Sandra Jesus

Immune response elicited by an intranasally delivered HBsAg low-dose adsorbed to poly-ε-caprolactone based nanoparticles

Among new strategies to increase hepatitis B virus (HBV) vaccination, especially in developing countries, the development of self-administered vaccines is considered one of the most valuable. Nasal vaccination using polymeric nanoparticles (NPs) constitutes a valid approach to this issue. In detail, poly-ε-caprolactone (PCL)/chitosan NPs present advantages as a mucosal vaccine delivery system: the high resistance of PCL against degradation in biological fluids and the mucoadhesive and immunostimulatory properties of chitosan. In vitro studies revealed these NPs were retained in a mucus-secreting pulmonary epithelial cell line and were capable of entering into differentiated epithelial cells. The intranasal (IN) administration of 3 different doses of HBsAg (1.5 μg, 5 μg and 10 μg) adsorbed on a fixed amount of PCL/chitosan NPs (1614 μg) generated identical titers of serum anti-HBsAg IgG and anti-HBsAg sIgA in mice nasal secretions. Besides other factors, the NP surface characteristics, particularly, zeta potential differences among the administered formulations are believed to be implicated in the outcome of the immune response generated.

Synthesis and controlled curcumin supramolecular complex release from pH-sensitive modified gum-arabic-based hydrogels

Curcumin (CUR) is a polyphenolic compound including a beta-diketone moiety, which is associated with numerous pharmacological activities, but applications are limited due to its low water solubility. Thus, in this work some inclusion complexes of CUR with alpha-cyclodextrin (α-CD) and beta-cyclodextrin (β-CD) were prepared using different host : guest proportions to improve drug solubilization in biological fluids. The formation of these complexes was confirmed by 1H NMR and thermogravimetric analysis. The stoichiometries of the CUR/α-CD and CUR/β-CD complexes were 1 : 1 and 1 : 2 and the association constants were 344 mol−1 L and 7.2 × 107 mol−2 L2 for α-CD and β-CD, respectively. The major stability of the CUR/β-CD complex is justified by an inclusion of the aromatic ring inside the CD cavity, whilst in the case of α-CD-complexes the interactions occur via H-bridges, showing the latter complexes’ slow exchange on the NMR time-scale. Even so, the solubility of curcumin complexes is clearly controlled by the solubility of CDs, showing the highest solubility for CUR/α-CD complexes. Hydrogels of modified gum arabic containing CUR/α-CD (1 : 4) were obtained and used for controlled release of CUR in simulated intestinal fluid (SIF) and simulated gastric fluid (SGF). The kinetics of release was pH-responsive and the percentage of CUR released was ca. 97% in SIF and 6.7% in SGF. For the toxicity studies on undifferentiated Caco-2 cells, IC50s of 63.4 ± 14.4 μg mL−1 and 85.2 ± 14.9 μg mL−1 for CUR and CUR/α-CD (1 : 4), respectively, were obtained. The toxicity of these samples on differentiated Caco-2 cells was lower than on undifferentiated cells. Additionally, the CUR incorporated into hydrogels showed no toxic effects on differentiated and undifferentiated Caco-2 cells, indicating the pharmaceutical potential of three-dimensional matrices of GAm for controlled release of CUR complexed with cyclodextrin.

Poly-ϵ-caprolactone/chitosan nanoparticles provide strong adjuvant effect for hepatitis B antigen

AIM This work aims to investigate the adjuvant effect of poly-ϵ-caprolactone/chitosan nanoparticles (NPs) for hepatitis B surface antigen (HBsAg) and the plasmid DNA encoding HBsAg (pRC/CMV-HBs). METHODS Both antigens were adsorbed onto preformed NPs. Vaccination studies were performed in C57BL/6 mice. Transfection efficiency was investigated in A549 cell line. RESULTS HBsAg-adsorbed NPs generated strong anti-HBsAg IgG titers, mainly of IgG1 isotype, and induced antigen-specific IFN-γ and IL-17 secretion by spleen cells. The addition of pRC/CMV-HBs to the HBsAg-adsorbed NPs inhibited IL-17 secretion but had minor effect on IFN-γ levels. Lastly, pRC/CMV-HBs-loaded NPs generated a weak serum antibody response. CONCLUSION Poly-ϵ-caprolactone/chitosan NPs provide a strong humoral adjuvant effect for HBsAg and induce a Th1/Th17-mediated cellular immune responses worth explore for hepatitis B virus vaccination.

Chitosan-based nanoparticles as a hepatitis B antigen delivery system.

The design of antigen delivery systems, particularly for mucosal surfaces, has been a focus of interest in recent years. In this chapter, we describe the preparation of chitosan-based particles as promising antigen delivery systems for mucosal surfaces already tested by our group with hepatitis B surface antigen. The final proof of the concept is always carried out with immunization studies performed in an appropriate animal model. However, before these important studies, it is advisable that the delivery system should be submitted to a variety of in vitro tests. Among several tests, the characterization of the particles (size, morphology, and zeta potential), the studies of antigen adsorption onto particles, the evaluation of toxicity of the particles, and the studies of particle uptake into lymphoid organs are the most important and will be described in this chapter.

Poly-ε-caprolactone/Chitosan and Chitosan Particles: Two Recombinant Antigen Delivery Systems for Intranasal Vaccination.

Several evidences converge on the idea that among the mucosal administration routes, the nasal mucosa is the most attractive site for the delivery of vaccines. Mucoadhesive particulate adjuvants should be able to increase the residence time of antigens in nasal cavity in order to increase their probability of being taken up by nasopharynx-associated lymphoid tissue (NALT) cells and subsequently to initiate the innate and adaptive immune response. Focusing on chitosan, a mucoadhesive biopolymer, we describe in this chapter a method to prepare antigen loaded chitosan nanoparticles and a second method to prepare antigen loaded poly-ε-caprolactone/chitosan nanoparticles. Additionally the methodology for the assessment of mucoadhesivity of the delivery system is also described. The two critical procedures in mice intranasal immunization experiments include challenges in the intranasal administration itself due to the small mouse nose, and the other is related with the collection of mucosal secretions to assess the sIgA. The techniques are difficult to perform without advanced training. Therefore, protocols followed in our laboratory, as well as some tips, are described in this chapter.

The Inclusion of Chitosan in Poly-ε-caprolactone Nanoparticles: Impact on the Delivery System Characteristics and on the Adsorbed Ovalbumin Secondary Structure

This report extensively explores the benefits of including chitosan into poly-ε-caprolactone (PCL) nanoparticles (NPs) to obtain an improved protein/antigen delivery system. Blend NPs (PCL/chitosan NPs) showed improved protein adsorption efficacy (84%) in low shear stress and aqueous environment, suggesting that a synergistic effect between PCL hydrophobic nature and the positive charges of chitosan present at the particle surface was responsible for protein interaction. Additionally, thermal analysis suggested the blend NPs were more stable than the isolated polymers and cytotoxicity assays in a primary cell culture revealed chitosan inclusion in PCL NPs reduced the toxicity of the delivery system. A quantitative 6-month stability study showed that the inclusion of chitosan in PCL NPs did not induce a change in adsorbed ovalbumin (OVA) secondary structure characterized by the increase in the unordered conformation (random coil), as it was observed for OVA adsorbed to chitosan NPs. Additionally, the slight conformational changes occurred, are not expected to compromise ovalbumin secondary structure and activity, during a 6-month storage even at high temperatures (45°C). In simulated biological fluids, PCL/chitosan NPs showed an advantageous release profile for oral delivery. Overall, the combination of PCL and chitosan characteristics provide PCL/chitosan NPs valuable features particularly important to the development of vaccines for developing countries, where it is difficult to ensure cold chain transportation and non-parenteral formulations would be preferred.

Chitosan:β-glucan particles as a new adjuvant for the hepatitis B antigen

Graphical abstract Figure. No caption available. ABSTRACT The development of new vaccine adjuvants is urgently needed not only to enable new routes of vaccine administration but mostly to go beyond protective humoral immunity, often insufficient to fight infectious diseases. The association of two or more immunopotentiators or mimicking pathogen physicochemical properties are strategies that can favor powerful and more balanced Th1/Th2 immune responses. Therefore, the present work aimed to combine both chitosan and &bgr;‐glucan biopolymers in the same particle, preferably with surface &bgr;‐glucan localization to simulate the cell wall of some pathogens and to stimulate the immune cells expressing the Dectin‐1 receptor. Chitosan:&bgr;‐glucan particles (ChiGluPs) were developed through a chitosan precipitation method. The chitosan was precipitated into a &bgr;‐glucan alkaline solution followed by genipin crosslink. The optimized method produced particles with a mean diameter of 837 nm for ChiPs and 1274 nm for ChiGluPs. &bgr;‐glucan surface location was confirmed by zeta potential measurements (+24 mV for ChiGluPs and +36 mV for ChiPs) and zeta potential titration. These new particles showed high antigen loading efficacy and low cytotoxicity. Mice vaccination studies revealed that both ChiPs and ChiGluPs had an adjuvant effect for the hepatitis B surface antigen (HBsAg), with ChiGluPs resulting in serum anti‐HBsAg total IgG 16‐fold higher than ChiPs, when administered with 1.5 &mgr;g HBsAg per dose. Specifically, IgG1 subclass was 5‐fold higher and IgG3 subclass was 4‐fold higher for ChiGluPs comparing to ChiPs. Overall, the preparation method developed allowed the advantageous combination of &bgr;‐glucan with chitosan, without chemical functionalization, which represents an additional step toward tailor‐made adjuvants production using simple precipitation techniques.