Retinoic acid-loaded liposomes induce human mucosal CD103+ dendritic cells that inhibit Th17 cells and drive regulatory T-cell development in vitro.

The active vitamin A metabolite, all-trans-retinoic acid (RA), primes precursor dendritic cells (DCs) into a mucosal phenotype with tolerogenic properties characterized by the expression of integrin CD103. CD103+ DCs can counteract pathogenic Th1 and Th17 in inflammatory bowel disease (IBD) or celiac disease (CD). Tolerogenic manipulation of DCs using nanoparticles carrying tolerogenic adjuvants and disease-specific antigens is a valuable treatment strategy to induce antigen-specific mucosal tolerance in vivo. Here, we investigated the effects of RA-loaded liposomes on human DC phenotype and function, including DC-driven T-cell development, both during the generation of monocyte-derived DCs (moDCs) as well as by priming immature moDCs. RA liposomes drove CD103+ DC differentiation as well as ALDH1A2 expression in DCs. Neutrophil-dependent Th17 cell development was reduced by RA-liposome-differentiated and RA-liposome-primed DCs. Moreover, RA liposome treatment shifted T-cell development toward a Th2 cell profile. Importantly, RA liposomes induced the development of IL-10-producing and FoxP3+ regulatory T cells (Tregs) of various Treg subsets, including ICOS+ Tregs, that were potent inhibitors of bystander memory T-cell proliferation. Taken together, RA-loaded liposomes could be a novel treatment avenue for IBD or CD patients.

Intradermal Vaccination with PLGA Nanoparticles via Dissolving Microneedles and Classical Injection Needles.

PURPOSE: A dissolving microneedle array (dMNA) is a vaccine delivery device with several advantages over conventional needles. By incorporating particulate adjuvants in the form of poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) into the dMNA, the immune response against the antigen might be enhanced. This study aimed to prepare PLGA-NP-loaded dMNA and to compare T-cell responses induced by either intradermally injected aqueous-PLGA-NP formulation or PLGA-NP-loaded dMNA in mice. METHODS: PLGA NPs were prepared with microfluidics, and their physicochemical characteristics with regard to encapsulation efficiencies of ovalbumin (OVA) and CpG oligonucleotide (CpG), zeta potentials, polydispersity indexes, and sizes were analysed. PLGA NPs incorporated dMNA was produced with three different dMNA formulations by using the centrifugation method, and the integrity of PLGA NPs in dMNAs was evaluated. The immunogenicity was evaluated in mice by comparing the T-cell responses induced by dMNA and aqueous formulations containing ovalbumin and CpG (OVA/CpG) with and without PLGA NP. RESULTS: Prepared PLGA NPs had a size of around 100 nm. The dMNA formulations affected the particle integrity, and the dMNA with poly(vinyl alcohol) (PVA) showed almost no aggregation of PLGA NPs. The PLGA:PVA weight ratio of 1:9 resulted in 100% of penetration efficiency and the fastest dissolution in ex-vivo human skin (< 30 min). The aqueous formulation with soluble OVA/CpG and the aqueous-PLGA-NP formulation with OVA/CpG induced the highest CD4 + T-cell responses in blood and spleen cells. CONCLUSIONS: PLGA NPs incorporated dMNA was successfully fabricated and the aqueous formulation containing PLGA NPs induce superior CD4+ and CD8+ T-cell responses.

Tuning the Cross-Linking Density and Cross-Linker in Core Cross-Linked Polymeric Micelles and Its Effects on the Particle Stability in Human Blood Plasma and Mice.

Core cross-linked polymeric micelles (CCPMs) are designed to improve the therapeutic profile of hydrophobic drugs, reduce or completely avoid protein corona formation, and offer prolonged circulation times, a prerequisite for passive or active targeting. In this study, we tuned the CCPM stability by using bifunctional or trifunctional cross-linkers and varying the cross-linkable polymer block length. For CCPMs, amphiphilic thiol-reactive polypept(o)ides of polysarcosine-block-poly(S-ethylsulfonyl-l-cysteine) [pSar-b-pCys(SO2Et)] were employed. While the pCys(SO2Et) chain lengths varied from Xn = 17 to 30, bivalent (derivatives of dihydrolipoic acid) and trivalent (sarcosine/cysteine pentapeptide) cross-linkers have been applied. Asymmetrical flow field-flow fraction (AF4) displayed the absence of aggregates in human plasma, yet for non-cross-linked PM and CCPMs cross-linked with dihydrolipoic acid at [pCys(SO2Et)]17, increasing the cross-linking density or the pCys(SO2Et) chain lengths led to stable CCPMs. Interestingly, circulation time and biodistribution in mice of non-cross-linked and bivalently cross-linked CCPMs are comparable, while the trivalent peptide cross-linkers enhance the circulation half-life from 11 to 19 h.

Biological recognition and cellular trafficking of targeted RNA-lipid nanoparticles.

Lipid nanoparticles (LNPs) have unlocked the potential of ribonucleic acid (RNA) therapeutics and vaccines. Production and large-scale manufacturing methods for RNA-LNPs have been established and rapidly accelerate. Despite this, basic research on LNPs is still required, due to their high assembly complexity and fairly new development, including research on lipid organization, transfection optimization, and in vivo behavior. Understanding fundamental aspects of LNPs that is, how lipid composition and physicochemical properties affect their biodistribution, cell recognition, and transfection, could propel their clinical development and facilitate overcoming current challenges. Herein, we review recent developments in the field of LNP technology and summarize the main findings focusing on nano-bio interactions.

Lipid nanoparticle-based mRNA candidates elicit potent T cell responses.

The induction of a potent T cell response is essential for successful tumor immunotherapy and protection against many infectious diseases. In the past few years, mRNA vaccines have emerged as potent immune activators and inducers of a robust T cell immune response. The recent approval of the Moderna and the Pfizer/BioNTech vaccines based on lipid nanoparticles (LNP) encapsulating antigen-encoding mRNA has revolutionized the field of vaccines. The advantages of LNPs are their ease of design and formulation resulting in potent, effective, and safe vaccines. However, there is still plenty of room for improvement with respect to LNP efficacy, for instance, by optimizing the lipid composition and tuning LNP for specific purposes. mRNA delivery is known to be strongly dependent on the lipid composition of LNPs and the efficiency is mainly determined by the ionizable lipids. Besides that, cholesterol and helper lipids also play important roles in mRNA transfection potency. Here, a panel of LNP formulations was studied by keeping the ionizable lipids constant, replacing cholesterol with ß-sitosterol, and changing the fusogenic helper lipid DOPE content. We studied the ability of this LNP library to induce antigen presentation and T cell proliferation to identify superior LNP candidates eliciting potent T cell immune responses. We hypothesize that using ß-sitosterol and increasing DOPE content would boost the mRNA transfection on immune cells and result in enhanced immune responses. Transfection of immortal immune cell lines and bone marrow dendritic cells (BMDCs) with LNPs was studied. Delivery of mRNA coding for the model antigen ovalbumin (OVA-mRNA) to BMDCs with a number of LNP formulations, resulted in a high level of activation, as evidenced by the upregulation of the co-stimulatory receptors (CD40 and CD86) and IL-12 in BMDCs. The enhancement of BMDC activation and T cell proliferation induced by the introduction of ß-sitosterol and fusogenic DOPE lipids were cell dependent. Four LNP formulations (C12-200-cho-10%DOPE, C12-200-sito-10%DOPE, cKK-E12-cho-10%DOPE and cKK-E12-sito-30%DOPE) were identified that induced robust T cell proliferation and enhanced IFN-γ, TNF-α, IL-2 expression. These results demonstrate that T cell proliferation is strongly dependent on LNP composition and promising LNP-mRNA vaccine formulations were identified.

Liposomes loaded with vitamin D3 induce regulatory circuits in human dendritic cells.

Introduction: Nanomedicine provides a promising platform for manipulating dendritic cells (DCs) and the ensuing adaptive immune response. For the induction of regulatory responses, DCs can be targeted in vivo with nanoparticles incorporating tolerogenic adjuvants and auto-antigens or allergens. Methods: Here, we investigated the tolerogenic effect of different liposome formulations loaded with vitamin D3 (VD3). We extensively phenotyped monocyte-derived DCs (moDCs) and skin DCs and assessed DC-induced regulatory CD4+ T cells in coculture. Results: Liposomal VD3 primed-moDCs induced the development of regulatory CD4+ T cells (Tregs) that inhibited bystander memory T cell proliferation. Induced Tregs were of the FoxP3+ CD127low phenotype, also expressing TIGIT. Additionally, liposome-VD3 primed moDCs inhibited the development of T helper 1 (Th1) and T helper 17 (Th17) cells. Skin injection of VD3 liposomes selectively stimulated the migration of CD14+ skin DCs. Discussion: These results suggest that nanoparticulate VD3 is a tolerogenic tool for DC-mediated induction of regulatory T cell responses.

The Use of a Staggered Herringbone Micromixer for the Preparation of Rigid Liposomal Formulations Allows Efficient Encapsulation of Antigen and Adjuvant.

Anionic liposomal formulations have previously shown to have intrinsic tolerogenic capacity and these properties have been related to the rigidity of the particles. The combination of highly rigid anionic liposomes to deliver tolerogenic adjuvants and antigen peptides has potential applications for the treatment of autoimmune and inflammatory diseases. However, the preparation of these highly rigid anionic liposomes using traditional methods such as lipid film hydration presents problems in terms of scalability and loading efficiency of some costly tolerogenic adjuvants like 1-α,25-dihydroxyvitaminD3. Here we propose the use of an off-the-shelf staggered herringbone micromixer for the preparation of these formulations and performed a systematic study on the effect of temperature and flow conditions on the size and polydispersity index of the formulations. Furthermore, we show that the system allows for the encapsulation of a wide variety of peptides and significantly higher loading efficiency of 1-α,25-dihydroxyvitaminD3 compared to the traditional lipid film hydration method, without compromising their non-inflammatory interaction with dendritic cells. Therefore, the microfluidics method presented here is a valuable tool for the preparation of highly rigid tolerogenic liposomes in a fast, size-tuneable and scalable manner.

Uptake Kinetics Of Liposomal Formulations of Differing Charge Influences Development of in Vivo Dendritic Cell Immunotherapy.

Dendritic cells (DCs) control adaptive immunity and are therefore attractive for in vivo targeting to either induce immune activation or tolerance, depending on disease. Liposomes, nanoparticles comprised of a lipid bi-layer, provide a nanoplatform for loading disease-relevant antigen, adjuvant and DC-targeting molecules simultaneously. However, it is yet not fully understood how liposomal formulations affect uptake by DCs and DC function. Here, we examined monocyte-derived DC (moDC) and skin DC uptake of six different liposomal formulations, together with their DC-modulating effect. Contrary to literature, we show using imaging flow cytometry that anionic or neutral liposomes are taken up more efficiently than cationic liposomes by moDCs, or by skin DCs after intradermal injection. None of the formulations yielded significant modulation of DC function as determined by the upregulation of maturation markers and cytokine production. These results suggest that anionic liposomes would be more suitable as vaccine carriers for a dermal application.

Structural Fine-Tuning of Desmuramylpeptide NOD2 Agonists Defines Their In Vivo Adjuvant Activity.

We report on the design, synthesis, and biological evaluation of a series of nucleotide-binding oligomerization-domain-containing protein 2 (NOD2) desmuramylpeptide agonists with improved in vitro and in vivo adjuvant properties. We identified two promising compounds: 68, a potent nanomolar in vitro NOD2 agonist, and the more lipophilic 75, which shows superior adjuvant activity in vivo. Both compounds had immunostimulatory effects on peripheral blood mononuclear cells at the protein and transcriptional levels, and augmented dendritic-cell-mediated activation of T cells, while 75 additionally enhanced the cytotoxic activity of peripheral blood mononuclear cells against malignant cells. The C18 lipophilic tail of 75 is identified as a pivotal structural element that confers in vivo adjuvant activity in conjunction with a liposomal delivery system. Accordingly, liposome-encapsulated 75 showed promising adjuvant activity in mice, surpassing that of muramyl dipeptide, while achieving a more balanced Th1/Th2 immune response, thus highlighting its potential as a vaccine adjuvant.

Complement Receptor Targeted Liposomes Encapsulating the Liver X Receptor Agonist GW3965 Accumulate in and Stabilize Atherosclerotic Plaques.

Atherosclerosis is characterized by the retention of lipids in foam cells in the arterial intima. The liver X receptor (LXR) agonist GW3965 is a promising therapeutic compound, since it induces reverse cholesterol transport in foam cells. However, hepatic LXR activation increases plasma and liver lipid levels, inhibiting its clinical development. Herein, a formulation that specifically enhances GW3965 deposition in the atherosclerotic lesion is aimed to be developed. GW3965 is encapsulated in liposomes functionalized with the cyclic peptide Lyp-1 (CGNKRTRGC), which binds the p32 receptor expressed on foam cells. These liposomes show preferential uptake by foam cells in vitro and higher accumulation in atherosclerotic plaques in mice compared to non-targeted liposomes as determined by in vivo imaging. Flow cytometry analysis of plaques reveals increased retention of Lyp-1 liposomes in atherosclerotic plaque macrophages compared to controls (p < 0.05). Long term treatment of established plaques in LDLR -/- mice with GW3965-containing Lyp-1 liposomes significantly reduces plaque macrophage content by 50% (p < 0.01). Importantly, GW3965-containing Lyp-1 liposomes do not increase plasma or hepatic lipid content. Thus, GW3965-containing Lyp-1 liposomes successfully target the atherosclerotic macrophages allowing plaque stabilization without commonly observed side effects of LXR agonists.

Atomic force microscopy measurements of anionic liposomes reveal the effect of liposomal rigidity on antigen-specific regulatory T cell responses.

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 OVA323-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.

Anionic 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG) liposomes induce antigen-specific regulatory T cells and prevent atherosclerosis in mice.

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.

Development of PLGA nanoparticle loaded dissolving microneedles and comparison with hollow microneedles in intradermal vaccine delivery.

Skin is an attractive but also very challenging immunisation site for particulate subunit vaccines. The aim of this study was to develop hyaluronan (HA)-based dissolving microneedles (MNs) loaded with PLGA nanoparticles (NPs) co-encapsulating ovalbumin (OVA) and poly(I:C) for intradermal immunisation. The NP:HA ratio used for the preparation of dissolving MNs appeared to be critical for the quality of MNs and their dissolution in ex vivo human skin. Asymmetrical flow field-flow fractionation and dynamic light scattering were used to analyse the NPs released from the MNs in vitro. Successful release of the NPs depended on the drying conditions during MN preparation. The delivered antigen dose from dissolving MNs in mice was determined to be 1 µg OVA, in NPs or as free antigen, by using near-infrared fluorescence imaging. Finally, the immunogenicity of the NPs after administration of dissolving MNs (NP:HA weight ratio 1:4) was compared with that of hollow MN-delivered NPs in mice. Immunization with free antigen in dissolving MNs resulted in equally strong immune responses compared to delivery by hollow MNs. However, humoral and cellular immune responses evoked by NP-loaded dissolving MNs were inferior to those elicited by NPs delivered through a hollow MN. In conclusion, we identified several critical formulation parameters for the further development of NP-loaded dissolving MNs.

Intradermal vaccination with hollow microneedles: A comparative study of various protein antigen and adjuvant encapsulated nanoparticles.

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.

Characterization of Inner and Outer Membrane Proteins from Francisella tularensis Strains LVS and Schu S4 and Identification of Potential Subunit Vaccine Candidates.

Francisella tularensis is the causative agent of tularemia and a potential bioterrorism agent. In the present study, we isolated, identified, and quantified the proteins present in the membranes of the virulent type A strain, Schu S4, and the attenuated type B strain, LVS (live vaccine strain). Spectral counting of mass spectrometric data showed enrichment for membrane proteins in both strains. Mice vaccinated with whole LVS membranes encapsulated in poly (lactic-co-glycolic acid) (PLGA) nanoparticles containing the adjuvant polyinosinic-polycytidylic acid [poly(I·C)] showed significant protection against a challenge with LVS compared to the results seen with naive mice or mice vaccinated with either membranes or poly(I·C) alone. The PLGA-encapsulated Schu S4 membranes with poly(I·C) alone did not significantly protect mice from a lethal intraperitoneal challenge with Schu S4; however, this vaccination strategy provided protection from LVS challenge. Mice that received the encapsulated Schu S4 membranes followed by a booster of LVS bacteria showed significant protection with respect to a lethal Schu S4 challenge compared to control mice. Western blot analyses of the sera from the Schu S4-vaccinated mice that received an LVS booster showed four immunoreactive bands. One of these bands from the corresponding one-dimensional (1D) SDS-PAGE experiment represented capsule. The remaining bands were excised, digested with trypsin, and analyzed using mass spectrometry. The most abundant proteins present in these immunoreactive samples were an outer membrane OmpA-like protein, FopA; the type IV pilus fiber building block protein; a hypothetical membrane protein; and lipoproteins LpnA and Lpp3. These proteins should serve as potential targets for future recombinant protein vaccination studies.IMPORTANCE The low infectious dose, the high potential mortality/morbidity rates, and the ability to be disseminated as an aerosol make Francisella tularensis a potential agent for bioterrorism. These characteristics led the Centers for Disease Control (CDC) to classify F. tularensis as a Tier 1 pathogen. Currently, there is no vaccine approved for general use in the United States.

Adjuvant effect of cationic liposomes and CpG depends on administration route.

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.

PLGA, PLGA-TMC and TMC-TPP nanoparticles differentially modulate the outcome of nasal vaccination by inducing tolerance or enhancing humoral immunity.

Development of vaccines in autoimmune diseases has received wide attention over the last decade. However, many vaccines showed limited clinical efficacy. To enhance vaccine efficacy in infectious diseases, biocompatible and biodegradable polymeric nanoparticles have gained interest as antigen delivery systems. We investigated in mice whether antigen-encapsulated PLGA (poly-lactic-co-glycolic acid), PLGA-TMC (N-trimethyl chitosan) or TMC-TPP (tri-polyphosphate) nanoparticles can also be used to modulate the immunological outcome after nasal vaccination. These three nanoparticles enhanced the antigen presentation by dendritic cells, as shown by increased in vitro and in vivo CD4(+) T-cell proliferation. However, only nasal PLGA nanoparticles were found to induce an immunoregulatory response as shown by enhanced Foxp3 expression in the nasopharynx associated lymphoid tissue and cervical lymph nodes. Nasal administration of OVA-containing PLGA particle resulted in functional suppression of an OVA-specific Th-1 mediated delayed-type hypersensitivity reaction, while TMC-TPP nanoparticles induced humoral immunity, which coincided with the enhanced generation of OVA-specific B-cells in the cervical lymph nodes. Intranasal treatment with Hsp70-mB29a peptide-loaded PLGA nanoparticles suppressed proteoglycan-induced arthritis, leading to a significant reduction of disease. We have uncovered a role for PLGA nanoparticles to enhance CD4(+) T-cell mediated immunomodulation after nasal application. The exploitation of this differential regulation of nanoparticles to modulate nasal immune responses can lead to innovative vaccine development for prophylactic or therapeutic vaccination in infectious or autoimmune diseases.

Administration routes affect the quality of immune responses: A cross-sectional evaluation of particulate antigen-delivery systems.

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.

Nasal vaccination with N-trimethyl chitosan and PLGA based nanoparticles: nanoparticle characteristics determine quality and strength of the antibody response in mice against the encapsulated antigen.

Nasal vaccination is a promising, needle-free alternative to classical vaccination. Nanoparticulate delivery systems have been reported to overcome the poor immunogenicity of nasally administered soluble antigens, but the characteristics of the ideal particle are unknown. This study correlates differences in physicochemical characteristics of nanoparticles to their adjuvant effect, using ovalbumin (OVA)-loaded poly(lactic-co-glycolic acid) nanoparticles (PLGA NP), N-trimethyl chitosan (TMC) based NP (TMC NP) and TMC-coated PLGA NP (PLGA/TMC NP). PLGA NP and PLGA/TMC NP were prepared by emulsification/solvent extraction and TMC NP by ionic complexation. The NP were characterized physicochemically. Their toxicity and interaction with and stimulation of monocyte derived dendritic cells (DC) were tested in vitro. Furthermore, the residence time and the immunogenicity (serum IgG titers and secretory IgA levels in nasal washes) of the nasally applied OVA formulations were assessed in Balb/c mice. All NP were similar in size, whereas only PLGA NP carried a negative zeta potential. The NP were non-toxic to isolated nasal epithelium. Only TMC NP increased the nasal residence time of OVA compared to OVA administered in PBS and induced DC maturation. After i.m. administration all NP systems induced higher IgG titers than OVA alone, PLGA NP and TMC NP being superior to PLGA/TMC NP. Nasal immunization with the slow antigen releasing particles, PLGA NP and PLGA/TMC NP, did not induce detectable antibody titers. In contrast, nasal immunization with the positively charged, fast antigen releasing TMC NP led to high serum antibody titers and sIgA levels. In conclusion, particle charge and antigen release pattern of OVA-loaded NP has to be adapted to the intended route of administration. For nasal vaccination, TMC NP, releasing their content within several hours, being mucoadhesive and stimulating the maturation of DC, were superior to PLGA NP and PLGA/TMC NP which lacked some or all of these characteristics.

Mechanistic study of the adjuvant effect of biodegradable nanoparticles in mucosal vaccination.

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.