Preparation and evaluation of PEG-coated zein nanoparticles for oral drug delivery purposes.

The aim was to produce PEG-coated nanoparticles (NP-PEG), with mucus-permeating properties, for oral drug delivery purposes by using simple procedures and regulatory-approved compounds in order to facilitate a potential clinical development. For this purpose, zein nanoparticles were prepared by desolvation and, then, coated by incubation with PEG 35,000. The resulting nanocarriers displayed a mean size of about 200 nm and a negative zeta potential. The presence of PEG on the surface of nanoparticles was evidenced by electron microscopy and confirmed by FTIR analysis. Likely, the hydrophobic surface of zein nanoparticles (NP) was significantly reduce by their coating with PEG. This increase of the hydrophilicity of PEG-coated nanoparticles was associated with an important increase of their mobility in pig intestinal mucus. In laboratory animals, NP-PEG (fluorescently labelled with Lumogen® Red 305) displayed a different behavior when compared with bare nanoparticles. After oral administration, NP appeared to be trapped in the mucus mesh, whereas NP-PEG were capable of crossing the protective mucus layer and reach the epithelium. Finally, PEG-coated zein nanoparticles, prepared by a simple and reproducible method without employing reactive reagents, may be adequate carriers for promoting the oral bioavailability of biomacromolecules and other biologically active compounds with low permeability properties.

Preparation, radiolabeling with 99mTc and 67Ga and biodistribution studies of albumin nanoparticles covered with polymers. / Preparación, radiomarcaje con 99mTc y 67Ga y estudios de biodistribución de nanopartículas de albúmina con recubrimientos poliméricos.

OBJECTIVE: To optimize radiolabeling with 99mTc and 67Ga of albumin nanoparticles coated with 4 differents synthetic polymers and to evaluate their stability in vivo and in vitro, as well as their biodistribution in vivo after intravenous administration. MATERIAL AND METHODS: The nanoparticles were prepared using albumin and NOTA-modified albumin by the desolvation method and coated with 4 different polymers; HPMC, GMN2, GPM2 and GTM2. They were purified, lyophilized and characterized. Radiolabelling with 99mTc was perfomed with 74 MBq of 99mTc sodium pertechnetate, previously reduced with and acid solution of tin chloride at different concentrations (0.003, 0.005, 0.007, 0.01, 0.05 and 0.1mg/ml) and at different times (5, 10, 15, 30 and 60minutes) and temperatures (room temperature, 40°C and 60°C). Radiolabelling with 67Ga was perfomed by incubation of the nanoparticles with 37 MBq of 67Gallium chloride (obtained from commercial gallium-67 citrate) at different times (10 and 30minutes) and temperatures (room temperature, 30°C and 60°C), and posterior purification with microconcentrators. The radiochemical purity was evaluated by TLC. Stability studies of radiolabeled nanoparticles in physiological serum and blood plasma were perfomed. Biodistribution studies of nanoparticles coated with GPM2 polymer were carried out in Wistar rats after intravenous administration of the nanoparticles. Control animals were carried out with 99mTc sodium pertechnetate and 67Ga chloride. To do so, the animals were killed and activity in organs was measured in a gamma counter. RESULTS: 99mTc labeling was carried out optimally with a tin concentration of 0.007mg/ ml for the GPM2 nanoparticles and 0.005mg / ml for the rest of the formulations, with a radiolabelling time of 10minutes at room temperature. In the case of 67Ga the label was optimized at 30° C temperature and 30minutes of incubation. In both cases the radiochemical purity obtained was greater than 97%. The nanoparticles showed high stability in vitro after 48hours of labeling (70% nanoparticles labeled with 99mTc and 90% those labeled with 67Ga). Biodistribution studies of nanoparticles 99mTc -GPM2 and 67Ga -NOTA-GPM2 showed a high accumulation of activity in the liver at 2 and 24hours after intravenous administration. CONCLUSION: The labeling procedure with 99mTc and 67Ga of albumin and albumin modified with NOTA nanoparticles allows obtaining nanoparticles with high labeling yields and adequate in vitro stability, allowing their use for in vivo studies.

Soybean protein-based microparticles for oral delivery of probiotics with improved stability during storage and gut resistance.

The present work describes the encapsulation of probiotics using a by-product as wall material and a process feasible to be scaled-up: coacervation of soybean protein concentrate (SPC) by using calcium salts and spray-drying. SPC was extracted from soybean flour, produced during the processing of soybean milk, by alkaline extraction following isoelectric precipitation. Two probiotic strains were selected for encapsulation (Lactobacillus plantarum CECT 220 and Lactobacillus casei CECT 475) in order to evaluate the ability of SPC to encapsulate and protect bacteria from stress conditions. The viability of these encapsulated strains under in vitro gastrointestinal conditions and shelf-life during storage were compared with the most common forms commercialized nowadays. Results show that SPC is a feasible material for the development of probiotic microparticles with adequate physicochemical properties and enhanced significantly both probiotic viability and tolerance against simulated gastrointestinal fluids when compared to current available commercial forms.

A combination of nanosystems for the delivery of cancer chemoimmunotherapeutic combinations: 1-Methyltryptophan nanocrystals and paclitaxel nanoparticles.

IDO is an enzyme that tumors use to create a state of immunosupression. 1-d-methyltryptophan (1-MT) is an IDO pathway inhibitor. After being successfully evaluated in preclinical studies, current clinical trials are actually analyzing its efficacy as monotherapy or in combination with multiple chemotherapeutic agents such as paclitaxel. 1-MT very poor solubility in water and many other solvents precludes its ease parenteral administration. It is currently administered by oral route because high daily doses were well-tolerated and effectively inhibited the IDO activity although only 25% of dose was recovered in plasma. The present work describes the preparation and characterization of 1-MT nanocrystals in order to enhance its solubility, dissolution rate, biodisponibility as well as facilitate its administration by parenteral route. A bottom-down approach of nanoprecipitation with an antisolvent was used for the fabrication of the nanocrystals and the choice of stabilizers was critical for reducing the size. Thermal analysis and x-ray diffraction indicated modifications in the drug crystalline state by the process. Through the reduction size and crystalline state modifications the dissolution characteristics of raw material were significantly increased. In a Lewis Lung cancer mice model, the nanocrystals strategy facilitated the sc administration and its antitumoral activity was similar to that of i.v. paclitaxel. The best efficacy was achieved when sc 1-MT nanocrystals were administered in combination with oral paclitaxel loaded in poly(anhydride) nanoparticles. Take together, 1-MT nanocrystals delivery performs a nanotechnological strategy suitable to modify the current route and schedule for its administration.

Genotoxic evaluation of poly(anhydride) nanoparticles in the gastrointestinal tract of mice.

Gantrez® AN 119-based NPs have been developed as oral drug carriers due to their strong bioadhesive interaction with components of the gastrointestinal mucosa and to their adaptable surface. The use of mannosamine to coat Gantrez® AN 119-based NPs results in a high mucus-permeable carrier, able to reach the gastrointestinal epithelium. Although their efficacy to transport a therapeutic agent has been demonstrated, their safety has not yet been thoroughly studied. They have proved to be non-cytotoxic, non-genotoxic and non-mutagenic in vitro; however, the in vivo toxicity profile has not yet been determined. In this study, the in vivo genotoxic potential of Gantrez® AN 119 NPs coated with mannosamine (GN-MA-NP) has been assessed using the in vivo comet assay in combination with the enzyme formamidopyrimidine DNA glycosylase in mice, following the OECD test guideline 489. To determine the relevant organs to analyse and the sampling times, an in vivo biodistribution study was also carried out. Results showed a statistically significant induction of DNA strand breaks and oxidized bases in the duodenum of animals exposed to 2000 mg/kg bw. However, this effect was not observed at lower doses (i.e. 500 and 1000 mg/kg which are closer to the potential therapeutic doses) or in other organs. In conclusion, GN-MA-NP are promising nanocarriers as oral drug delivery systems.

In vitro evaluation of the genotoxicity of poly(anhydride) nanoparticles designed for oral drug delivery.

In the last years, the development of nanomaterials has significantly increased due to the immense variety of potential applications in technological sectors, such as medicine, pharmacy and food safety. Focusing on the nanodevices for oral drug delivery, poly(anhydride) nanoparticles have received extensive attention due to their unique properties, such as their capability to develop intense adhesive interactions within the gut mucosa, their modifiable surface and their biodegradable and easy-to-produce profile. However, current knowledge of the possible adverse health effects as well as, toxicological information, is still exceedingly limited. Thus, we investigated the capacity of two poly(anhydride) nanoparticles, Gantrez® AN 119-NP (GN-NP) and Gantrez® AN 119 covered with mannosamine (GN-MA-NP), and their main bulk material (Gantrez® AN 119-Polymer), to induce DNA damage and thymidine kinase (TK+/-) mutations in L5178Y TK+/- mouse lymphoma cells after 24h of exposure. The results showed that GN-NP, GN-MA-NP and their polymer did not induce DNA strand breaks or oxidative damage at concentrations ranging from 7.4 to 600µg/mL. Besides, the mutagenic potential of these nanoparticles and their polymer revealed no significant or biologically relevant gene mutation induction at concentrations up to 600µg/mL under our experimental settings. Considering the non-genotoxic effects of GN-NP and GN-MA-NP, as well as their exceptional properties, these nanoparticles are promising nanocarriers for oral medical administrations.

Evaluation of the cytotoxicity, genotoxicity and mucus permeation capacity of several surface modified poly(anhydride) nanoparticles designed for oral drug delivery.

The main concerns with drugs designed for oral administration are their inactivation or degradation in the harsh conditions of the gastrointestinal tract, their poor solubility through the gastrointestinal mucus gel layer, the poor intestinal epithelium permeability that limits their absorption, and their toxicity. In this context, poly(anhydride) nanoparticles are capable of protecting the drug from the harsh environment, reduce the drug's toxicity and, by virtue of surface modification, to enhance or reduce their mucus permeability and the bioadhesion to specific target cells. The copolymer between methyl vinyl ether and maleic anhydride (commercialized as Gantrez® AN 119) are part of the poly(anhydride) nanoparticles. These biocompatible and biodegradable nanoparticles (NPs) can be modified by using different ligands. Their usefulness as drug carriers and their bioadhesion with components of the intestinal mucosa have been described. However, their toxicity, genotoxicity and mucus permeation capacity has not been thoroughly studied. The aim of this work was to evaluate and compare the in vitro toxicity, cell viability and in vitro genotoxicity of the bioadhesive empty Gantrez® AN 119 NPs modified with dextran, aminodextran, 2-hydroxypropyl-ß-cyclodextrin, mannosamine and poly-ethylene glycol of different molecular weights. Results showed that, in general, coated NPs exhibit better mucus permeability than the bare ones, those coated with mannosamine being the most permeable ones. The NPs studied did not affect cell metabolism, membrane integrity or viability of Caco-2 cells at the different conditions tested. Moreover, they did not induce a relevant level of DNA strand breaks and FPG-sensitive sites (as detected with the comet assay).

Toxicity evaluation of nanocarriers for the oral delivery of macromolecular drugs.

Oral administration is the most commonly used and accepted route for drug administration. However, two of the main concerns are the poor intestinal epithelium permeability and rapid degradation, which limit absorption of drugs. In this context, nanocarriers have shown great potential for oral drug delivery. Nevertheless, special importance should be given to the possible toxic effect of these nanocarriers, such as their bioaccumulation in different tissues of the body, as well as, the different physicochemical parameters influencing their properties and so their potential toxic effect. This review describes first some aspects related to the behavior of nanosystems within the gastrointestinal tract and then some aspects of nanotoxicology and its evaluation, including the most popular techniques and approaches used for in vitro and in vivo toxicity studies. It also reviews the physicochemical characteristics of polymeric nanoparticles that may influence the development of toxicological effects, and finally it summarizes the toxicity results that have been published regarding polymeric nanocarriers.

Interactions of poly (anhydride) nanoparticles with macrophages in light of their vaccine adjuvant properties.

Understanding how nanoparticles are formed and how those processes ultimately determine the nanoparticles' properties and their impact on their capture by immune cells is key in vaccination studies. Accordingly, we wanted to evaluate how the previously described poly (anhydride)-based nanoparticles of the copolymer of methyl vinyl ether and maleic anhydride (NP) interact with macrophages, and how this process depends on the physicochemical properties derived from the method of preparation. First, we studied the influence of the desolvation and drying processes used to obtain the nanoparticles. NP prepared by the desolvation of the polymers in acetone with a mixture of ethanol and water yielded higher mean diameters than those obtained in the presence of water (250nm vs. 180nm). In addition, nanoparticles dried by lyophilization presented higher negative zeta potentials than those dried by spray-drying (-47mV vs. -35mV). Second, the influence of the NP formulation on the phagocytosis by J774 murine macrophage-like cell line was investigated. The data indicated that NPs prepared in the presence of water were at least three-times more efficiently internalized by cells than NPs prepared with the mixture of ethanol and water. Besides, lyophilized nanoparticles appeared to be more efficiently taken up by J744 cells than those dried by spray-drying. To further understand the specific mechanisms involved in the cellular internalization of NPs, different pharmacological inhibitors were used to interfere with specific uptake pathways. Results suggest that the NP formulations, particularly, nanoparticles prepared by the addition of ethanol:water, are internalized by the clathrin-mediated endocytosis, rather than caveolae-mediated mechanisms, supporting their previously described vaccine adjuvant properties.

Mimicking microbial strategies for the design of mucus-permeating nanoparticles for oral immunization.

Dealing with mucosal delivery systems means dealing with mucus. The name mucosa comes from mucus, a dense fluid enriched in glycoproteins, such as mucin, which main function is to protect the delicate mucosal epithelium. Mucus provides a barrier against physiological chemical and physical aggressors (i.e., host secreted digestive products such as bile acids and enzymes, food particles) but also against the potentially noxious microbiota and their products. Intestinal mucosa covers 400m(2) in the human host, and, as a consequence, is the major portal of entry of the majority of known pathogens. But, in turn, some microorganisms have evolved many different approaches to circumvent this barrier, a direct consequence of natural co-evolution. The understanding of these mechanisms (known as virulence factors) used to interact and/or disrupt mucosal barriers should instruct us to a rational design of nanoparticulate delivery systems intended for oral vaccination and immunotherapy. This review deals with this mimetic approach to obtain nanocarriers capable to reach the epithelial cells after oral delivery and, in parallel, induce strong and long-lasting immune and protective responses.

Development and validation of a HPLC method for the determination of cyclosporine a in new bioadhesive nanoparticles for oral administration.

A simple and reliable high performance liquid chromatography method was developed and validated for the rapid determination of cyclosporine A in new pharmaceutical dosage forms based on the use of poly (methylvinylether-co-maleic anhydride) nanoparticles. The chromatographic separation was achieved using Ultrabase C18 column (250×4.6 mm, 5 µm), which was kept at 75°. The gradient mobile phase consisted of acetonitrile and water with a flow rate of 1 ml/min. The effluent was monitored at 205 nm using diode array detector. The method exhibited linearity over the assayed concentration range (22-250 µg/ml) and demonstrated good intraday and interday precision and accuracy (relative standard deviations were less than 6.5% and the deviation from theoretical values is below 5.5%). The detection limit was 1.36 µg/ml. This method was also applied for quantitative analysis of cyclosporine A released from poly (methylvinylether-co-maleic anhydride) nanoparticles.

Nanoparticle-based vaccine for mucosal protection against Shigella flexneri in mice.

Shigellosis is one of the leading causes of diarrhea worldwide with more than 130 million cases annually. Hence, the research of an effective vaccine is still a priority. Unfortunately, a safe and efficacious vaccine is not available yet. We have previously demonstrated the capacity of outer membrane vesicles (OMVs) to protect mice against an experimental infection with Shigella flexneri. Now, we present results on the capacity of this antigenic complex to confer a longer-term protection by oral or nasal routes when encapsulated into nanoparticles. OMVs were encapsulated in poly(anhydride) nanoparticles (NP) prepared by a solvent displacement method with the copolymer poly methyl vinyl ether/maleic anhydride. OMVs loaded into nanoparticles (NP-OMVs) were homogeneous and spherical in shape, with a size of 148nm (PdI=0.2). BALB/c mice were immunized with OMVs either free or encapsulated in nanoparticles by nasal (20µg or 10µg of OMVs) or oral route (100µg or 50µg of OMVs). All immunized animals remained in good health after administration. Challenge infection was performed intranasally on week 8th with a lethal dose of 5×10(7)CFU/mouse of S. flexneri 2a. The number of dead mice after challenge was recorded daily. Results confirmed the value of OMVs as a vaccine. By oral route, the OMV-vaccine was able to protect independently either the dose or the formulation. When vaccine was delivered by nasal route, encapsulation into NPs resulted beneficial in increasing protection from 40% up to 100% when low dose was administered. These results are extraordinary promising and put in relevance the positive effect of nanoencapsulation of the OMV subcellular vaccine.

[Mucopenetrating nanoparticles: vehicles for the oral administration of paclitaxel]. / Nanoparticules mucopénétrantes: véhicules pour l'administration orale du paclitaxel.

Paclitaxel is an anticancer drug used as solution for perfusion for the treatment of certain types of cancers. In the last years, a number of strategies have been proposed for the development of an oral formulation of this drug. However, this task is quite complicated due to the poor aqueous solubility of paclitaxel as well as the fact that this compound is substrate of the intestinal P-glycoprotein and the cytochrome P450 enzymatic complex. In this work, we have developed pegylated nanoparticles with mucopenetrating properties in order to conduct paclitaxel onto the surface of the enterocyte. These nanoparticles displayed a size of about 180 nm and a drug loading close to 15% by weight. The pharmacokinetic study in mice has shown that these nanoparticles were capable to offer therapeutic plasma levels of paclitaxel up to 72 hours. In addition, the oral relative bioavailability of paclitaxel when loaded in nanoparticles pegylated with poly(ethylene glycol) 2000 (PEG) was found to be 85%. In a subcutaneous model of tumour in mice, these pegylated nanoparticles administered orally every 3 days have demonstrated a similar efficacy than Taxol® administered intravenously every day during 9 days. All of these results suggested that these pegylated nanoparticles were capable to cross the mucus layer of the gut and, then, reach the surface of the enterocytes. The PEG molecules would facilitate the adhesion of nanoparticles to this epithelial surface, minimise the pre-systemic metabolism of paclitaxel and, thus, promote its absorption.

[Radiolabeling and biodistribution studies of polymeric nanoparticles as adjuvants for ocular vaccination against brucellosis]. / Radiomarcaje y estudios de biodistribución de nanopartículas poliméricas como adyuvantes para la vacunación oftálmica frente a la brucelosis.

PURPOSE: To optimize radiolabeling with (99m)Tc of mannosylated Gantrez(®) nanoparticles loaded with the Brucella Ovis antigen (Man-NP-HS) and to carry out biodistribution studies in mice after ocular administration of the nanoparticles. MATERIAL AND METHODS: Man-NP-HS nanoparticles were prepared by the solvent displacement method. They were purified, lyophilized and characterized. Following this, they were radiolabeled with 74 MBq of (99m)TcO4(-) previously reduced with an acidic stannous chloride solution, working in absence of oxygen and at a final pH of 4. Radiolabeling yield was evaluated by TLC. Biodistribution studies were carried out in mice after ocular administration of the formulation and control of free (99m)TcO4(-). To do so, the animals were humanely killed at 2 and 24hours after the ocular administration and activity in organs was measured in a Gamma counter. RESULTS: Radiolabeling yield obtained was greater than 90%. Biodistribution studies of (99m)Tc-Man-NP-HS showed radioactivity accumulated at 2 and 24hours in nasal and ocular mucosa and gastrointestinal tract, in contrast to biodistribution of free (99m)TcO4(-) that remained concentrated in the skin around the eye and gastrointestinal tract. CONCLUSION: Biodistribution studies of (99m)Tc-Man-NP-HS after ocular instillation have made it possible to demonstrate its biodistribution in nasal mucosa and gastrointestinal tract. This characteristic is essential as an antigenic delivery system throughout the ocular mucosa. This, together with its elevated immune response, effective protection and intrinsic avirulence make them a suitable anti-Brucella vaccine candidate.

Towards a non-living vaccine against Shigella flexneri: from the inactivation procedure to protection studies.

Shigellosis is one of the leading causes of diarrhea worldwide with more than 165 million cases annually. Hence, a vaccine against this disease is a priority, but no licensed vaccine is still available. Considering target population as well as intrinsic risks of live attenuated vaccines, non-living strategies appear as the most promising candidates. Remarkably, the preservation of antigenic properties is a major concern since inactivation methods of bacteria affect these qualities. We previously reported the use of a subcellular antigen complex for vaccination against shigellosis, based on outer membrane vesicles (OMVs) released from Shigella flexneri. Now, we describe in more detail the employment of binary ethylenimine (BEI) for inactivation of Shigella and its subsequent effect on the antigenic conservation of the vaccinal product. Results demonstrate the effectiveness of BEI treatment to completely inactivate Shigella cells without disturbing the antigenicity and immunogenicity of the OMVs. Thus, OMVs harvested after BEI inactivation were able to protect mice against an experimental infection with S. flexneri.

Innovative lead compounds and formulation strategies as newer kinetoplastid therapies.

The protozoan diseases leishmaniasis, human African trypanosomiasis (HAT) and Chagas disease (CD) are responsible for substantial global morbidity and mortality in tropical and subtropical regions. Environmental changes, drug resistance and immunosuppression are contributing to the emergence and spread of these diseases. In the absence of safe and efficient vaccines, chemotherapy, together with vector control, remains the most important measure to control kinetoplastid diseases. Nevertheless, the current chemotherapeutic treatments are clearly inadequate because of their toxic effects, generation of resistances as well as route and schedules of administration not adapted to the field-conditions. This review overlooks the strategies that can be addressed to meet immediately the patient needs such as the reconsideration of current regimens of administration and the rational combination of drugs in use. In the medium-long term, due to new methodologies of medicinal-chemistry, the screening from natural products and the identification of new therapeutic targets, new lead compounds have great chance to advance through the drug development pipeline to clinic. Modern pharmaceutical formulation strategies and nanomedicines also merit a place in view of the benefits of a single dose of liposomal Amphotericin B (AmBisome®) against visceral leishmaniasis. BBB-targeted nanodevices could be suited for selective delivery of drugs against HAT encephalitic phase. Bioadhesive nanoparticles can be proposed to enhance the bioavailability of drugs after oral administration by reason of improving the drug solubility, and permeability across the intestinal epithelia.

[New pharmaceutical dosage forms for allergy treatment]. / Nuevas formas farmacéuticas para el tratamiento de enfermedades alérgicas.

Specific immunotherapy involves certain drawbacks which could be minimized by the use of appropriate adjuvants, capable of amplifying the right immune response with minimal side effects. In this context, we review different types of immunotherapy vehicles and coadjuvants. We describe previous studies by our group in which we demonstrated the adjuvant capacity of Gantrez® AN nanoparticles, which can effectively enhance the immune response. We employed two types of nanoparticles (with and without LPS of Brucella ovis as immunomodulator) within capsulated ovoalbumin and Lollium perenne extract, tested on a model of mice sensitized to this allergenic mixture. In the challenge experiment involving the sensitized mice, differences in the mortality rate and in the MCP-1 levels were found between the treated groups and the control. Under the experimental conditions of this model of mice pre-sensitized to L. perenne, Gantrez®AN nanoparticles appeared to be a good strategy for immunotherapy. We finally tested these carriers administered by the oral route and found that they were able to protect a model of mice sensitized to ovalbumin from anaphylactic shock.

Gantrez AN nanoparticles for ocular delivery of memantine: in vitro release evaluation in albino rabbits.

AIM: To prepare and evaluate the in vitro release of memantine-loaded poly(anhydride) (Gantrez®) nanoparticles (NPs). The clinical safety and retinal toxicity caused by unloaded NPs after sub-Tenon and intravitreal ocular injections were also evaluated. METHODS: Preparation and characterization of this type of NP as well as the in vitro release study are described. Twenty-three healthy New Zealand rabbits were used for clinical and histological assessment after sub-Tenon and intravitreal ocular injections of unloaded NPs. RESULTS: The amount of drug associated with NPs was 55 µg of memantine/mg of NP. The release profile of memantine from this type of NPs was characterized by an initial burst effect, followed by continuous release of the drug for at least 15 days. No relevant complications were found during the clinical follow-up. The histological evaluation suggested that Gantrez NPs are well tolerated after sub-Tenon ocular injection and that signs of inflammation during the first days after intravitreal ocular injections can be considered a normal reaction of the eye's defence mechanism.

Mucosal immunization with Shigella flexneri outer membrane vesicles induced protection in mice.

Vaccination appears to be the only rational prophylactic approach to control shigellosis. Unfortunately, there is still no safe and efficacious vaccine available. We investigated the protection conferred by a new vaccine containing outer membrane vesicles (OMVs) from Shigella flexneri with an adjuvant based on nanoparticles in an experimental model of shigellosis in mice. OMVs were encapsulated in poly(anhydride) nanoparticles prepared by a solvent displacement method with the copolymer PMV/MA. OMVs loaded into NPs (NP-OMVs) were homogeneous and spherical in shape, with a size of 197nm (PdI=0.06). BALB/c mice (females, 9-week-old, 20±1g) were immunized by intradermal, nasal, ocular (20µg) or oral route (100µg) with free or encapsulated OMV. Thirty-five days after administration, mice were infected intranasally with a lethal dose of S. flexneri (1×10(7)CFU). The new vaccine was able to protect fully against infection when it was administered via mucosa. By intradermal route the NP-OMVs formulation increased the protection from 20%, obtained with free extract, to 100%. Interestingly, both OMVs and OMV-NP induced full protection when administered by the nasal and conjuntival route. A strong association between the ratio of IL-12p40/IL-10 and protection was found. Moreover, low levels of IFN-γ correlate with protection. Under the experimental conditions used, the adjuvant did not induce any adverse effects. These results place OMVs among promising candidates to be used for vaccination against Shigellosis.