A lipophilic adjuvant carrier system for antigenic peptides

A lipoamino acid based synthetic peptide, (Lipid Core Peptide, LCP) derived from the conserved region of group A streptococci (GAS) was evaluated as potential candidate in a vaccine to prevent GAS-associated diseases, including rheumatic heart disease and post-streptococcal acute glomerulonephritis. Multiple copies of a peptide sequence from the bacterial surface M protein were incorporated into a lipid core and it was used to immunize mice with and without the application of adjuvant. The LCP construct had significantly enhanced immunogenicity compared with the monomeric peptide epitope. Furthermore, the peptides incorporated into the LCP system generated antibodies without the use of any conventional adjuvant.

Lipoamino acid-based adjuvant carrier system: Enhanced immunogenicity of group a streptococcal peptide epitopes

Lipoamino acid-based synthetic peptides (lipid core peptides, LCP) derived from the type-specific and conserved region determinants of group A streptococci (GAS) were evaluated as potential candidate sequences in a vaccine to prevent GAS-associated diseases, including rheumatic heart, disease and poststreptococcal acute glomerulonephritis. The LCP peptides had significantly enhanced immunogenicity as compared with the monomeric peptide epitopes. Furthermore, the peptides incorporated into the LCP system generated epitope-specific antibodies without the use of any conventional adjuvant.

N,N,N-trimethyl chitosan nanoparticles as a vitamin carrier system

There is considerable interest in incorporating stabilized vitamins into biopolymeric nanoparticles, especially in the development of carriers and active systems for pharmaceutical and food applications. Amongst biopolymer, chitosan is highly desirable owing to its good biocompatibility, biodegradability and ability to be chemically modified. In this paper, nanoparticles from three kinds of water-soluble derivative chitosan (N,N,N-trimethyl chitosan, TMC) have successfully been synthesized by ionic gelation with tripolyphosphate (TPP) anions. Combinations of concentrations of TMC and TPP have resulted in nanoparticles with varying sizes for which the capability for loading with vitamins was investigated. Zeta potential measurement and particle size analysis demonstrated that the size of the nanoparticles wasoptimized (196±8nm) when the lowest TMC and TPP amounts were used, i.e., 0.86mgmL -1 and 0.114mgmL -1 respectively. As the TMC and/or the TPP concentrations increase, the resulting size of the nanoparticles increases considerably. Three different vitamins (B9, B12 and C) were tested as additives and the final system characterized in relation to size, morphology, spectroscopic and zeta potential properties. In general, the incorporation of vitamins increased all the TMC-TPP original nanoparticle sizes, reaching a maximum diameter of 534±20nm when loaded with vitamin C. The presence of vitamins also decreases the zeta potential, with one exception observed when using vitamin C. The preliminary results of this study suggested that all TMC/TPP nanoparticles can be successfully used as a stable medium to incorporate and transport vitamins, with potential applications in foodstuffs. © 2011 Elsevier Ltd.

Physicochemical Characterization and Development of a Microemulsion System for Transdermal Use

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Development and characterization of biocompatible isotropic and anisotropic oil-in-water colloidal dispersions as a new delivery system for methyl dihydrojasmonate antitumor drug

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Synthesis and immunological evaluation of M protein targeted tetra-valent and tri-valent group A streptococcal vaccine candidates based on the lipid-core peptide system

Group A streptococcus (GAS) is responsible for causing many clinical complications including the relatively benign streptococcal pharyngitis and impetigo. However. if left untreated. these conditions may lead to more severe diseases such as rheumatic fever (RF) and rheumatic heart disease (RHD). These diseases exhibit high morbidity and mortality, Particularly in developing countries and in indigenous populations of affluent countries. Only ever occur following GAS infection, a vaccine offers Promise for their Prevention. As stich, we have investigated the Use of the lipid-core peptide (LCP) system for the development of multi-valent Prophylactic GAS vaccines. The current study has investigated the capacity of this system to adjuvant LIP to four different GAS peptide epitopes. Presented are the synthesis and immunological assessment of tetra-valent and tri-valent GAS LCP systems. We demonstrated their capacity to elicit systemic IgG antibody responses in B10.BR mice to all GAS peptide epitopes. The data also showed that the LCP systems Were self-adjuvanting. These findings are particularly encouraging for the development of multi-valent LCP-based GAS vaccines.

Method for the synthesis of highly pure vaccines using the lipid core peptide system

Traditional vaccines consisting of whole attenuated microorganisms, killed microorganisms, or microbial components, administered with an adjuvant (e.g. alum), have been proved to be extremely successful. However, to develop new vaccines, or to improve upon current vaccines, new vaccine development techniques are required. Peptide vaccines offer the capacity to administer only the minimal microbial components necessary to elicit appropriate immune responses, minimizing the risk of vaccination associated adverse effects, and focusing the immune response toward important antigens. Peptide vaccines, however, are generally poorly immunogenic, necessitating administration with powerful, and potentially toxic adjuvants. The attachment of lipids to peptide antigens has been demonstrated as a potentially safe method for adjuvanting peptide epitopes. The lipid core peptide (LCP) system, which incorporates a lipidic adjuvant, carrier, and peptide epitopes into a single molecular entity, has been demonstrated to boost immunogenicity of attached peptide epitopes without the need for additional adjuvants. The synthesis of LCP systems normally yields a product that cannot be purified to homogeneity. The current study describes the development of methods for the synthesis of highly pure LCP analogs using native chemical ligation. Because of the highly lipophilic nature of the LCP lipid adjuvant, difficulties (e.g. poor solubility) were experienced with the ligation reactions. The addition of organic solvents to the ligation buffer solubilized lipidic species, but did not result in successful ligation reactions. In comparison, the addition of approximately 1% (w/v) sodium dodecyl sulfate (SDS) proved successful, enabling the synthesis of two highly pure, tri-epitopic Streptococcus pyogenes LCP analogs. Subcutaneous immunization of B10.BR (H-2(k)) mice with one of these vaccines, without the addition of any adjuvant, elicited high levels of systemic IgG antibodies against each of the incorporated peptides. Copyright (c) 2006 European Peptide Society and John Wiley & Sons, Ltd.

Chitosan Nanoparticles Loaded The Herbicide Paraquat: The Influence Of The Aquatic Humic Substances On The Colloidal Stability And Toxicity.

Polymeric nanoparticles have been developed for several applications, among them as carrier system of pesticides. However, few studies have investigated the fate of these materials in the environment in relation to colloidal stability and toxicity. In nature, humic substances are the main agents responsible for complexation with metals and organic compounds, as well as responsible for the dynamics of these nanoparticles in aquatic and terrestrial environments. In this context, the evaluation of the influence of aquatic humic substances (AHS) on the colloidal stability and toxicity of polymeric nanoparticles of chitosan/tripolyphosphate with or without paraquat was performed. In this study, the nanoparticles were prepared by the ionic gelation method and characterized by size distribution measurements (DLS and NTA), zeta potential, infrared and fluorescence spectroscopy. Allium cepa genotoxicity studies and ecotoxicity assays with the alga Pseudokirchneriella subcapitata were used to investigate the effect of aquatic humic substances (AHS) on the toxicity of this delivery system. No changes were observed in the physical-chemical stability of the nanoparticles due to the presence of AHS using DLS and NTA techniques. However some evidence of interaction between the nanoparticles and AHS was observed by infrared and fluorescence spectroscopies. The ecotoxicity and genotoxicity assays showed that humic substances can decrease the toxic effects of nanoparticles containing paraquat. These results are interesting because they are important for understanding the interaction of these nanostructured carrier systems with species present in aquatic ecosystems such as humic substances, and in this way, opening new perspectives for studies on the dynamics of these carrier systems in the ecosystem.

Quercetin in w/o microemulsion: In vitro and in vivo skin penetration and efficacy against UVB-induced skin damages evaluated in vivo

The present study evaluated the potential of a w/o microemulsion as a topical carrier system for delivery of the antioxidant quercetin. Topical and transdermal delivery of quercetin were evaluated in vitro Using porcine car skin mounted on a Franz diffusion cell and in vivo on hairless-skin mice. Skin irritation by topical application of the microemulsion containing quercetin, and the protective effect of the formulation on UVB-induced decrease of endogenous reduced glutathione levels and increase of cutaneous proteinase secretion/activity were also investigated. The w/o microemulsion increased the penetration of quercetin into the stratum corneum and epidermis plus dermis at 3, 6. 9 and 12 h post-application in vitro and in vivo at 6 h post-application. No transdermal delivery of quercetin Occurred. By evaluating established endpoints of skin irritation (erythema formation, epidermis thickening and infiltration of inflammatory cells), the Study demonstrated that the daily application of the w/o microemulsion for up to 2 days did not cause skin irritation. W/o microemulsion containing quercetin significantly prevented the UVB irradiation-induced GSH depletion and secretion/activity of metalloproteinases. (C) 2008 Elsevier B.V. All rights reserved.

Functional analysis of the murine T lymphocyte immune response to a protozoan parasite, Leishmania tropica

The results presented in this review summarize a seirs of experiments designed to characterize the murine T cell imune response to the protozoan parasite Leishmania tropica. Enriched T cell populations and T cell clones specific for L. tropica antigens were derived from lymph nodes of primed mice and maintained in continous culture in vitro. These T lymphocytes were shown (A) to express the Lyt 1+ 3- cell surface phenotype, (B) to proliferate specifically in vitro in response to parasite antigens, together with a source of irradiated syngeneic macrophages, (C) to transfer antigen-specific delayed-type hypersensitivity (DTH) responses to normal syngeneic mice, (D) to induce specific activation of parasitized macrophages in vitro resulting in the destruction of intracellular parasites, (E) to provide specific helper activity for antibody responses in vitro in a hapten-carrier system. Protection studies using these defiened T cell populations should allow the characterization of parasite antigen(s) implicated in the induction of cellular immune responses beneficial for the host.

Fate of TLR-1/TLR-2 agonist functionalised pDNA nanoparticles upon deposition at the human bronchial epithelium in vitro.

BACKGROUND: Plasmid DNA vaccination is a promising approach, but studies in non-human primates and humans failed to achieve protective immunity. To optimise this technology further with focus on pulmonary administration, we developed and evaluated an adjuvant-equipped DNA carrier system based on the biopolymer chitosan. In more detail, the uptake and accompanying immune response of adjuvant Pam3Cys (Toll-like receptor-1/2 agonist) decorated chitosan DNA nanoparticles (NP) were explored by using a three-dimensional (3D) cell culture model of the human epithelial barrier. Pam3Cys functionalised and non-functionalised chitosan DNA NP were sprayed by a microsprayer onto the surface of 3D cell cultures and uptake of NP by epithelial and immune cells (blood monocyte-derived dendritic cells (MDDC) and macrophages (MDM)) was visualised by confocal laser scanning microscopy. In addition, immune activation by TLR pathway was monitored by analysis of interleukin-8 and tumor necrosis factor-α secretions (ELISA). RESULTS: At first, a high uptake rate into antigen-presenting cells (MDDC: 16-17%; MDM: 68-75%) was obtained. Although no significant difference in uptake patterns was observed for Pam3Cys adjuvant functionalised and non-functionalised DNA NP, ELISA of interleukin-8 and tumor necrosis factor-α demonstrated clearly that Pam3Cys functionalisation elicited an overall higher immune response with the ranking of Pam3Cys chitosan DNA NPâeuro0/00>âeuro0/00chitosan DNA NPâeuro0/00=âeuro0/00DNA unloaded chitosan NPâeuro0/00>âeuro0/00control (culture medium). CONCLUSIONS: Chitosan-based DNA delivery enables uptake into abluminal MDDC, which are the most immune competent cells in the human lung for the induction of antigen-specific immunity. In addition, Pam3Cys adjuvant functionalisation of chitosan DNA NP enhances significantly an environment favoring recruitment of immune cells together with a Th1 associated (cellular) immune response due to elevated IL-8 and TNF-α levels. The latter renders this DNA delivery approach attractive for potential DNA vaccination against intracellular pathogens in the lung (e.g., Mycobacterium tuberculosis or influenza virus).

In vitro antitumor activity of methotrexate via pH-sensitive chitosan nanoparticles

Nanoparticles with pH-sensitive behavior may enhance the success of chemotherapy in many cancers by efficient intracellular drug delivery. Here, we investigated the effect of a bioactive surfactant with pH-sensitive properties on the antitumor activity and intracellular behavior of methotrexate-loaded chitosan nanoparticles (MTX-CS-NPs). NPs were prepared using a modified ionotropic complexation process, in which was included the surfactant derived from Nα,Nε-dioctanoyl lysine with an inorganic lithium counterion. The pH-sensitive behavior of NPs allowed accelerated release of MTX in an acidic medium, as well as membrane-lytic pH-dependent activity, which facilitated the cytosolic delivery of endocytosed materials. Moreover, our results clearly proved that MTX-CSNPs were more active against the tumor HeLa and MCF-7 cell lines than the free drug. The feasibilty of using NPs to target acidic tumor extracellular pH was also shown, as cytotoxicity against cancer cells was greater in a mildly acidic environment. Finally, the combined physicochemical and pH-sensitive properties of NPs generally allowed the entrapped drug to induce greater cell cycle arrest and apoptotic effects. Therefore, our overall results suggest that pH-sensitive MTX-CS-NPs could be potentially useful as a carrier system for tumor and intracellular drug delivery in cancer therapy.