Desenvolvimento e caracterização de nanopartículas lipídicas sólidas para administração cutânea de trans-resveratrol

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Recent advances in nanoparticle carriers for coordination complexes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Nanotechnology-based drug delivery systems for the treatment of Alzheimer's disease

Alzheimer's disease is a neurological disorder that results in cognitive and behavioral impairment. Conventional treatment strategies, such as acetylcholinesterase inhibitor drugs, often fail due to their poor solubility, lower bioavailability, and ineffective ability to cross the blood-brain barrier. Nanotechnological treatment methods, which involve the design, characterization, production, and application of nanoscale drug delivery systems, have been employed to optimize therapeutics. These nanotechnologies include polymeric nanoparticles, solid lipid nanoparticles, nanostructured lipid carriers, microemulsion, nanoemulsion, and liquid crystals. Each of these are promising tools for the delivery of therapeutic devices to the brain via various routes of administration, particularly the intranasal route. The objective of this study is to present a systematic review of nanotechnology-based drug delivery systems for the treatment of Alzheimer's disease.

Nanopartículas lipídicas como sistemas carreadores para ivermectina e metopreno visando aplicações em veterinária

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The Use of Nanotechnology in Cosmetic Formulations: The Influence of Vehicle in the Vitamin A Skin Penetration

Despite the efficacy of topical retinoic acid, skin reactions have limited its acceptance by patients. Other retinoids, like Retinyl Palmitate (RP), are considerably less irritating, but they are also less effective. In order to enhance the performance of retinoids, in this work RP has been added to cosmetic formulations such as nanoemulsions, which can provide better penetration of this active substance. Because the vehicle can directly influence the skin penetration and the effectiveness of RP, two skin care products containing 5000 UI RP have been developed and investigated, namely a nanoemulsifying system and a classic gel cream. In vitro penetration tests were conducted by using Franz diffusion cells and placing porcine ear skin and iso-propanol in the receptor compartment. The RP concentration in the skin layers was analyzed by high performance liquid chromatography, and a Zeta-Sizer system was employed for measurement of the the particle size distribution. The penetration tests revealed a large difference between the vehicles in terms of the RP concentrations in each skin layer. The classic gel cream furnished better RP penetration in both the stratum corneum and the epidermis without stratum corneum + dermis, as compared to the self-nanoemulsifying system. The two vehicles displayed the same particle size (between 100 and 200 nm). Better understanding of RP skin delivery using different vehicles has been acquired, and the importance of evaluating the efficacy of nanocosmetics. Results from the present study should also contribute to the assessment of commercial self-nanoemulsifying systems with potential application in the facile production of nanoemulsions.

Nanopartículas lipídicas sólidas e vesículas cataniônicas contendo ftalocianina de cloro alumínio aplicadas nos processos fotodinâmicos

O trabalho apresentado foi realizado em duas etapas independentes e baseou-se no estudo de diferentes sistemas nanométricos para viabilizar a aplicação da ftalocianina de cloro alumínio (ClAlPc) na terapia fotodinâmica (TFD) para o tratamento do câncer de pele do tipo melanoma. O fármaco fotossensibilizante (FS) utilizado apresenta propriedades físico-químicas que lhe permitem exercer sua atividade fotodinâmica com excelência, sem a interferência do cromóforo endógeno melanina existente nas células melanocíticas. Para driblar sua elevada hidrofobicidade, ClAlPc foi encapsulada em sistemas nanométricos para administração em meio fisiológico. Inicialmente nanopartículas lipídicas sólidas (NLS) foram desenvolvidas por emulsificação direta, após um estudo de elaboração do diagrama de fases. O compritol foi o lipídio sólido escolhido para compor as NLS, com diferentes concentrações de ClAlPc. Todas as formulações desenvolvidas foram devidamente caracterizadas, com tamanho médio entre 100 e 200 nm, baixa polidispersão, potencial zeta adequadamente negativo (~|30| mV), drug loading de ClAlPc entre 76-94% (com pequena redução após 24 meses) e alta eficiência de encapsulação (E.E.). A morfologia arredondada das nanopartículas foi confirmada por microscopia eletrônica de transmissão e de força atômica. A estabilidade das NLS foi de 24 meses. A avaliação da cristalinidade do lipídio revelou a integração da ClAlPc à matriz lipídica da NLS, presença de estruturas polimórficas e grau de cristalinidade adequado, sem alterações após 24 meses. Nos estudos de difusão in vitro, observou-se que ftalocianina encapsulada nas NLS acumulam-se preferencialmente na epiderme e derme do que no estrato córneo, sem traços de permeação do ativo. Foi confirmado o caráter biocompatível das NLS sobre fibroblastos NIH-3T3. A ftalocianina encapsulada nas NLS não foi tóxica na linhagem de melanoma B16-F10 na ausência de luz, porém, apresentou excelente efeito fototóxico (0,75 ?g mL-1 de ClAlPc nanoencapsulada e irradiação entre 0,5 e 2,0 J cm-2), com redução da viabilidade celular de 87%. O segundo sistema de veiculação estudado foram as vesículas cataniônicas (VesCat), que se formam espontaneamente em água com o tensoativo TriCat 12. A obtenção das vesículas contendo ClAlPc envolve uma etapa adicional, para remoção de solvente orgânico, que foi aprimorada, reduzindo o tempo de produção em 55%. As VesCat/ClAlPc obtidas mantiveram suas propriedades físico-químicas e morfologia arredondada (confirmada por microscopia eletrônica de varredura), drug loading de 47% e alta E.E. Os resultados comprovaram que a aplicação desses dois sistemas nanométricos é altamente eficiente para aplicação da TFD no tratamento do câncer de pele do tipo melanoma ou outras doenças cutâneas, apresentando características favoráveis para avanços nos estudos de fase clínica e pré-clínica.

Sistemas semissólidos à base de nanopartículas lipídicas

As nanopartículas lipídicas foram desenvolvidas no início dos anos 90 e, atendendo às vantagens que apresentam comparativamente a outros sistemas coloidais, têm-se demonstrado muito promissoras, tanto para uso cosmético como farmacêutico. No entanto, atualmente apenas existem comercializados produtos cosméticos à base de nanopartículas lipídicas, o que pode ser justificado pelas restrições regulamentares relacionadas com a introdução de medicamentos no mercado. Existem dois tipos de nanopartículas lipídicas, as nanopartículas de lípidos sólidos (Solid Lipid Nanoparticles, SLN) e os vetores lipídicos nanoestruturados (Nanostructured Lipid Carriers, NLC), consistindo ambos em dispersões aquosas de nanopartículas sólidas. A baixa viscosidade destes sistemas dificulta a sua aplicação tópica. Neste contexto, têm sido desenvolvidas várias formulações semissólidas à base de nanopartículas lipídicas para aplicação tópica, nomeadamente cutânea, ocular, nasal e vaginal. A primeira parte desta dissertação consiste na revisão bibliográfica relativa ao estado da arte dos sistemas semissólidos à base de nanopartículas lipídicas, para uso farmacêutico e cosmético, baseada nos estudos realizados por diversos autores, entre 2012 e 2016. Na segunda parte, são apresentados resultados do trabalho experimental relativo ao desenvolvimento e caraterização de uma formulação semissólida à base de nanopartículas lipídicas.

Drug Delivery through the Blood-Brain Barrier through Use of Vascular Cell Adhesion Molecule 1- Targeted Nanoparticles

The blood brain barrier (BBB) is a semi-permeable membrane separating the brain from the bloodstream, preventing many drugs that treat neurological diseases, such as Alzheimer’s and Parkinson’s, from reaching the brain. Our project aimed to create a novel drug delivery system targeting the brain during neural inflammation. We developed a cationic solid lipid nanoparticle (CSLN) complex composed of cationic nanoparticles, biotin, streptavidin, and anti-vascular cell adhesion molecule-1 (anti- VCAM-1) antibodies. The anti-VCAM-1 antibody is used to target VCAM-1, a cell adhesion protein found on the BBB endothelium. VCAM-1 expression is elevated in the presence of inflammatory molecules, such as tumor necrosis factor-alpha (TNF- α). Through the use of a simple BBB model, results showed that our novel drug delivery system experienced some level of success in targeting the brain inflammation due to increasing TNF-α concentrations. This is promising for drug delivery research and provides support for VCAM-1 targeting using more robust and complex BBB models.

Grafting of Dispersants on MoS2 Nanoparticles in Base Oil Lubrication of Steel

Solid lubricant nanoparticles in suspension in oil are good lubricating options for practical machinery. In this article, we select a range of dispersants, based on their polar moieties, to suspend 50-nm molybdenum disulfide particles in an industrial base oil. The suspension is used to lubricate a steel on steel sliding contact. A nitrogen-based polymeric dispersant (aminopropyl trimethoxy silane) with a free amine group and an oxygen-based polymeric dispersant (sorbital monooleate) when grafted on the particle charge the particle negatively and yield an agglomerate size which is almost the same as that of the original particle. Lubrication of the contact by these suspensions gives a coefficient of friction in the similar to 0.03 range. The grafting of these surfactants on the particle is shown here to be of a chemical nature and strong as the grafts survive mechanical shear stress in tribology. Such grafts are superior to those of other silane-based test surfactants which have weak functional groups. In the latter case, the particles bereft of strong grafts agglomerate easily in the lubricant and give a coefficient of friction in the 0.08-0.12 range. This article investigates the mechanism of frictional energy dissipation as influenced by the chemistry of the surfactant molecule.

Theranostic applications of fluorescent liquid crystalline nanoparticles

Lipid liquid crystalline nanoparticles can find application as nanocarriers in several fields of the daily life but, very likely, the pharmaceutical arena is the most relevant. Indeed, several problems encountered in drugs administration (e.g. critical sideeffects from antitumor drugs) require alternative, less invasive, but simultaneously efficient therapeutic routes to be explored. Novel fields of personalized nanomedicine are developing in this direction. One of the most interesting is theranostic, which calls for the design of platforms capable of combining therapeutic and diagnostic functionalities. In this optic, we explored the potential of monoolein-based cubosomes and hexosomes as nanocarriers for theranostic purposes. Our work focussed on the design of lipid nanoparticles able to deliver antineoplastic drugs and imaging probes for fluorescent optical in vitro and in vivo imaging. We developed cubosome formulations loaded with antineoplastic drugs and useful for the fluorescence imaging of cells. Such formulations were also actively targeted to cancer cells and coupled with a NIR-emitting fluorophore, which was the promise for in vivo applications. We also investigated hexosomes with encouraging results encapsulating in their lipid matrix a BODIPY derivative with solvatochromic properties, helpful for the understanding of the dye localization. Importantly, we reported (manuscript submitted) the first proof-of-principle for in vivo fluorescence optical imaging application using monoolein-based cubosomes in a healthy mouse animal model. Finally, since relatively little is known about the interaction of cubosomes with biological systems, their effects on lipid droplets, mitochondria and lipid profile of HeLa cells were deeply studied. This thesis is divided in two main parts. The introduction section reports on the essential background of the research field, and it is followed by the publications (published or submitted) resulting from these three years of work.

Functionalization of lamellar molybdenum disulphide nanocomposite with gold nanoparticles

This work explores the functionalization of an organic-inorganic MoS2 lamellar compound, prepared by a Chemical Liquid Deposition Method (CLD), that has an interlamellar distance of ~5.2 nm, using clusters of gold nanoparticles. The gold nanoparticles have a mean diameter of 1.2 nm, a stability of ~85 days, and a zeta potential measured to be ζ = -6.8 mV (solid). The nanoparticles are localized in the hydrophilic zones, defined by the presence of amine groups of the surfactant between the lamella of MoS2. SEM, TEM, EDAX and electron diffraction provide conclusive evidence of the interlamellar insertion of the gold nanoparticles in the MoS2.

Nanoparticles increase the efficacy of cancer chemopreventive agents in cells exposed to cigarette smoke condensate

Lung cancer is a leading cause of death in developed countries. Although smoking cessation is a primary strategy for preventing lung cancer, former smokers remain at high risk of cancer. Accordingly, there is a need to increase the efficacy of lung cancer prevention. Poor bioavailability is the main factor limiting the efficacy of chemopreventive agents. The aim of this study was to increase the efficacy of cancer chemopreventive agents by using lipid nanoparticles (NPs) as a carrier. This study evaluated the ability of lipid NPs to modify the pharmacodynamics of chemopreventive agents including N-acetyl-L-cysteine, phenethyl isothiocyanate and resveratrol (RES). The chemopreventive efficacy of these drugs was determined by evaluating their abilities to counteract cytotoxic damage (DNA fragmentation) induced by cigarette smoke condensate (CSC) and to activate protective apoptosis (annexin-V cytofluorimetric staining) in bronchial epithelial cells both in vitro and in ex vivo experiment in mice. NPs decreased the toxicity of RES and increased its ability to counteract CSC cytotoxicity. NPs significantly increased the ability of phenethyl isothiocyanate to attenuate CSC-induced DNA fragmentation at the highest tested dose. In contrast, this potentiating effect was observed at all tested doses of RES, NPs dramatically increasing RES-induced apoptosis in CSC-treated cells. These results provide evidence that NPs are highly effective at increasing the efficacy of lipophilic drugs (RES) but are not effective towards hydrophilic agents (N-acetyl-L-cysteine), which already possess remarkable bioavailability. Intermediate effects were observed for phenethyl isothiocyanate. These findings are relevant to the identification of cancer chemopreventive agents that would benefit from lipid NP delivery.

The role of dendritic cells (DC) in Friend retrovirus-induced immunosuppression and immunotherapeutic applications of functionalized nanoparticles

Friend murine leukemia Virus (FV) infection of immunocompetent mice is a well- established model to acquire further knowledge about viral immune suppression mechanisms, with the aim to develop therapeutics against retrovirus-induced diseases. Interestingly, BALB/c mice are infected by low doses of FV and die from FV-induced erythroleukemia, while C57/BL6 mice are infected by FV only at high viral dose, and remain persistently infected for their whole life. Due to the central role of dendritic cells (DC) in the induction of anti-viral responses, we asked for their functional role in the genotype-dependent sensitivity towards FV infection. In my PhD study I showed that bone marrow (BM)-derived DC differentiated from FV-infected BM cells obtained from FV-inoculated BALB/c (FV susceptible) and C57BL/6 (FV resistant) mice showed an increased endocytotic activity and lowered expression of MHCII and of costimulatory receptors as compared with non-infected control BMDC. FV-infected BMDC from either mouse strain were partially resistant towards stimulation-induced upregulation of MHCII and costimulators, and accordingly were poor T cell stimulators in vitro and in vivo. In addition, FV-infected BMDC displayed an altered expression profile of proinflammator cytokines and favoured Th2 polarization. Ongoing work is focussed on elucidating the functional role of proteins identified as differentially expressed in FV-infected DC in a genotype-dependent manner, which therefore may contribute to the differential course of FV infection in vivo in BALB/c versus C57BL/6 mice. So far, more than 300 proteins have been identified which are differently regulated in FV-infected vs. uninfected DC from both mouse strains. One of these proteins, S100A9, was strongly upregulated specifically in BMDC derived from FV-infected C57BL/6 BM cells. S100A9-/- mice were more sensitive towards inoculation with FV than corresponding wild type (WT) mice (both C57BL/6 background), which suggests a decisive role of this factor for anti-viral defense. In addition, FV-infected S100A9-/- BMDC showed lower motility than WT DC. The future work is aimed to further elucidate the functional importance of S100A9 for DC functions. To exploit the potential of DC for immunotherapeutic applications, in another project of this PhD study the usability of different types of functionalized nanoparticles

Nanopartículas lipídicas na promoção da absorção oral de fármacos

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Nanopartículas lipídicas

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas