Modulating the immune system through nanotechnology.

Nowadays, nanotechnology-based modulation of the immune system is presented as a cutting-edge strategy, which may lead to significant improvements in the treatment of severe diseases. In particular, efforts have been focused on the development of nanotechnology-based vaccines, which could be used for immunization or generation of tolerance. In this review, we highlight how different immune responses can be elicited by tuning nanosystems properties. In addition, we discuss specific formulation approaches designed for the development of anti-infectious and anti-autoimmune vaccines, as well as those intended to prevent the formation of antibodies against biologicals.

Mauritian cynomolgus macaques with M3M4 MHC genotype control SIVmac251 infection.

BACKGROUND: Understanding natural HIV control may lead to new preventative or therapeutic strategies. Several protective major histocompatibility complex (MHC) genotypes were found in humans and rhesus macaques. Here, we report a simian immunodeficiency virus (SIV) controller MHC genotype in Mauritian cynomolgus macaques (MCMs). METHODS: Twelve MHC-genotyped MCMs were infected with SIVmac251 and monitored for viral loads and CD4+ T-cell counts. RESULTS: Two macaques with M3M4 genotype exhibited the lowest peak viral loads (log plasma SIV RNA copies/mL), nearly 3 logs lower than those in most macaques with other MHC haplotype combinations, and set point viral loads below the level of detection limit by RT-qPCR (<2 log RNA copies/mL). They maintained healthy CD4+ T-cell counts of >500 cells/µL blood, while CD4 counts in the vast majority of other macaques were below this level. CONCLUSIONS: The M3M4 MHC genotype may confer enhanced control of SIV replication in MCMs.

Controlling the protein corona of polymeric nanocapsules: effect of polymer shell on protein adsorption.

Understanding the interactions between nanocarriers and plasma proteins is essential for controlling their biological fate. Based on the reported potential of polymeric nanocapsules (NCs) for the targeted delivery of oncological drugs, the main objective of this work has been to investigate how the surface chemical composition influences their protein corona fingerprint. Thus, we developed six NC prototypes with different polymer shells and physicochemical properties and quantified the amount of protein adsorbed upon incubation in human plasma. Using sequential window acquisition of all theoretical mass spectra (SWATH-MS) and following the Minimum Information about Nanomaterial Biocorona Experiments (MINBE) guidelines, we identified different protein corona patterns. As expected, the presence of polyethylene glycol (PEG) in the polymer shell reduced the protein corona, particularly the adsorption of immunoglobulins. However, by comparing the different prototypes, we concluded that the protein adsorption pattern was not exclusively driven by PEG. In fact, a highly PEGylated prototype exhibited intense apolipoprotein IV adsorption. On the other hand, we also observed that polymeric NCs containing 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) exhibited high adsorption of vitronectin, a protein that is known for enhancing the uptake of nanosystems by lung epithelium and several cancer cells. Overall, the gathered information allowed us to identify promising polymeric NCs with an expected prolonged circulation time, enhanced tumor targeting, liver accumulation, and preferential uptake by the immune system. In this sense, the analyses of the protein corona performed along this work will hopefully contribute to advancing a new generation of rationally designed nanometric drug delivery systems.

Biofunctionalization of 3D printed collagen with bevacizumab-loaded microparticles targeting pathological angiogenesis.

Pathological angiogenesis is a crucial attribute of several chronic diseases such as cancer, age-related macular degeneration, and osteoarthritis (OA). In the case of OA, pathological angiogenesis mediated by the vascular endothelial growth factor (VEGF), among other factors, contributes to cartilage degeneration and to implants rejection. In line with this, the use of the anti-VEGF bevacizumab (BVZ) has been shown to prevent OA progression and support cartilage regeneration. The aim of this work was to functionalize a medical grade collagen with poly (lactic-co-glycolic acid) (PLGA) microparticles containing BVZ via three-dimensional (3D) printing to target pathological angiogenesis. First, the effect of several formulation parameters on the encapsulation and release of BVZ from PLGA microparticles was studied. Then, the anti-angiogenic activity of released BVZ was tested in a 3D cell model. The 3D printability of the microparticle-loaded collagen ink was tested by evaluating the shape fidelity of 3D printed structures. Results showed that the release and the encapsulation efficiency of BVZ could be tuned as a function of several formulation parameters. In addition, the released BVZ was observed to reduce vascularization by human umbilical vein endothelial cells. Finally, the collagen ink with embedded BVZ microparticles was successfully printed, leading to shape-stable meniscus-, nose- and auricle-like structures. Taken altogether, we defined the conditions for the successful combination of BVZ-loaded microparticles with the 3D printing of a medical grade collagen to target pathological angiogenesis.

Mannose-modified hyaluronic acid nanocapsules for the targeting of tumor-associated macrophages.

Tumor-associated macrophages (TAMs), a class of immune cells that play a key role in tumor immunosuppression, are recognized as important targets to improve cancer prognosis and treatment. Consequently, the engineering of drug delivery nanocarriers that can reach TAMs has acquired special relevance. This work describes the development and biological evaluation of a panel of hyaluronic acid (HA) nanocapsules (NCs), with different compositions and prepared by different techniques, designed to target macrophages. The results showed that plain HA NCs did not significantly influence the polarization of M0 and M2-like macrophages towards an M1-like pro-inflammatory phenotype; however, the chemical functionalization of HA with mannose (HA-Man) led to a significant increase of NCs uptake by M2 macrophages in vitro and to an improved biodistribution in a MN/MNCA1 fibrosarcoma mouse model with high infiltration of TAMs. These functionalized HA-Man NCs showed a higher accumulation in the tumor compared to non-modified HA NCs. Finally, the pre-administration of the liposomal liver occupying agent Nanoprimer™ further increased the accumulation of the HA-Man NCs in the tumor. This work highlights the promise shown by the HA-Man NCs to target TAMs and thus provides new options for the development of nanomedicine and immunotherapy-based cancer treatments.

Polymeric nanocapsules loaded with poly(I:C) and resiquimod to reprogram tumor-associated macrophages for the treatment of solid tumors.

Background: In the tumor microenvironment (TME), tumor-associated macrophages (TAMs) play a key immunosuppressive role that limits the ability of the immune system to fight cancer. Toll-like receptors (TLRs) ligands, such as poly(I:C) or resiquimod (R848) are able to reprogram TAMs towards M1-like antitumor effector cells. The objective of our work has been to develop and evaluate polymeric nanocapsules (NCs) loaded with poly(I:C)+R848, to improve drug stability and systemic toxicity, and evaluate their targeting and therapeutic activity towards TAMs in the TME of solid tumors. Methods: NCs were developed by the solvent displacement and layer-by-layer methodologies and characterized by dynamic light scattering and nanoparticle tracking analysis. Hyaluronic acid (HA) was chemically functionalized with mannose for the coating of the NCs to target TAMs. NCs loaded with TLR ligands were evaluated in vitro for toxicity and immunostimulatory activity by Alamar Blue, ELISA and flow cytometry, using primary human monocyte-derived macrophages. For in vivo experiments, the CMT167 lung cancer model and the MN/MCA1 fibrosarcoma model metastasizing to lungs were used; tumor-infiltrating leukocytes were evaluated by flow cytometry and multispectral immunophenotyping. Results: We have developed polymeric NCs loaded with poly(I:C)+R848. Among a series of 5 lead prototypes, protamine-NCs were selected based on their physicochemical properties (size, charge, stability) and in vitro characterization, showing good biocompatibility on primary macrophages and ability to stimulate their production of T-cell attracting chemokines (CXCL10, CCL5) and to induce M1-like macrophages cytotoxicity towards tumor cells. In mouse tumor models, the intratumoral injection of poly(I:C)+R848-protamine-NCs significantly prevented tumor growth and lung metastasis. In an orthotopic murine lung cancer model, the intravenous administration of poly(I:C)+R848-prot-NCs, coated with an additional layer of HA-mannose to improve TAM-targeting, resulted in good antitumoral efficacy with no apparent systemic toxicity. While no significant alterations were observed in T cell numbers (CD8, CD4 or Treg), TAM-reprogramming in treated mice was confirmed by the relative decrease of interstitial versus alveolar macrophages, having higher CD86 expression but lower CD206 and Arg1 expression in the same cells, in treated mice. Conclusion: Mannose-HA-protamine-NCs loaded with poly(I:C)+R848 successfully reprogram TAMs in vivo, and reduce tumor progression and metastasis spread in mouse tumors.

Quantification of the actual composition of polymeric nanocapsules: a quality control analysis.

Nanocapsules (NCs) are drug delivery nanosystems that contain an oily core, stabilized by a surfactant, and surrounded by a polymeric shell. The assembling of the components is based on physical and physicochemical forces, and, hence, usually, only a fraction of each component is finally part of the NCs' structure, while the remaining amount might be solubilized or forming micelles in the NCs' suspending medium. Usually, reports on the characterization of nanostructures simply indicate the association efficiency of the loaded drugs instead of their complete final composition. In this work, we have developed a liquid chromatography (LC) mass spectrometry (MS) methodology that allows the quantification of all the components of a series of NCs prepared by different techniques, namely DL-α-tocopherol; D-α-tocopherol polyethylene glycol 1000 succinate; benzethonium; lecithin; hexadecyltrimethylammonium; 1,2-dioleoyl-3-trimethylammoniumpropane; caprylic/capric triglycerides; macrogol 15-hydroxystearate; polysorbate 80; polysialic acid; hyaluronic acid; and polyethylene glycol polyglutamic acid. The LC-MS method was validated in terms of linearity (0.9383 < r2 < 0.9997), quantification limits, and recoveries of the isolated NCs' and waste fractions. The final composition of the isolated NCs was found to strongly depend on their composition and preparation technique. In our view, the rigorous quantification of the exact composition of nanosystems is essential for the progress of nanotechnology. This quantitative analysis will allow researchers to draw more accurate conclusions about the influence of the nanosystems' composition on their biological performance.

Development of a nanocapsule-loaded hydrogel for drug delivery for intraperitoneal administration.

Intraperitoneal (IP) drug delivery of chemotherapeutic agents, administered through hyperthermal intraperitoneal chemotherapy (HIPEC) and pressurized intraperitoneal aerosolized chemotherapy (PIPAC), is effective for the treatment of peritoneal malignancies. However, these therapeutic interventions are cumbersome in terms of surgical practice and are often associated with the formation of peritoneal adhesions, due to the catheters inserted into the peritoneal cavity during these procedures. Hence, there is a need for the development of drug delivery systems that can be administered into the peritoneal cavity. In this study, we have developed a nanocapsule (NCs)-loaded hydrogel for drug delivery in the peritoneal cavity. The hydrogel has been developed using poly(ethylene glycol) (PEG) and thiol-maleimide chemistry. NCs-loaded hydrogels were characterized by rheology and their resistance to dilution and drug release were determined in vitro. Using IVIS® to measure individual organ and recovered gel fluorescence intensity, an in vivo imaging study was performed and demonstrated that NCs incorporated in the PEG gel were retained in the IP cavity for 24 h after IP administration. NCs-loaded PEG gels could find potential applications as biodegradable, drug delivery systems that could be implanted in the IP cavity, for example at a the tumour resection site to prevent recurrence of microscopic tumours.

Engineering Anisotropic Meniscus: Zonal Functionality and Spatiotemporal Drug Delivery.

Human meniscus is a fibrocartilaginous structure that is crucial for an adequate performance of the human knee joint. Degeneration of the meniscus is often followed by partial or total meniscectomy, which enhances the risk of developing knee osteoarthritis. The lack of a satisfactory treatment for this condition has triggered a major interest in drug delivery (DD) and tissue engineering (TE) strategies intended to restore a bioactive and fully functional meniscal tissue. The aim of this review is to critically discuss the most relevant studies on spatiotemporal DD and TE, aiming for a multizonal meniscal reconstruction. Indeed, the development of meniscal tissue implants should involve a provision for adequate active molecules and scaffold features that take into account the anisotropic ultrastructure of human meniscus. This zonal differentiation is reflected in the meniscus biochemical composition, collagen fiber arrangement, and cell distribution. In this sense, it is expected that a proper combination of advanced DD and zonal TE strategies will play a key role in the future trends in meniscus regeneration. Impact statement Meniscus degeneration is one of the main causes of knee pain, inflammation, and reduced mobility. Currently used suturing procedures and meniscectomy are far from being ideal solutions to the loss of meniscal function. Therefore, drug delivery (DD) and tissue engineering (TE) strategies are currently under investigation. DD systems aim at an in situ controlled release of growth factors, whereas TE strategies aim at mimicking the anisotropy of native meniscus. The goal of this review is to discuss these two main approaches, as well as synergies between them that are expected to lead to a real breakthrough in the field.

Asymmetric flow field-flow fractionation as a multifunctional technique for the characterization of polymeric nanocarriers.

The importance of polymeric nanocarriers in the field of drug delivery is ever-increasing, and the accurate characterization of their properties is paramount to understand and predict their behavior. Asymmetric flow field-flow fractionation (AF4) is a fractionation technique that has gained considerable attention for its gentle separation conditions, broad working range, and versatility. AF4 can be hyphenated to a plurality of concentration and size detectors, thus permitting the analysis of the multifunctionality of nanomaterials. Despite this potential, the practical information that can be retrieved by AF4 and its possible applications are still rather unfamiliar to the pharmaceutical scientist. This review was conceived as a primer that clearly states the "do's and don'ts" about AF4 applied to the characterization of polymeric nanocarriers. Aside from size characterization, AF4 can be beneficial during formulation optimization, for drug loading and drug release determination and for the study of interactions among biomaterials. It will focus mainly on the advances made in the last 5 years, as well as indicating the problematics on the consensus, which have not been reached yet. Methodological recommendations for several case studies will be also included.

Carboxymethyl-ß-glucan/chitosan nanoparticles: new thermostable and efficient carriers for antigen delivery.

In the last few decades, nanotechnology has emerged as an important tool aimed at enhancing the immune response against modern antigens. Nanocarriers designed specifically for this purpose have been shown to provide protection, stability, and controlled release properties to proteins, peptides, and polynucleotide-based antigens. Polysaccharides are particularly interesting biomaterials for the construction of these nanocarriers given their wide distribution among pathogens, which facilitates their recognition by antigen-presenting cells (APCs). In this work, we focused on an immunostimulant beta-glucan derivative, carboxymethyl-ß-glucan, to prepare a novel nanocarrier in combination with chitosan. The resulting carboxymethyl-ß-glucan/chitosan nanoparticles exhibited adequate physicochemical properties and an important protein association efficiency, with ovalbumin as a model compound. Moreover, thermostability was achieved through the optimization of a lyophilized form of the antigen-loaded nanoparticles, which remained stable for up to 1 month at 40 ºC. Biodistribution studies in mice showed an efficient drainage of the nanoparticles to the nearest lymph node following subcutaneous injection, and a significant co-localization with dendritic cells. Additionally, subcutaneous immunization of mice with a single dose of the ovalbumin-loaded nanoparticles led to induced T cell proliferation and antibody responses, comparable to those achieved with alum-adsorbed ovalbumin. These results illustrate the potential of these novel nanocarriers in vaccination.

Zebrafish Models for the Safety and Therapeutic Testing of Nanoparticles with a Focus on Macrophages.

New nanoparticles and biomaterials are increasingly being used in biomedical research for drug delivery, diagnostic applications, or vaccines, and they are also present in numerous commercial products, in the environment and workplaces. Thus, the evaluation of the safety and possible therapeutic application of these nanomaterials has become of foremost importance for the proper progress of nanotechnology. Due to economical and ethical issues, in vitro and in vivo methods are encouraged for the testing of new compounds and/or nanoparticles, however in vivo models are still needed. In this scenario, zebrafish (Danio rerio) has demonstrated potential for toxicological and pharmacological screenings. Zebrafish presents an innate immune system, from early developmental stages, with conserved macrophage phenotypes and functions with respect to humans. This fact, combined with the transparency of zebrafish, the availability of models with fluorescently labelled macrophages, as well as a broad variety of disease models offers great possibilities for the testing of new nanoparticles. Thus, with a particular focus on macrophage-nanoparticle interaction in vivo, here, we review the studies using zebrafish for toxicological and biodistribution testing of nanoparticles, and also the possibilities for their preclinical evaluation in various diseases, including cancer and autoimmune, neuroinflammatory, and infectious diseases.

Nanotechnologies for the delivery of biologicals: Historical perspective and current landscape.

Biological macromolecule-based therapeutics irrupted in the pharmaceutical scene generating a great hope due to their outstanding specificity and potency. However, given their susceptibility to degradation and limited capacity to overcome biological barriers new delivery technologies had to be developed for them to reach their targets. This review aims at analyzing the historical seminal advances that shaped the development of the protein/peptide delivery field, along with the emerging technologies on the lead of the current landscape. Particularly, focus is made on technologies with a potential for transmucosal systemic delivery of protein/peptide drugs, followed by approaches for the delivery of antigens as new vaccination strategies, and formulations of biological drugs in oncology, with special emphasis on mAbs. Finally, a discussion of the key challenges the field is facing, along with an overview of prospective advances are provided.

Effect of mesoporous silica and its combination with hydroxyapatite on the regeneration of rabbit's bone defects: A pilot study.

BACKGROUND: Bone volume augmentation is a routine technique used in oral implantology and periodontology. Advances in the surgical techniques and the biomaterials field have allowed a greater accessibility to these treatments. Nevertheless, dehiscence and fenestrations incidence during dental implant procedures are still common in patients with bone loss. OBJECTIVE: The main objective is to evaluate in a pilot experimental study the biological response to mesoporous silica (MS) hybrid scaffolds and its regenerative capacity in different formulations. METHODS: Two defects per rabbit tibia were performed (one for control and other for test) and the biomaterials tested in this study have been used to fill the bone defects, prepared in two different formulations (3D hybrid scaffolds or powdered material, in 100% pure MS form, or 50% MS with 50% hydroxyapatite (HA). Euthanasia was performed 4 months after surgery for bone histopathological study and radiographic images were acquired by computerized microtomography. RESULTS: Results showed that radiographically and histopathologically pure MS formulations lead to a lower biological response, e.g when formulated with HA, the osteogenic response in terms of osteoconduction was greater. CONCLUSIONS: We observed tolerance and lack of toxicity of the MS and HA, without registering any type of local or systemic allergic reaction.

Unveiling the pitfalls of the protein corona of polymeric drug nanocarriers.

The protein corona is a natural protein layer spontaneously formed around nanomaterials when exposed to biological media. This layer can alter the nanosystems' biological performance, particularly their tissue accumulation, cellular uptake, clearance by the immune system, toxicity, and even the release profile of their payloads. Hence, the characterization of this protein layer has become a critical step when developing a new nanomedicine. The modification of the nanosystem fate by the protein corona, systematically ignored in the vast majority of the nanotechnology-based research, may have contributed to the low in vitro/in vivo correlation. Actually, the protein corona of polymeric nanosystems has been scarcely studied in the literature, and most studies have been focused instead on metallic nanoparticles and liposomes. In this review, we analyzed the influence of the physicochemical properties and composition of the polymeric nanosystems on the protein layer deposited around them. In addition, we present some recommendations on how to perform the protein corona studies of polymeric nanoparticles, which, hopefully, will contribute to obtain more reliable and reproducible data in the future. Graphical abstract.

Tuning the PEG surface density of the PEG-PGA enveloped Octaarginine-peptide Nanocomplexes.

One of the main limitations of protein drugs is their restricted capacity to cross biological barriers. We have previously reported nanostructured complexes of insulin and modified octaarginine (C12-r8), enveloped by a polyethyleneglycol-polyglutamic acid (PEG-PGA) protective shell, and showed their capacity to overcome different barriers associated to the oral modality of administration. The objective of this work was to produce the said nanocomplexes with structurally diverse PEG-PGA shells, i.e. with different chain lengths and PEG substitution degrees, and comparatively analyze their PEG surface density and subsequent impact on their interaction with mucus glycoproteins and Caco-2 cells. The new PEG-PGA enveloped C12-r8-insulin nanocomplexes (ENCPs) exhibited a narrow size distribution (average size of 210-239 nm), a neutral surface charge and a 100% insulin association efficiency (final insulin loading of 16.5-29.6% w/w). Proton nuclear magnetic resonance (1H NMR) analysis indicated the possibility to modulate the PEG density on the ENCPs from 6.7 to 44.5 PEG chains per 100 nm2. This increase in the ENCPs PEG surface density resulted in their reduced interaction with mucins in vitro, while their interaction with Caco-2 cells in vitro remained unaltered. Overall, these data indicate the capacity to tune the surface characteristics of the ENCPS in order to maximize the capacity of these nanocarriers to overcome barriers associated to mucosal surfaces.

Arginine-Based Poly(I:C)-Loaded Nanocomplexes for the Polarization of Macrophages Toward M1-Antitumoral Effectors.

Background: Tumor-associated macrophages (TAMs), with M2-like immunosuppressive profiles, are key players in the development and dissemination of tumors. Hence, the induction of M1 pro-inflammatory and anti-tumoral states is critical to fight against cancer cells. The activation of the endosomal toll-like receptor 3 by its agonist poly(I:C) has shown to efficiently drive this polarization process. Unfortunately, poly(I:C) presents significant systemic toxicity, and its clinical use is restricted to a local administration. Therefore, the objective of this work has been to facilitate the delivery of poly(I:C) to macrophages through the use of nanotechnology, that will ultimately drive their phenotype toward pro-inflammatory states. Methods: Poly(I:C) was complexed to arginine-rich polypeptides, and then further enveloped with an anionic polymeric layer either by film hydration or incubation. Physicochemical characterization of the nanocomplexes was conducted by dynamic light scattering and transmission electron microscopy, and poly(I:C) association efficiency by gel electrophoresis. Primary human-derived macrophages were used as relevant in vitro cell model. Alamar Blue assay, ELISA, PCR and flow cytometry were used to determine macrophage viability, polarization, chemokine secretion and uptake of nanocomplexes. The cytotoxic activity of pre-treated macrophages against PANC-1 cancer cells was assessed by flow cytometry. Results: The final poly(I:C) nanocomplexes presented sizes lower than 200 nm, with surface charges ranging from +40 to -20 mV, depending on the envelopment. They all presented high poly(I:C) loading values, from 12 to 50%, and great stability in cell culture media. In vitro, poly(I:C) nanocomplexes were highly taken up by macrophages, in comparison to the free molecule. Macrophage treatment with these nanocomplexes did not reduce their viability and efficiently stimulated the secretion of the T-cell recruiter chemokines CXCL10 and CCL5, of great importance for an effective anti-tumor immune response. Finally, poly(I:C) nanocomplexes significantly increased the ability of treated macrophages to directly kill cancer cells. Conclusion: Overall, these enveloped poly(I:C) nanocomplexes might represent a therapeutic option to fight cancer through the induction of cytotoxic M1-polarized macrophages.

Advanced nanomedicine characterization by DLS and AF4-UV-MALS: Application to a HIV nanovaccine.

Nanoformulations are complex systems where physicochemical properties determine their therapeutic efficacy and safety. In the case of nanovaccines, particle size and shape play a crucial role on the immune response generated. Furthermore, the antigen's integrity is also a key aspect to control when producing a nanovaccine. The determination of all those physicochemical properties is still an analytical challenge and the lack of well-established methods hinders the access of new therapeutics to the market. In this work, robust methods for the characterization of a novel HIV nanoparticle-based vaccine produced in good manufacturing practice (GMPs)-like environment were developed. With slightly polydisperse particles (< 0.2) close to 180 nm of size, batch-mode Dynamic Light Scattering (DLS) was validated to be used as a quality control technique in the pilot production plant. In addition, a high size resolution method using Asymmetrical Flow Field Flow Fractionation (AF4) demonstrated its ability to determine not only size and size distribution but also shape modification across the size and accurate quantification of the free active ingredient. Results showed a monomodal distribution of particles from 60 to 700 nm, most of them (> 90%) with size lower than 250 nm, consistent with more traditional techniques, and revealed a slight change in the structure of the particles induced by the presence of the antigen. Finally, a batch to batch variability lower than 20% was obtained by both DLS and AF4 methods indicating that preparation method was highly reproducible.

Syzygium aromaticum (clove) and Thymus zygis (thyme) essential oils increase susceptibility to colistin in the nosocomial pathogens Acinetobacter baumannii and Klebsiella pneumoniae.

The discovery of new antibiotics that are effective against Acinetobacter baumannii and Enterobacteralesis a research priority. Several essential oils (EOs) have displayed some antimicrobial activity and could potentially act as antibiotic adjuvants. Research in this area aims to develop new therapeutic alternatives to treat infections caused by these pathogens. MICs of different EOs were determined against A. baumannii and Klebsiella pneumoniae. Combined disk diffusion tests and checkerboard assays were used to study the synergy between the EOs and antibiotics. The fractional inhibitory concentration index (FICindex) was calculated in order to categorize the interaction. Time-kill assays were also performed. The EOs that displayed the highest levels of antimicrobial activity were clove (Syzygium aromaticum L.) and thyme (Thymus zygis L.). Combined disk diffusion tests and checkerboard assays revealed synergy between these EOs and colistin. Addition of either clove or thyme EO decreased the MIC of colistin by 8- to 64-fold and 8- to 128-fold in the colistin-resistant A. baumannii and K. pneumoniae strains, respectively (FICindex ≤ 0.5, synergy). MICs were also reduced in the colistin-susceptible strains. Time-kill assays also indicated the strong activity of the combined therapy. In summary, the use of clove or thyme EO in combination with colistin could improve the efficacy of the antibiotic and significantly reduce the concentrations needed to inhibit growth of A. baumannii and K. pneumoniae.

Design of Polymeric Nanocapsules for Intranasal Vaccination against Mycobacterium Tuberculosis: Influence of the Polymeric Shell and Antigen Positioning.

Tuberculosis (TB) is the leading cause of death from a single infectious microorganism and Bacillus Calmette Guerin (BCG), the only authorized vaccine, does not confer protection against pulmonary TB. Based on the hypothesis that mucosal protection could help to prevent the infection at the site of entrance, the objective of this work was to develop an intranasal vaccine against Mycobacterium tuberculosis (Mtb), the microorganism that causes TB. Our approach consisted of the use of polymeric nanocapsules (NCs) with an oily core and a polymer shell made of chitosan (CS) or inulin/polyarginine (INU/pArg). The immunostimulant Imiquimod, a Toll-like receptor-7 (TLR-7) agonist, was encapsulated in the oily core and a fusion protein, formed by two antigens of Mtb, was absorbed either onto the NC surface (CS:Ag and INU:pArg:Ag) or between two polymer layers (INU:Ag:pArg) in order to assess the influence of the antigen positioning on the immune response. Although CS NCs were more immunostimulant than the INU/pArg NCs in vitro, the in vivo experiments showed that INU:pArg:Ag NCs were the only prototype inducing an adequate immunoglobulin A (IgA) response. Moreover, a previous immunization with BCG increased the immune response for CS NCs but, conversely, decreased for INU/pArg NCs. Further optimization of the antigen and the vaccination regime could provide an efficacious vaccine, using the INU:pArg:Ag NC prototype as nanocarrier.