and its effect on DC maturation was assessed in vitro and its immunogenicity was investigated in mice. We found that with the SPDP method a reducible covalent bond between TMC and OVA could be introduced, without disrupting the protein's antigenicity and structure. Uptake of TMC-OVA conjugate by dendritic cells was similar to the uptake of TMC/OVA nanoparticles, over 5-fold increased compared to a solution of OVA and TMC. Mice immunized with TMC-OVA conjugate produced 1000-fold higher OVA specific IgG titers than mice immunized with either OVA or a physical mixture of TMC and OVA. Moreover, these antibody titers were slightly elevated compared to the titers obtained with TMC/OVA nanoparticles. Conjugation of the antigen to an adjuvant is therefore a viable strategy to increase the immunogenicity of subunit vaccines and may provide an alternative to the use of particles., (Copyright 2010 Elsevier B.V. All rights reserved.)

The function of N-trimethyl chitosan (TMC) in dermal immunisation is unknown. Therefore we investigated the immunogenicity of both antigen-containing TMC nanoparticles and TMC/antigen solutions after intradermal injection. Nanoparticles were prepared with a size around 200 nm and a positive zetapotential. In vitro, TMC nanoparticles increased the uptake of OVA by dendritic cells (DCs) and both nanoparticles and TMC/OVA mixtures were able to induce upregulation of MHC-II, CD83 and CD86. These activated DCs could induce a Th2 biased T cell proliferation. A solution of plain OVA did not induce DC maturation or T cell proliferation. In vivo, mice were injected thrice with TMC based formulations containing either OVA or diphtheria toxoid (DT), a more relevant antigen. All TMC-containing formulations were able to increase the IgG titres compared to unadjuvanted antigen and induced a Th2 biased immune response. When using DT-containing TMC formulations, IgG titres and neutralising antibody titres could match up to those obtained after subcutaneous injection of DT-Alum. In conclusion, both soluble TMC/antigen mixtures and TMC nanoparticles are able to induce DC maturation and enhance immune responses after intradermal injection. This demonstrates that TMC functions as an immune potentiator for antigens delivered via the skin., ((c) 2009 Elsevier B.V. All rights reserved.)

For oral vaccination, incorporation of antigens into nanoparticles has been shown to protect the antigen from degradation, but may also increase its uptake through the intestinal epithelium via M-cells. The aim of this study was to understand the mechanisms by which oral administration of antigen-loaded nanoparticles induces an immune response and to analyze the effect of the nanoparticle composition on these mechanisms. Nanoparticles made from chitosan (CS) and its N-trimethylated derivative, TMC, loaded with a model antigen ovalbumin (OVA) were prepared by ionic gelation with tripolyphosphate. Intraduodenal vaccination with OVA-loaded nanoparticles led to significantly higher antibody responses than immunization with OVA alone. TMC nanoparticles induced anti-OVA antibodies after only a priming dose. To explain these results, the interaction of nanoparticles with the intestinal epithelium was explored, in vitro, using a follicle associated epithelium model and visualized, ex vivo, using confocal laser scanning microscopy. The transport of FITC-OVA-loaded TMC nanoparticles by Caco-2 cells or follicle associated epithelium model was higher than FITC-OVA-loaded CS or PLGA nanoparticles. The association of nanoparticles with human monocyte derived dendritic cells and their effect on their maturation were determined with flow cytometry. TMC nanoparticles but not CS or PLGA nanoparticles had intrinsic adjuvant effect on DCs. In conclusion, depending on their composition, nanoparticles can increase the M-cell dependent uptake and enhance the association of the antigen with DC. In this respect, TMC nanoparticles are a promising strategy for oral vaccination.