Insights into Immune Exhaustion in Chronic Hepatitis B: A Review of Checkpoint Receptor Expression.

Hepatitis B, caused by the hepatitis B virus (HBV), often progresses to chronic infection, leading to severe complications, such as cirrhosis, liver failure, and hepatocellular carcinoma. Chronic HBV infection is characterized by a complex interplay between the virus and the host immune system, resulting in immune cell exhaustion, a phenomenon commonly observed in chronic viral infections and cancer. This state of exhaustion involves elevated levels of inhibitory molecules, cells, and cell surface receptors, as opposed to stimulatory counterparts. This review aims to elucidate the expression patterns of various co-inhibitory and co-stimulatory receptors on immune cells isolated from chronic hepatitis B (CHB) patients. By analyzing existing data, the review conducts comparisons between CHB patients and healthy adults, explores the differences between HBV-specific and total T cells in CHB patients, and examines variations between intrahepatic and peripheral immune cells in CHB patients. Understanding the mechanisms underlying immune exhaustion in CHB is crucial for developing novel immunotherapeutic approaches. This detailed analysis sheds light on the immune exhaustion observed in CHB and lays the groundwork for future combined immunotherapy strategies aimed at leveraging checkpoint receptors to restore immune function and improve clinical outcomes.

Exploring the immunomodulatory properties of glucan particles in human primary cells.

The immunomodulatory properties of ß-glucans have sparked interest among various medical fields. As vaccine adjuvants, glucan particles offer additional advantages as antigen delivery systems. This study reported the immunomodulatory properties of glucan particles with different size and chemical composition. The effect of glucan microparticles (GPs) and glucan nanoparticles (Glu 130 and 355 NPs) was evaluated on human immune cells. While GPs and Glu 355 NPs demonstrated substantial interaction with Dectin-1 receptor on monocytes, Glu 130 NPs exhibited reduced activation of this receptor. This observation was substantiated by blocking Dectin-1, resulting in inhibition of reactive oxygen species production induced by GPs and Glu 355 NPs. Notably, monocyte-derived dendritic cells (moDCs) stimulated by Glu 355 NPs exhibited phenotypic and functional maturation, essential for antigen cross-presentation. The immunomodulatory efficacy was investigated using an autologous mixed lymphocyte reaction (AMLR), resulting in considerable rates of lymphocyte proliferation and an intriguing profile of cytokine and chemokine release. Our findings highlight the importance of meticulously characterizing the size and chemical composition of ß-glucan particles to draw accurate conclusions regarding their immunomodulatory activity. This in vitro model mimics the human cellular immune response, and the results obtained endorse the use of ß-glucan-based delivery systems as future vaccine adjuvants.

Endotoxin contamination of nanoparticle formulations: A concern in vaccine adjuvant mechanistic studies.

The increasing awareness of endotoxin contamination has raised important questions during the study of the mechanism of action of the vaccine adjuvants. The endotoxins or lipopolysaccharides (LPS) can contaminate vaccine formulations contributing to result misinterpretations of the in vitro and in vivo studies. In this short communication, we considered the suitability of the Limulus amebocyte lysate (LAL) assay to quantify chitosan (Chit) nanoparticle (NP) endotoxin contamination to use them in a comparative in vitro immunotoxicology study using both LPS-free (LF) and non-LF Chit NPs. It was shown that chit NPs had a masking effect on endotoxin levels, hampering a reliable conclusion about the effect of their contamination. Neither non-LF nor LF Chit NPs induced the production of ROS in RAW 264.7 cells or IL-6 and TNF-α in PBMCs. The lack of effect of non-LF NPs was not expected and likely due to the NPs masking effect, more evident for higher deacetylation degree Chit. Overall, to prevent questionable results, nanomaterials should be produced under endotoxin-free conditions.

The Effect of Curcumin-Loaded Glucan Nanoparticles on Immune Cells: Size as a Critical Quality Attribute.

Curcumin is known for its multiple health benefits, largely due to its antioxidant and anti-inflammatory properties. It has been extensively studied as a therapeutic agent, however, it does not have good clinical efficacy due to its poor water solubility and bioavailability. Despite accepting the encapsulation of this compound in polymeric particles as one of the most promising strategies to increase its therapeutic value, these nanoparticles have fallen short of expectations due to a lack of assessment of their possible adverse effects on the immune system. Therefore, in this work, we report on a new method to encapsulate curcumin into glucan nanoparticles and their effects on cells of the immune system were evaluated. Two different-sized curcumin-loaded glucan NPs (GluCur 100 and GluCur 380) were produced, each with an encapsulation efficiency close to 100%, and were characterized regarding their size distribution, surface properties, and morphology. The results revealed the greatest hemolytic effect and cytotoxicity for the smallest particles (100 nm) tested in human PBMCs and RAW 264.7 cells. Although GluCur 380 NPs showed a weaker ROS production, they were able to inhibit the production of NO by macrophages. Furthermore, we found that the coagulation time was not affected by both sized-particles as well as platelet function. Additionally, both nanoparticles induced lymphocyte proliferation and TNF-α secretion by Mo-DCs. In conclusion, this report emphasizes the importance of the immunotoxicity assessment and how this is dependent on the intrinsic properties of nanomaterials, hopefully contributing to increasing the safety of nanomedicines.

Trehalose-releasing nanogels: A step toward a trehalose delivery vehicle for autophagy stimulation.

Trehalose has been widely studied as a treatment for a variety of human disorders due to its ability to stimulate autophagy. Trehalose, however, is poorly adsorbed and is hydrolyzed in the intestinal mucosa, and oral delivery requires relatively high doses to induce autophagy. The parenteral injection of trehalose-releasing nanogels proposed in this study offers an alternative mode of delivery. This study aimed to develop stable colloidal dispersions of trehalose-rich nanogels that could sustainably release trehalose under physiologically relevant conditions. The nanogel design was based on the covalent incorporation of 6-O-acryloyl-trehalose within a polymer network. A series of nine trehalose-rich nanogels with highly conjugated trehalose (up to 59 % w/w) were synthesized and shown to sustainably release trehalose at a rate that is not dose dependent. The nanogels were optimized to keep colloidal stability in serum-enriched cell culture media. The stable nanogels were not cytotoxic to primary HUVECs. Two selected nanogels with opposite surface charges were subjected to extended in vitro characterization that included a cellular uptake study and a hemocompatibility assay. Both nanogels were efficiently taken up by HUVECs during a short incubation. They also proved not to be hemolytic to human RBCs in concentrations up to 2.0 mg/mL. Finally, an in vivo autophagy stimulation study employing transgenic zebrafish and Drosophila larvae demonstrated that prolonged exposure to a cationic trehalose-releasing nanogel can induce autophagic activity in in vivo systems without any detectable toxicity.

Glucan Particles: Choosing the Appropriate Size to Use as a Vaccine Adjuvant.

Beta-glucans are a group of polysaccharides with intrinsic immunostimulatory properties which makes the design of new particulate vaccine adjuvants based on ß-glucans very promising. The size of the particles and the antigen loading method, encapsulated into particles or adsorbed on its surface, will influence the toxicological and adjuvanticity properties of the particulate adjuvant. Herein we describe the production of glucan nanoparticles (NPs) with three different sizes, approximately 150 nm, 350 nm, and microparticles as shells (GPs) with approximately 3 µm. The association of the antigen to the particulate adjuvant is described using model protein antigens. The method can be easily adapted for real protein antigens.

Unravelling the Immunotoxicity of Polycaprolactone Nanoparticles-Effects of Polymer Molecular Weight, Hydrolysis, and Blends.

Poly-ε-caprolactone (PCL) is a biodegradable polyester that has FDA and CE approval as a medical device. Nonetheless, the lack of toxicity exhibited by the polymer cannot be extrapolated to its nanomaterial conformation. Despite PCL-based NPs being widely studied in the biomedical field for their advantages as controlled drug delivery systems, little data describe PCL NPs' toxicity, particularly immunotoxicity. This work assessed different PCL-based delivery systems intended for protein delivery regarding their immunotoxicity and hemocompatibility. Two different molecular weight PCL polymers were used, as well as blends with chitosan and glucan. Results showed that the presence of NaOH during the production of PCL2 NPs and PCL2/glucan NPs induced PCL alkali hydrolysis, generating more reactive groups (carboxyl and hydroxyl) that contributed to an increased toxicity of the NPs (higher reduction in peripheral blood mononuclear cell viability and lower hemocompatibility). PCL2/glucan NPs showed an anti-inflammatory activity characterized by the inhibition of LPS stimulated nitric oxide (NO) and TNF-α. In conclusion, generalizations among different PCL NP delivery systems must be avoided, and immunotoxicity assessments should be performed in the early stage of product development to increase the clinical success of the nanomedicine.

Chitosan-coated PLGA nanoparticles for the nasal delivery of ropinirole hydrochloride: In vitro and ex vivo evaluation of efficacy and safety.

Nose-to-brain delivery is an attractive route for direct drug delivery to the central nervous system (CNS), avoiding hepatic first-pass metabolism and solving blood-brain barrier passage issues. Therefore, the aim of the present study was the development of PLGA and PLGA/chitosan (chit) nanoparticles (NPs) with mucoadhesive properties, able to encapsulate ropinirole hydrochloride (RH), an anti-Parkinsonian dopaminergic agonist, and suitable to promote RH delivery across the nasal mucosa. NPs produced by nanoprecipitation showed spherical shape and a mean average size of 98.8 nm and 468.0 nm (PLGA and PLGA/chit, respectively). RH loaded PLGA/chit NPs showed a complete release of the drug in simulated nasal electrolyte solution (SNES) over the period of 24 h and increased the permeation of RH through sheep nasal mucosa by 3.22-fold in comparison to PLGA NPs. None of RH loaded NPs induced hemolysis in whole blood or the production of reactive oxygen species (ROS) in Raw 264.7 cells. On their turn, PLGA/chit NPs decreased cell viability of Raw 264.7 cells and Peripheral Blood Mononuclear Cells (PBMCs) in a concentration-dependent manner. These results revealed that, particularly PLGA/chit NPs, could be a valuable carrier for the delivery of RH to the CNS, opening a new path for Parkinson's disease therapy.

A Methodological Safe-by-Design Approach for the Development of Nanomedicines.

Safe-by-Design (SbD) concepts foresee the risk identification and reduction as well as uncertainties regarding human health and environmental safety in early stages of product development. The EU's NANoREG project and further on the H2020 ProSafe initiative, NanoReg2, and CALIBRATE projects have developed a general SbD approach for nanotechnologies (e.g., paints, textiles, etc.). Based on it, the GoNanoBioMat project elaborated a methodological SbD approach (GoNanoBioMat SbD approach) for nanomedicines with a focus on polymeric nanobiomaterials (NBMs) used for drug delivery. NBMs have various advantages such as the potential to increase drug efficacy and bioavailability. However, the nanoscale brings new challenges to product design, manufacturing, and handling. Nanomedicines are costly and require the combination of knowledge from several fields. In this paper, we present the GoNanoBioMat SbD approach, which allows identifying and addressing the relevant safety aspects to address when developing polymeric NBMs during design, characterization, assessment of human health and environmental risk, manufacturing and handling, and combines the nanoscale and medicine field under one approach. Furthermore, regulatory requirements are integrated into the innovation process.

How the Lack of Chitosan Characterization Precludes Implementation of the Safe-by-Design Concept.

Efficacy and safety of nanomedicines based on polymeric (bio)materials will benefit from a rational implementation of a Safe-by-Design (SbD) approach throughout their development. In order to achieve this goal, however, a standardization of preparation and characterization methods and their accurate reporting is needed. Focusing on the example of chitosan, a biopolymer derived from chitin and frequently used in drug and vaccine delivery vector preparation, this review discusses the challenges still to be met and overcome prior to a successful implementation of the SbD approach to the preparation of chitosan-based protein drug delivery systems.

Chitosan Nanoparticles: Shedding Light on Immunotoxicity and Hemocompatibility.

Nanoparticles (NPs) assumed an important role in the area of drug delivery. Despite the number of studies including NPs are growing over the last years, their side effects on the immune system are often ignored or omitted. One of the most studied polymers in the nano based drug delivery system field is chitosan (Chit). In the scientific literature, although the physicochemical properties [molecular weight (MW) or deacetylation degree (DDA)] of the chitosan, endotoxin contamination and appropriate testing controls are rarely reported, they can strongly influence immunotoxicity results. The present work aimed to study the immunotoxicity of NPs produced with different DDA and MW Chit polymers and to benchmark it against the polymer itself. Chit NPs were prepared based on the ionic gelation of Chit with sodium tripolyphosphate (TPP). This method allowed the production of two different NPs: Chit 80% NPs (80% DDA) and Chit 93% NPs (93% DDA). In general, we found greater reduction in cell viability induced by Chit NPs than the respective Chit polymers when tested in vitro using human peripheral blood monocytes (PBMCs) or RAW 264.7 cell line. In addition, Chit 80% NPs were more cytotoxic for PBMCs, increased reactive oxygen species (ROS) production (above 156 µg/mL) in the RAW 264.7 cell line and interfered with the intrinsic pathway of coagulation (at 1 mg/mL) when compared to Chit 93% NPs. On the other hand, only Chit 93% NPs induced platelet aggregation (at 2 mg/mL). Although Chit NPs and Chit polymers did not stimulate the nitric oxide (NO) production in RAW 264.7 cells, they induced a decrease in lipopolysaccharide (LPS)-induced NO production at all tested concentrations. None of Chit NPs and polymers caused hemolysis, nor induced PBMCs to secrete TNF-α and IL-6 cytokines. From the obtained results we concluded that the DDA of the Chit polymer and the size of Chit NPs influence the in vitro immunotoxicity results. As the NPs are more cytotoxic than the corresponding polymers, one should be careful in the extrapolation of trends from the polymer to the NPs, and in the comparisons among delivery systems prepared with different DDA chitosans.

Safe-by-Design of Glucan Nanoparticles: Size Matters When Assessing the Immunotoxicity.

Glucan (from Alcaligenes faecalis) is a polymer composed of ß-1,3-linked glucose residues, and it has been addressed in different medical fields, namely in nanotechnology, as a vaccine or a drug delivery system. However, due to their small size, nanomaterials may present new risks and uncertainties. Thus, this work aims to describe the production of glucan nanoparticles (NPs) with two different sizes, and to evaluate the influence of the NPs size on immunotoxicity. Results showed that, immediately after production, glucan NPs presented average sizes of 129.7 ± 2.5 and 355.4 ± 41.0 nm. Glucan NPs of 130 nm presented greater ability to decrease human peripheral blood mononuclear cells and macrophage viability and to induce reactive oxygen species production than glucan NPs of 355 nm. Both NP sizes caused hemolysis and induced a higher metabolic activity in lymphocytes, although the concentration required to observe such effect was lower for the 130 nm glucan NPs. Regarding pro-inflammatory cytokines, only the larger glucan NPs (355 nm) were able to induce the secretion of IL-6 and TNF-α, probably due to their recognition by dectin-1. This higher immunomodulatory effect of the larger NPs was also observed in its ability to stimulate the production of nitric oxide (NO) and IL-1ß. On the contrary, a small amount of Glu 130 NPs inhibited NO production. In conclusion, on the safe-by-design of glucan NPs, the size of the particles should be an important critical quality attribute to guarantee the safety and effectiveness of the nanomedicine.

Airborne environmental fine particles induce intense inflammatory response regardless of the absence of heavy metal elements.

Airborne environmental particles (EP) more commonly referred as particulate matter (PM) are an illustrative marker of air pollution that is associated with adverse effects on human health. Considering, PM is a complex mixture, not only in terms of its chemical composition, but also in the range of particle size, it is difficult to identify which attribute contributes more for the toxicity. Currently, there is no report about the immunotoxicological effects caused by PM with reduced content of heavy metals. This study intends to address this gap and provides a detailed characterization and immunotoxicity evaluation of PM collected in an urban area with heavy traffic congestion. Environmental particles were separated by different sizes though a sucrose gradient. This method allowed to achieve 4 sized fractions: EP f 15 % with a mean diameter of 284 nm ± 1.86 nm, EP f 25 % with a mean diameter of 461 nm ± 1.72 nm, EP f 35 % with a mean diameter of 1845 nm ± 251 nm and EP f 45 % with a mean diameter of 2204 nm ± 310 nm. Only the fractions with the smallest sizes (EP f 15 % and EP f 25 %) were subsequently studied. The chemical composition of both fractions was not substantially different, and the dominant elements were C, O, Ca and K. Only EP f 25 % showed to have a small amount of Fe. Therefore, the heavy metal elements were eliminated through centrifugation. Essentially, we found that the EP f 15 % was more cytotoxic in RAW 264.7 cells than EP f 25 %, which indicates the smaller size as the motive for the higher toxicity. In addition, both fractions of EP presented a good internalization in macrophages after 2 h exposure and induced the production of reactive oxygen species in a concentration-dependent manner. Moreover, EP f 15 % and EP f 25 % led to a strong secretion of proinflammatory cytokines (TNF-α and IL-6) in human peripheral blood mononuclear cells (hPBMCs) in the 3 concentrations tested. The inflammatory response observed was independent of the presence of heavy metals and endotoxins, since these last were suppressed by using polymyxin B sulfate. This report emphasizes the importance of an adequate physicochemical characterization and adequate controls in the experiments to achieve a right interpretation of the biological effects caused by PM.

Oral treatment with T6-loaded yeast cell wall particles reduces the parasitemia in murine visceral leishmaniasis model.

Yeast cell wall particles isolated from Saccharomyces cerevisiae (scYCWPs) have a rich constitution of ß-glucan derived from the cell wall. After removing intracellular contents, ß-glucan molecules are readily recognized by dectin-1 receptors, present on the cytoplasmic membrane surface of the mononuclear phagocytic cells and internalized. Leishmania spp. are obligate intracellular parasites; macrophages are its primary host cells. An experimental murine model of visceral leishmaniasis caused by L. infantum was used to evaluate the antileishmanial activity of oral administration of these particles. A low-water soluble thiophene previously studied in vitro against L. infantum was entrapped into scYCWPs to direct it into the host cell, in order to circumvent the typical pharmacokinetic problems of water-insoluble compounds. We found that scYCWPs + T6 reduced the parasitic burden in the liver and spleen. There was an increase in IFN-γ levels related to nitric oxide production, explaining the reduction of the L. infantum burden in the tissue. Histological analysis did not show signals of tissue inflammation and biochemical analysis from plasma did not indicate signals of cytotoxicity after scYCWPs + T6 treatment. These findings suggested that scYCWPs + T6 administered through oral route reduced the parasitic burden without causing toxic effects, satisfying requirements for development of new strategies to treat leishmaniasis.

Hazard Assessment of Polymeric Nanobiomaterials for Drug Delivery: What Can We Learn From Literature So Far.

The physicochemical properties of nanobiomaterials, such as their small size and high surface area ratio, make them attractive, novel drug-carriers, with increased cellular interaction and increased permeation through several biological barriers. However, these same properties hinder any extrapolation of knowledge from the toxicity of their raw material. Though, as suggested by the Safe-by-Design (SbD) concept, the hazard assessment should be the starting point for the formulation development. This may enable us to select the most promising candidates of polymeric nanobiomaterials for safe drug-delivery in an early phase of innovation. Nowadays the majority of reports on polymeric nanomaterials are focused in optimizing the nanocarrier features, such as size, physical stability and drug loading efficacy, and in performing preliminary cytocompatibility testing and proving effectiveness of the drug loaded formulation, using the most diverse cell lines. Toxicological studies exploring the biological effects of the polymeric nanomaterials, particularly regarding immune system interaction are often disregarded. The objective of this review is to illustrate what is known about the biological effects of polymeric nanomaterials and to see if trends in toxicity and general links between physicochemical properties of nanobiomaterials and their effects may be derived. For that, data on chitosan, polylactic acid (PLA), polyhydroxyalkanoate (PHA), poly(lactic-co-glycolic acid) (PLGA) and policaprolactone (PCL) nanomaterials will be evaluated regarding acute and repeated dose toxicity, inflammation, oxidative stress, genotoxicity, toxicity on reproduction and hemocompatibility. We further intend to identify the analytical and biological tests described in the literature used to assess polymeric nanomaterials toxicity, to evaluate and interpret the available results and to expose the obstacles and challenges related to the nanomaterial testing. At the present time, considering all the information collected, the hazard assessment and thus also the SbD of polymeric nanomaterials is still dependent on a case-by-case evaluation. The identified obstacles prevent the identification of toxicity trends and the generation of an assertive toxicity database. In the future, in vitro and in vivo harmonized toxicity studies using unloaded polymeric nanomaterials, extensively characterized regarding their intrinsic and extrinsic properties should allow to generate such database. Such a database would enable us to apply the SbD approach more efficiently.

Activity and Cell-Death Pathway in Leishmania infantum Induced by Sugiol: Vectorization Using Yeast Cell Wall Particles Obtained From Saccharomyces cerevisiae.

Visceral leishmaniasis, caused by Leishmania infantum, is a neglected tropical disease, to which efforts in the innovation of effective and affordable treatments remain limited, despite the rising incidence in several regions of the world. In this work, the antileishmanial effects of sugiol were investigated in vitro. This compound was isolated from the bark of Cupressus lusitanica and showed promising activity against L. infantum. In spite of the positive results, it is known that the compound is a poorly water-soluble diterpene molecule, which hinders further investigation, especially in preclinical animal studies. Thus, in an alternative delivery method, sugiol was entrapped in glucan-rich particles obtained from Saccharomyces cerevisiae yeast cell walls (YCWPs). To evaluate the activity of sugiol, the experiments were divided into two parts: (i) the in vitro investigation of antileishmanial activity of free sugiol against L. infantum promastigotes after 24, 48, and 72 h of treatment and (ii) the evaluation of antileishmanial activity of sugiol entrapped in glucan-rich particles against intracellular L. infantum amastigotes. Free sugiol induced the cell-death process in promastigotes, which was triggered by enhancing cytosolic calcium level and promoting the autophagy up to the first 24 h. Over time, the presence of autophagic vacuoles became rarer, especially after treatment with lower concentrations of sugiol, but other cellular events intensified, like ROS production, cell shrinkage, and phosphatidylserine exposure. Hyperpolarization of mitochondrial membrane potential was found at 72 h, induced by the mitochondria calcium uptake, causing an increase in ROS production and lipid peroxidation as a consequence. These events resulted in the cell death of promastigotes by secondary necrosis. Sugiol entrapped in glucan-rich particles was specifically recognized by dectin-1 receptor on the plasma membrane of macrophages, the main host cell of Leishmania spp. Electron micrographs revealed particles containing sugiol within the infected macrophages and these particles were active against the intracellular L. infantum amastigotes without affecting the host cell. Therefore, the YCWPs act like a Trojan horse to successfully deliver sugiol into the macrophage, presenting an interesting strategy to deliver water-insoluble drugs to parasitized cells.

Poly(D,L-Lactic Acid) Nanoparticle Size Reduction Increases Its Immunotoxicity.

Polylactic acid (PLA), a biodegradable and biocompatible polymer produced from renewable resources, has been widely used as a nanoparticulate platform for antigen and drug delivery. Despite generally regarded as safe, its immunotoxicological profile, when used as a polymeric nanoparticle (NP), is not well-documented. Thus, this study intends to address this gap, by evaluating the toxicity of two different sized PLA NPs (PLAA NPs and PLAB NPs), produced by two nanoprecipitation methods and extensively characterized regarding their physicochemical properties in in vitro experimental conditions. After production, PLAA NPs mean diameter (187.9 ± 36.9 nm) was superior to PLAB NPs (109.1 ± 10.4 nm). Interestingly, when in RPMI medium, both presented similar mean size (around 100 nm) and neutral zeta potential, possibly explaining the similarity between their cytotoxicity profile in PBMCs. On the other hand, in DMEM medium, PLAA NPs presented smaller mean diameter (75.3 ± 9.8 nm) when compared to PLAB NPs (161.9 ± 8.2 nm), which may explain its higher toxicity in RAW 264.7. Likewise, PLAA NPs induced a higher dose-dependent ROS production. Irrespective of size differences, none of the PLA NPs presented an inflammatory potential (NO production) or a hemolytic activity in human blood. The results herein presented suggest the hypothesis, to be tested in the future, that PLA NPs presenting a smaller sized population possess increased cytotoxicity. Furthermore, this study emphasizes the importance of interpreting results based on adequate physicochemical characterization of nanoformulations in biological medium. As observed, small differences in size triggered by the dispersion in cell culture medium can have repercussions on toxicity, and if not correctly evaluated can lead to misinterpretations, and subsequent ambiguous conclusions.

Glucan Particles Are a Powerful Adjuvant for the HBsAg, Favoring Antiviral Immunity.

The lack of vaccine adjuvants that are able to induce robust T cell responses fosters the search for more powerful options. Pathogen-like particles are a promising approach. The adjuvant activity of pathogen-like particles is highly influenced by size and surface composition. This study aimed to evaluate the adjuvant potential of two different ß-glucan-based particles, blend chitosan/ß-glucan particles (ChiGluPs), which are positively charged and have mean size of 1276 nm, and neutral yeast-derived glucan particles (GPs), with a mean size of 3 µm. Additionally, chitosan particles (ChiPs) were used to understand the effect of ß-glucan addition (ChiGluPs). Mouse spleen cells responded through the production of either TNF-α or RANTES, following in vitro stimulation with particles containing either ß-glucan (ChiGluPs and GPs) or chitosan (ChiGluPs and ChiPs). Human monocytes responded to all particles through TNF-α secretion. Subcutaneous vaccination of mice with the hepatitis B surface antigen (HBsAg) showed increased serum IgG for all particles compared to HBsAg alone (435-, 4500-, or 2500-fold increase for either ChiPs, ChiGluPs, or GPs). Interestingly, only GPs elicited the secretion of HBsAg-specific Th1, Th2, Th9, Th17, Th22, and Treg-related cytokines. This study demonstrates, for the first time, that GPs can have a significant role against the hepatitis B virus by favoring antiviral immunity.

Optimization of Chitosan-α-casein Nanoparticles for Improved Gene Delivery: Characterization, Stability, and Transfection Efficiency.

Among non-viral vectors, the cationic polymer chitosan has gained attention as a gene delivery system. We hypothesized that the addition of casein into the nanoparticle's structure would facilitate a proper gene transfer. The work herein presented aimed to optimize the production method of chitosan-casein nanoparticles (ChiCas NPs) and to test their ability as a gene delivery system. ChiCas NPs formulation optimization was carried out by analyzing several characteristics such as NP size, zeta potential, and chitosan and casein incorporation efficacy. The best formulation developed presented small and homogenous particle size (around 335 nm) and positive zeta potential (≈ + 38 mV), and showed to be stable for 34 weeks both, at 4°C and 20°C. The particles were further used to entrap or to adsorb DNA and form NPs-DNA complexes. In vitro transfection studies, carried out in COS-7 cells, suggested a low transfection efficiency of the different NPs:DNA ratios tested, comparatively to the positive control. Nonetheless, we could observe that the complexes with larger sizes presented better transfection results than those with smaller diameters. To conclude, ChiCas NPs have great technological potential since the preparation process is very simple, and the DNA incorporation efficacy is very high and shows to be physically very stable. The NPs:DNA ratio still needs to be optimized with the aim of achieving better transfection results and being able to anticipate a high gene expression on DNA-based vaccination studies.