Navegando por Autor "Lana, Gwenaelle Elza Nathalie Pound"

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Copolímeros de D,L-lactídeo e glicidil éteres funcionalizados : síntese, uso em nanoesferas, citotoxicidade e encapsulação de fármaco modelo.
(2016) Silva, Luan Silvestro Bianchini; Mosqueira, Vanessa Carla Furtado; Lana, Gwenaelle Elza Nathalie Pound; Silva, Gisele Rodrigues da; Gurgel, Leandro Vinícius Alves; Mosqueira, Vanessa Carla Furtado
O domínio das rotas sintéticas de polímeros biocompatíveis e funcionalizados para acoplamento de diferentes tipos de ligantes são focos de intensas pesquisas. Estes polímeros podem ser associados com bioativos e utilizados na terapêutica e no diagnóstico. A síntese de copolímeros estatísticos a partir da polimerização por abertura de anel do D,L-lactídeo na presença dos comonômeros alil glicidil éter (AGE) e benzil glicidil éter (BGE) permitiu avaliar a inserção destes grupos nas cadeias poliméricas. Os polímeros sintetizados foram caracterizados por ressonância magnética nuclear de hidrogênio e cromatografia de permeação em gel. Foram avaliadas as influências dos comonômeros, das condições de síntese e dos catalisadores utilizados na polimerização. Houve uma relação direta entre maiores quantidades de glicidil éter no meio reacional, maior inserção deste nas cadeias e a obtenção de copolímeros de massas molares mais baixas. O acréscimo do co-catalisador 4-(dimetilamino)piridina associado com o octanoato de estanho não proporcionou maiores inserções dos comonômeros nas cadeias poliméricas quando comparado ao uso isolado do octanoato de estanho. O BGE foi inserido em maior proporção do que o AGE nas cadeias. Foram selecionados para o preparo de nanoesferas por nanoprecipitação os polímeros que apresentaram massas molares próximas (≅ 23.000 g/mol), menores dispersidades (1,1-1,3) e com inserções de comonômeros por cadeia próximas (≅ 2,3 unidades por cadeia). Foram preparadas nanoesferas a partir dos polímeros sintetizados com e sem benznidazol como fármaco modelo. Foi observado um aumento no diâmetro hidrodinâmico médio e diminuição, em módulo, do potencial zeta, com valores de (91 a 92 nm) e (- 25 a - 32 mV), para as nanoesferas brancas, e (94 a 117 nm) e (- 17 a - 21 mV), para as nanoesferas com benznidazol, sendo todas monodispersas. Variações significativas destes parâmetros foram observadas devido à presença de fármaco associado ao vetor em relação às nanoesferas brancas. A porcentagem de encapsulação, eficiência de encapsulação e capacidade de carga das nanoesferas com benznidazol foram significativamente maiores para as nanoesferas de PLA-co-AGE para a formulação com 0,65 mg/mL de benznidazol, sugerindo maior associação do fármaco com este copolímero. As imagens de nanoesferas por microscopia eletrônica de varredura mostraram estruturas com estreita faixa de diâmetro (50 a 120 nm) e forma esférica. O teste de viabilidade celular em cultura de células J774A.1 e Vero evidenciaram uma baixa toxicidade das nanoesferas obtidas com os diferentes polímeros sintetizados mesmo em altas concentrações (500 μg/mL). A partir da obtenção de polímeros funcionalizados, este estudo abre perspectivas para o melhor entendimento da inserção de glicidil éteres em cadeias de poli(D,L-lactídeo) e das relações intermoleculares entre fármacos e polímeros no subsídio de novas alternativas terapêuticas.
Efeito da variação de dose e das características de superfície de nanocápsulas sobre suas concentrações plasmáticas e hepáticas por via intravenosa.
(2016) Paula, Mônica Auxiliadora de; Mosqueira, Vanessa Carla Furtado; Lana, Gwenaelle Elza Nathalie Pound; Mosqueira, Vanessa Carla Furtado; Lana, Gwenaelle Elza Nathalie Pound; Postacchini, Bruna Bueno; Leite, Elaine Amaral
A aplicação de nanopartículas como carreadores de fármacos para o tratamento de diversas patologias tem se mostrado extremamente promissora, mas ainda encontra grandes desafios relacionados ao estabelecimento de esquemas posológicos seguros. Diferentes formulações de nanopartículas (NP), encapsuladas com o marcador fluorescente ftalocianina de cloro-alumínio (AlClPc) foram obtidas pelo método de nanoprecipitação: nanocápsulas (NC) de ácido polilático (NC PLA), NC de ácido polilático em bloco com o polietilenoglicol (NC PLAPEG) e NC PLA revestidas de quitosana em duas concentrações distintas (NC PLAQUI 0,1% e NC PLAQUI 0,25% p/v), além de uma nanoemulsão equivalente à NC PLA, exceto na presença da parede polimérica. Todas foram caracterizadas quanto as suas propriedades físico-químicas. O tamanho e índice de polidispersão foram determinados em batelada por espalhamento dinâmico de luz e após fracionamento em sistema de fracionamento por campo de fluxo assimétrico acoplado a detectores de espalhamento dinâmico de luz e espalhamento de luz em multiângulos. Desse modo foi possível também determinar o fator de forma das NP pela relação entre o raio de giração/raio hidrodinâmico. A carga de superfície foi determinada por anemometria do laser Doppler. Todas as formulações apresentaram-se monodispersas, com o diâmetro hidrodinâmico entre 133 a 297 nm e potencial zeta positivo para as formulações com quitosana (+26 mV) e negativo para as demais (entre -35 e -44 mV). Os resultados para o fator de forma próximos a 1,0 confirmaram a estrutura vesicular das nanopartículas em todas as formulações. Estudos in vivo foram conduzidos com o objetivo de avaliar o impacto das características de superfície das partículas, bem como das doses crescentes de polímero, sobre a biodistribuição e a capacidade de saturação do sistema fagocitário mononuclear (SFM), em camundongos, pela via intravenosa. O estudo in vivo mostrou que a superfície das partículas tem uma maior influência no modo como elas se distribuem pelo organismo que o tamanho. Além disso, esse estudo evidencia a possibilidade de elevadas doses de partículas saturarem o SFM, uma vez que para todas as formulações, com o aumento da dose injetada, houve uma diminuição da porcentagem da dose encontrada no fígado.
Functional polylactide via ring-opening copolymerisation with allyl, benzyl and propargyl glycidyl ethers.
(2017) Lana, Gwenaelle Elza Nathalie Pound; Rabanel, Jean Michel; Hildgen, Patrice; Mosqueira, Vanessa Carla Furtado
A versatile and simple strategy is presented to synthesize reactive polylactide derivatives and their block copolymers with polyethylene glycol. Commercially available glycidyl ethers with an allyl, benzyl or propargyl functional group were copolymerised with D,L-lactide. Tin(II)-2- ethylhexanoate-catalysis produced polymers with up to 4.6, 5.9 and 2.3 allyl, benzyl or propargyl groups per chain, respectively. In contrast, less than one reactive group per chain was obtained with the organocatalyst 1,5,7-triazabicyclo[4.4.0]dec-5-ene. By increasing the polymerisation feed ratio in glycidyl ether polymers with a higher number of reactive groups per chain were obtained, however a decrease in molar mass was observed. An azidocoumarin was conjugated to the propargylated polymers via copper-catalysed azide-alkyne cycloaddition. These dye-labelled polymers produced nanospheres with fluorescent properties and diameters in the 100-nm sizerange, as characterised by asymmetric flow field flow fractionation hyphenated with fluorescence, static and dynamic light scattering detection. The functionalised polymers were obtained at gram-scale in one step from commercially available reagents; therefore providing a robust and easy to implement approach for the production of multifunctional nanomaterials.
Host-parasite interactions in chagas disease : genetically unidentical isolates of a single Trypanosoma cruzi strain identified In vitro via LSSP-PCR.
(2015) Paiva, Nívia Carolina Nogueira de; Vieira, Paula Melo de Abreu; Oliveri, Larissa Maris Rezende; Fonseca, Kátia da Silva; Lana, Gwenaelle Elza Nathalie Pound; Oliveira, Maykon Tavares de; Veloso, Vanja Maria; Reis, Alexandre Barbosa; Tafuri, Washington Luiz; Carneiro, Cláudia Martins
The present study aims at establishing whether the diversity in pathogenesis within a genetically diverse host population infected with a single polyclonal strain of Trypanosoma cruzi is due to selection of specific subpopulations within the strain. For this purpose we infected Swiss mice, a genetically diverse population, with the polyclonal strain of Trypanosoma cruzi Berenice-78 and characterized via LSSP-PCR the kinetoplast DNA of subpopulations isolated from blood samples collected from the animals at various times after inoculation (3, 6 and 12 months after inoculation). We examined the biological behavior of the isolates in acellular medium and in vitro profiles of infectivity in Vero cell medium. We compared the characteristics of the isolates with the inoculating strain and with another strain, Berenice 62, isolated from the same patient 16 years earlier. We found that one of the isolates had intermediate behavior in comparison with Berenice-78 and Berenice-62 and a significantly different genetic profile by LSSP-PCR in comparison with the inoculating strain. We hereby demonstrate that genetically distinct Trypanosoma cruzi isolates may be obtained upon experimental murine infection with a single polyclonal Trypanosoma cruzi strain.
Impact of dose and surface features on plasmatic and liver concentrations of biodegradable polymeric nanocapsules.
(2017) Oliveira, Líliam Teixeira; Paula, Mônica Auxiliadora de; Roatt, Bruno Mendes; Garcia, Giani Martins; Silva, Luan Silvestro Bianchini; Reis, Alexandre Barbosa; Paula, Carina Silva de; Vilela, José Mário Carneiro; Andrade, Margareth Spangler; Lana, Gwenaelle Elza Nathalie Pound; Mosqueira, Vanessa Carla Furtado
The effect of polymeric nanocapsule dose on plasmatic and liver concentrations 20 min after intravenous administration in mice was evaluated. Nanocapsules were prepared with different polymers, namely, poly(D,Llactide) (PLA), polyethylene glycol-block-poly(D,L-lactide) (PLA-PEG), and PLA with chitosan (PLA-Cs) and compared with a nanoemulsion. These formulations were labelled with a phthalocyanine dye for fluorescent detection. The nanostructures had narrow size distributions upon separation by asymmetric flow field flow fractionation with static and dynamic light scattering detection, with average hydrodynamic diameters in the 130–300 nm range, negative zeta potentials, except PLA-Cs nanocapsules, which had a positive zeta potential. Flow cytometry revealed uptake mostly by monocytes and neutrophils in mice and human blood. PLA nanocapsules and the nanoemulsion showed dose-dependent plasma concentrations, where the percentage of plasmatic fluorescence increased with increasing administered dose. In contrast, PLA-PEG nanocapsules led to a dose-independent plasmatic profile. PLA-Cs nanocapsules showed the lowest plasmatic and liver levels of fluorescence at all administered doses and significant intravenous toxicity in mice. This work demonstrates the importance of considering the nanocarrier dose when evaluating pharmacokinetic and biodistribution data and emphasizes the role of surface features in determining the plasmatic and liver concentrations of a poorly soluble lipophilic encapsulated compound.
Increased body exposure to new anti-trypanosomal through nanoencapsulation.
(2017) Branquinho, Renata Tupinambá; Lana, Gwenaelle Elza Nathalie Pound; Milagre, Matheus Marques; Guimarães, Dênia Antunes Saúde; Vilela, José Mário Carneiro; Andrade, Margareth Spangler; Lana, Marta de; Mosqueira, Vanessa Carla Furtado
Lychnopholide, a lipophilic sesquiterpene lactone, is efficacious in mice at the acute and chronic phases of Chagas disease. Conventional poly-ε-caprolactone (PCL) and long-circulating poly(D,L-lactide)-block-polyethylene glycol (PLA-PEG) nanocapsules containing lychnopholide were developed and characterized. Lychnopholide presented high association efficiency (>90%) with the nanocapsules. A new, fast and simple HPLC-UV-based bioanalytical method was developed, validated in mouse plasma and applied to lychnopholide quantification in in vitro release kinetics and pharmacokinetics. The nanocapsules had mean hydrodynamic diameters in the range of 100–250 nm, negative zeta potentials (−30 mV to −57 mV), with good physical stability under storage. Atomic force microscopy morphological analysis revealed spherical monodispersed particles and the absence of lychnopholide crystallization or aggregation. Association of lychnopholide to PLA-PEG nanocapsules resulted in a 16-fold increase in body exposure, a 26-fold increase in plasma half-life and a dramatic reduction of the lychnopholide plasma clearance (17-fold) in comparison with free lychnopholide. The improved pharmacokinetic profile of lychnopholide in long-circulating nanocapsules is in agreement with the previously reported improved efficacy observed in Trypanosoma cruzi-infected mice. The present lychnopholide intravenous dosage form showed great potential for further pre-clinical and clinical studies in Chagas disease and cancer therapies.
IR780-polymer conjugates for stable near-infrared labeling of biodegradable polyester-based nanocarriers.
(2019) Oliveira, Maria Alice de; Machado, Marina Guimarães Carvalho; Silva, Sabrina Emanuelle Dias; Nascimento, Thais Leite; Lima, Eliana Martins; Lana, Gwenaelle Elza Nathalie Pound; Mosqueira, Vanessa Carla Furtado
Near-infrared dyes are useful to monitor nanocarriers in vitro and in vivo and can serve as photosensitizers in cancer photodynamic therapy. However, strategies need to be developed to guarantee that the dye photophysical properties and loading within the drug delivery system remain stable for reliable tracking within biological systems. This work reports the facile chemical conjugation of the carbocyanine heptamethine near-infrared dye IR780 to polylactide for stable fluorescent labeling of biodegradable polyester nanocarriers. “Clickable” polylactide was synthesized via organocatalyzed ring opening polymerization of D,L-lactide with a cyclooctyne initiator. IR780 was derivatized and conjugated to polylactide via a one-pot copper-free azide-alkyne cycloaddition reaction. The synthetic strategy developed was effective to promote conjugation of the near-infrared fluorescent dye to polylactide, as confirmed by high performance liquid chromatography. Nanoparticles containing the dye–polymer conjugate were prepared by nanoprecipitation and characterized. Asymmetric flow field-flow fractionation with light scattering and fluorescence detection revealed that the near-infrared fluorescence of the nanoparticles remained stable and was not transferred to serum proteins. In contrast, significant transfer of the dye to serum proteins was evidenced when the dye was merely encapsulated in similar nanoparticles through physical entrapment. Confocal microscopy and fluorescence tomography imaging showed that the polymer-dye conjugate confers fluorescence properties to the NP suitable for further in vitro and in vivo pre-clinical studies.
Labeling PLA-PEG nanocarriers with IR780 : physical entrapment versus covalent attachment to polylactide.
(2020) Machado, Marina Guimarães Carvalho; Lana, Gwenaelle Elza Nathalie Pound; Oliveira, Maria Alice de; Lanna, Elisa Gomes; Fialho, Márcia Célia Pacheco; Brito, Ana Carolina Ferreira de; Barboza, Ana Paula Moreira; Soares, Rodrigo Dian de Oliveira Aguiar; Mosqueira, Vanessa Carla Furtado
Near-infrared fluorescent dyes, such as IR780, are promising theranostics, acting as photosensitizers for photodynamic therapy and in vivo tracers in image-guided diagnosis. This work compared the uptake by macrophage-like cells of IR780 either physically associated or covalently attached to poly(D,L-lactide) (PLA) formulated as polymeric nanocapsules (NC) from a blend of PLA homopolymer and PLA-PEG block copolymer. The physicochemical characterization of both NC was conducted using asymmetric flow field-flow fractionation (AF4) analysis with static and dynamic light scattering and atomic force micros copy. The interaction of IR780 with serum proteins was evidenced by AF4 with fluorescence detection and flow cytometry in cell uptake studies. The average diameters of NC were around 120 nm and zeta potentials close to -40 mV for all NC. NC uptake by cells in different media and experimental conditions shows significantly lower fluorescence intensities for IR780 covalently linked to PLA and correspondingly low quantitative uptake. Different mechanisms of internalization were evidenced depending on the IR780 type of association to NC. Serum proteins mediate IR780 interaction with cells in a dose-dependent manner. Our results show that non-covalently linked IR780 was released from NC and accumulated in macrophage cells. Oppositely, IR780 conjugated to PLA provides stable association with NC, and its fluorescence is representative of cell uptake of the nanocarrier itself. This work strongly reinforces the importance of covalent attachment of a fluorescence dye such as IR780 to the nanocarrier to study their interaction with cells in vitro and to obtain reliable tracking in image-guided therapy.
Mechanisms of interaction of biodegradable polyester nanocapsules with non-phagocytic cells.
(2018) Trindade, Izabel Cristina; Lana, Gwenaelle Elza Nathalie Pound; Pereira, Douglas Gualberto Sales; Oliveira, Laser Antônio Machado de; Andrade, Margareth Spangler; Vilela, José Mário Carneiro; Postacchini, Bruna Bueno; Mosqueira, Vanessa Carla Furtado
The interaction of polymer nanocapsules (NC) prepared from four biodegradable polyesters with variable polymer hydrophobicity (PCL, PLA, PLGA and PLA-PEG) was investigated in the non-phagocytic Vero, Caco-2 and HepG2 cell lines. The NC, labeled with the highly lipophilic fluorescent indocarbocyanine dye DIL, had very similar sizes (approx. 140 nm) and negative zeta-potentials. Asymmetric flow field-flow fractionation evidenced NC colloidal stability and negligible transfer of the dye to serum proteins in the incubation medium. The cytotoxicity of the NC was evaluated via MTT assay over a large polymer concentration range (1–1000 μg/mL) and time of exposure (2, 24 and 48 h). The NC were safe in vitro up to a concentration of approx. 100 μg/mL or higher, depending on the cell line and nature of the polymer. Vero cells were more sensitive to the NC, in particular NC of the more hydrophobic polymer. The cells were exposed to endocytosis inhibitors, incubated with NC, and the cell-associated fluorescence was quantified by spectrofluorometry. HepG2 cells presented a 1.5–2-fold higher endocytic capacity than Caco-2 and Vero cells. The main mechanism of NC uptake was caveolin-mediated endocytosis in HepG2 and Vero cells, and macropinocytosis in Caco-2 cells. Polymer hydrophobicity had an effect on the level of NC associated to HepG2 cells and to a lesser extent on the endocytosis mechanisms in Vero and Caco-2 cells. The NC uptake levels and endocytosis mechanisms differed significantly between cell lines tested.
Mechanistic insights into the intracellular release of doxorubicin from pH-sensitive liposomes.
(2021) Reis, Samara Bonesso dos; Silva, Juliana de Oliveira; Fossa, Fernanda Garcia; Leite, Elaine Amaral; Souza, Angelo Malachias de; Lana, Gwenaelle Elza Nathalie Pound; Mosqueira, Vanessa Carla Furtado; Oliveira, Mônica Cristina de; Barros, André Luís Branco de; Jesus, Marcelo Bispo de
pH-sensitive liposomes are interesting carriers for drug-delivery, undertaking rapid bilayer destabilization in response to pH changes, allied to tumor accumulation, a desirable behavior in the treatment of cancer cells. Previously, we have shown that pH-sensitive liposomes accumulate in tumor tissues of mice, in which an acidic environment accelerates drug delivery. Ultimately, these formulations can be internalized by tumor cells and take the endosome-lysosomal route. However, the mechanism of doxorubicin release and intracellular traffic of pH-sensitive liposomes remains unclear. To investigate the molecular mechanisms underlying the intracellular release of doxorubicin from pH-sensitive liposomes, we followed HeLa cells viability, internalization, intracel lular trafficking, and doxorubicin’s intracellular delivery mechanisms from pH-sensitive (SpHL-DOX) and non pH-sensitive (nSpHL-DOX) formulations. We found that SpHL-DOX has faster internalization kinetics and intracellular release of doxorubicin, followed by strong nuclear accumulation compared to nSpHL-DOX. The increased nuclear accumulation led to the activation of cleaved caspase-3, which efficiently induced apoptosis. Remarkably, we found that chloroquine and E64d enhanced the cytotoxicity of SpHL-DOX. This knowledge is paramount to improve the efficiency of pH-sensitive liposomes or to be used as a rational strategy for developing new formulations to be applied in vivo.
Nanobastões de ouro no desenvolvimento de um método de diagnóstico de paracoccidioidomicose.
(2016) Dias, Bruna de Paula; Silva, Breno de Mello; Ferreira, Cyntia Silva; Lana, Gwenaelle Elza Nathalie Pound; Oliveira, Laser Antônio Machado de; Silva, Breno de Mello
A paracoccidioidomicose (PCM) é uma micose granulomatosa sistêmica causada por duas espécies de um fungo termodimórfico: Paracoccidioides brasiliensis e Paracoccidioides lutzii. A doença é endêmica na América Latina e apresenta o maior número de casos no Brasil. Embora a maioria das formas clínicas seja assintomática, podem ocorrer infecções severas e progressivas, envolvendo o tecido pulmonar, o sistema nervoso central e pode levar a óbito dependendo da gravidade. Assim, o diagnóstico precoce da doença torna-se essencial para dar início ao tratamento. Apesar disso, ainda não existe um diagnóstico rápido e preciso com custo apropriado para as áreas endêmicas. Nesse sentido, uma alternativa é representada pelo uso de nanobastões de ouro (AuNBs), os quais possuem uma forte absorção característica no espectro visível (ressonância de plasmon) que pode ser monitorada por espectrofotometria. Considerando o uso dos AuNBs como biosensores, os mesmos foram funcionalizados com polietilenoimina e bioconjugados com anticorpos anti-gp43. Tais conjugados foram testados na presença dos alvos purificados e também desafiados com soro de camundongos infectados. A ligação efetiva das biomoléculas em questão foi observada através do deslocamento do pico de absorção plasmônica longitudinal dos AuNBs, sendo satisfatórias quando empregadas as biomoléculas purificadas. Em amostras de soros o sensor não se comportou como esperado. Assim, o deslocamento observado para as biomoléculas purificadas mostram que o sistema deve ser aprimorado para a detecção de antígenos em amostras complexas e pode justificar em parte, o sucesso do sistema de detecção proposto. Além disso, a proposta representa um avanço biotecnológico para o diagnóstico de doenças negligenciadas.
Paclitaxel-loaded pH-sensitive liposome : new insights on structural and physicochemical characterization.
(2018) Monteiro, Liziane Oliveira Fonseca; Souza, Angelo Malachias de; Lana, Gwenaelle Elza Nathalie Pound; Paniago, Rogerio Magalhães; Mosqueira, Vanessa Carla Furtado; Oliveira, Mônica Cristina de; Barros, André Luís Branco de; Leite, Elaine Amaral
A long-circulating and pH-sensitive liposome containing paclitaxel (SpHL-PTX) was recently developed by our group. Once in an acidic environment, for example, tumors, these liposomes undergo destabilization, releasing the encapsulated drug. In this way, the aim of this study was to evaluate the molecular and supramolecular interactions between the lipid bilayer and PTX in similar biological environment conditions. High-sensitivity analyses of SpHL-PTX structures were obtained by the small-angle X-ray scattering technique combined with other techniques such as dynamic light scattering, asymmetric flow field-flow fractionation, transmission electron microscopy, and high-performance liquid chromatography. The results showed that PTX incorporation in the liposomal bilayer clearly leads to changes in supramolecular organization of dioleoylphosphatidylethanolamine (DOPE) molecules, inducing the formation of more ordered structures. Changes in supramolecular organization were observed at lower pH, indicating that pH sensitivity was preserved even in the presence of fetal bovine serum proteins. Furthermore, morphological and physicochemical characterization of SpHL-PTX evidenced the formation of nanosized dispersion suitable for intravenous administration. In conclusion, a stable nanosized dispersion of PTX was obtained at pH 7.4 with suitable parameters for intravenous administration. At lower pH conditions, the pH sensitivity of the system was clearly evidenced by changes in the supramolecular organization of DOPE molecules, which is crucial for the delivery of PTX into the cytoplasm of the targeted cells. In this way, the results obtained by different techniques confirm the feasibility of SpHL as a promising tool to PTX delivery in acidic environments, such as tumors.
Photodynamic therapy with the dual-mode association of IR780 to PEG-PLA nanocapsules and the effects on human breast cancer cells.
(2022) Machado, Marina Guimarães Carvalho; Oliveira, Maria Alice de; Lanna, Elisa Gomes; Siqueira, Raoni Pais; Lana, Gwenaelle Elza Nathalie Pound; Branquinho, Renata Tupinambá; Mosqueira, Vanessa Carla Furtado
IR780 is a near-infrared fluorescent dye, which can be applied as a photosensitizer in photodynamic (PDT) and photothermal (PTT) therapies and as a biodistribution tracer in imaging techniques. We investigated the growth and migration inhibition and mechanism of death of breast tumor cells, MCF-7 and MDA-MB-231, exposed to polymeric nanocapsules (NC) comprising IR780 covalently linked to the biodegradable polymer PLA (IR-PLA) and IR780 physically encapsulated (IR780-NC) in vitro. Both types of NC had mean diameters around 120 nm and zeta potentials around − 40 mV. IR-PLA-NC was less cytotoxic than IR780 NC to a non-tumorigenic mammary epithelial cell line, MCF-10A, which is an important aspect of selectivity. Free-IR780 was more cytotoxic than IR- PLA-NC for MCF-7 and MDA-MB-231 cells after illumination with a 808 nm laser. IR-PLA NC was effective to inhibit colony formation (50%) and migration (30–40%) for both cancer cell lines. MDA-MB-231 cells were less sensitive to all IR780 formulations compared to MCF-7 cells. Cell uptake was higher with IR-PLA-NC than with IR780-NC and free-IR780 in both cancer cell lines (p < 0.05). NC uptake was higher in MCF-7 than in MDA-MB- 231 cells. IR-PLA-NC induced a higher percentage of apoptosis upon illumination in MDA-MB-231 than in MCF-7 cells. The necrosis mechanism of death predominated in treatments with free-IR780 and with encapsulated IR780 NC, suggestive of damages at the plasma membrane. IR780 conjugated with PLA increased the apoptotic pathway and demonstrated potential as a multifunctional theranostic agent for breast cancer treatment with increased cellular uptake, photodynamic activity and more reliable tracking in cell-image studies.
Phthalocyanine photosensitizer in polyethylene glycol-block-poly(lactide-co-benzyl glycidyl ether) nanocarriers : probing the contribution of aromatic donor-acceptor interactions in polymeric nanospheres.
(2019) Lana, Gwenaelle Elza Nathalie Pound; Garcia, Giani Martins; Trindade, Izabel Cristina; Oliveira, Patricia Capelari de; Pontífice, Thaís Godinho; Vilela, José Mário Carneiro; Andrade, Margareth Spangler; Nottelet, Benjamin; Postacchini, Bruna Bueno; Mosqueira, Vanessa Carla Furtado
For best photosensitizer activity phthalocyanine dyes used in photodynamic therapy should be molecularly dispersed. Polyethylene glycol-block-polylactide derivatives presenting benzyl side-groups were synthesized to encapsulate a highly lipophilic phthalocyanine dye (AlClPc) and evaluate the effect of π-π interactions on the nanocarrier colloidal stability and dye dispersion. Copolymers with 0, 1, 2 and 6 mol% of benzyl glycidyl ether (BGE) were obtained via polyethylene glycol initiated ring-opening copolymerization of D,L-lactide with BGE. The block copolymers formed stable, monodisperse nanospheres with low in vitro cytotoxicity. AlClPc loading increased the nanosphere size and affected their colloidal stability. The photo-physical properties of the encapsulated dye, studied in batch and after separation by field flow fractionation, demonstrated the superiority of plain PEG-PLA over BGE-containing copolymers in maintaining the dye in its monomeric (non-aggregated) form in aqueous suspension. High dye encapsulation and sustained dye release suggest that these nanocarriers are good candidates for photodynamic therapy.
Physical and biological effects of paclitaxel encapsulation on disteraroylphosphatidylethanolamine-polyethyleneglycol polymeric micelles.
(2020) Oda, Caroline Mari Ramos; Gasperini, Antonio Augusto Malfatti; Souza, Angelo Malachias de; Lana, Gwenaelle Elza Nathalie Pound; Mosqueira, Vanessa Carla Furtado; Fernandes, Renata Salgado; Oliveira, Mônica Cristina de; Barros, André Luís Branco de; Leite, Elaine Amaral
Simple size observations of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-mPEG2000) polymeric micelles (PM) with different compositions including or not paclitaxel (PTX) are unable to evidence changes on the nanocarrier structure. In such system a detailed characterization using highly sensitive techniques such as X-ray scattering and asymmetric flow field flow fractionation coupled to multi-angle laser light scattering and dynamic light scattering (AF4-MALS-DLS) is mandatory to observe effects that take place by the addition of PTX and/or more lipid-polymer at PM, leading to complex changes on the structure of micelles, as well as in their supramolecular organization. SAXS and AF4-MALS-DLS suggested that PM can be found in the medium separately and highly organized, forming clusters of PM in the latter case. SAXS fitted parameters showed that adding the drug does not change the average PM size since the increase in core radius is compensated by the decrease in shell radius. SAXS observations indicate that PEG conformation takes place, changing from brush to mushroom depending on the PM composition. These findings directly reflect in in vivo studies of blood clearance that showed a longer circulation time of blank PM when compared to PM containing PTX.
Ravuconazole self-emulsifying delivery system : in vitro activity against Trypanosoma cruzi amastigotes and in vivo toxicity.
(2017) Spósito, Pollyanna Álvaro; Mazzeti, Ana Lia; Faria, Caroline de Oliveira; Urbina, Julio Alberto; Lana, Gwenaelle Elza Nathalie Pound; Bahia, Maria Terezinha; Mosqueira, Vanessa Carla Furtado
Self-emulsifying drug delivery systems (SEDDSs) are lipid-based anhydrous formulations composed of an isotropic mixture of oil, surfactant, and cosurfactants usually presented in gelatin capsules. Ravuconazole (Biopharmaceutics Classification System [BCS] Class II) is a poorly water-soluble drug, and a SEDDS type IIIA was designed to deliver it in a predissolved state, improving dissolution in gastrointestinal fluids. After emulsification, the droplets had mean hydrodynamic diameters ,250 nm, zeta potential values in the range of -45 mV to -57 mV, and showed no signs of ravuconazole precipitation. Asymmetric flow field-flow fractionation with dynamic and multiangle laser light scattering was used to characterize these formulations in terms of size distribution and homogeneity. The fractograms obtained at 37°C showed a polydisperse profile for all blank and ravuconazole–SEDDS formulations but no large aggregates. SEDDS increased ravuconazole in vitro dissolution extent and rate (20%) compared to free drug (3%) in 6 h. The in vivo toxicity of blank SEDDS comprising Labrasol® surfactant in different concentrations and preliminary safety tests in repeated-dose oral administration (20 days) showed a dose-dependent Labrasol toxicity in healthy mice. Ravuconazole–SEDDS at low surfactant content (10%, v/v) in Trypanosoma cruzi-infected mice was safe during the 20-day treatment. The anti-T. cruzi activity of free ravuconazole, ravuconazole–SEDDS and each excipient were evaluated in vitro at equivalent ravuconazole concentrations needed to inhibit 50% or 90% (IC50 and IC90), respectively of the intracellular amastigote form of the parasite in a cardiomyocyte cell line. The results showed a clear improvement of the ravuconazole anti-T. cruzi activity when associated with SEDDS. Based on our results, the repurposing of ravuconazole in SEDDS dosage form is a strategy that deserves further in vivo investigation in preclinical studies for the treatment of human T. cruzi infections.
Release, transfer and partition of fluorescent dyes from polymeric nanocarriers to serum proteins monitored by asymmetric flow field-flow fractionation.
(2021) Oliveira, Maria Alice de; Lana, Gwenaelle Elza Nathalie Pound; Oliveira, Patricia Capelari de; Pontífice, Thaís Godinho; Silva, Sabrina Emanuelle Dias; Machado, Marina Guimarães Carvalho; Postacchini, Bruna Bueno; Mosqueira, Vanessa Carla Furtado
Fluorescent probes are used in drug nanocarrier pre-clinical studies or as active compounds in theranostics and photodynamic therapy. In the biological medium, nanoparticles interact with proteins, which can result in the off-target release of their cargo. The present study used asymmetric flow field-flow fractionation with online multi-angle laser light scattering and fluorescence detection (AF4-MALLS-FLD) to study the release, transfer, and partition of fluorescent dyes from polymeric nanoparticles (NP). NP formulations containing the dyes Rose Bengal, Rhodamine B, DiI, 3-(α-azidoacetyl)coumarin and its polymer conjugate, Nile Red, and IR780 and its polymer conjugate were prepared. NP suspensions were incubated in a medium with serum proteins and then analyzed by AF4. AF4 allowed efficient separation of proteins (< 10 nm) from fluorescently labeled NP (range of 54 – 180 nm in diameters). The AF4 analyses showed that some dyes, such as Rose Bengal, IR780, and Coumarin were transferred to a high extent (68-77%) from NP to proteins. By contrast, for DiI and dye-polymer conjugates, transfer occured to a lower extent. The studies of dye release kinetics showed that the transfer of IR780 from NP to proteins occurs at a high extent (~50%) and rate, while Nile Red was slowly released from the NP over time with reduced association with proteins (~20%). This experiment assesses the stability of fluorescence labeling of nanocarriers and probes the transfer of fluorescent dyes from NP to proteins, which is otherwise not accessible with commonly used techniques of separation, such as dialysis and ultrafiltration/centrifugation employed in drug encapsulation and release studies of nanocarriers. Determining the interaction and transfer of dyes to proteins is of utmost importance in the pre-clinical evaluation of drug nanocarriers for improved correlation between in vitro and in vivo studies.
Repositioning of tamoxifen in surface-modified nanocapsules as a promising oral treatment for visceral leishmaniasis.
(2021) Silva, Débora Faria; Reis, Levi Eduardo Soares; Machado, Marina Guimarães Carvalho; Dophine, Douglas Daniel; Andrade, Vinicius Roberto de; Lima, Wanderson Geraldo de; Andrade, Margareth Spangler; Vilela, José Mário Carneiro; Reis, Alexandre Barbosa; Lana, Gwenaelle Elza Nathalie Pound; Rezende, Simone Aparecida; Mosqueira, Vanessa Carla Furtado
Standards of care for human visceral leishmaniasis (VL) are based on drugs used parenter- ally, and oral treatment options are urgently needed. In the present study, a repurposing strategy was used associating tamoxifen (TMX) with polyethylene glycol-block-polylactide nanocapsules (NC) and its anti-leishmanial efficacy was reported in vivo. Stable surface modified-NC (5 mg/mL of TMX) exhibited 200 nm in size, +42 mV of zeta potential, and 98% encapsulation efficiency. Atomic force microscopy evidenced core-shell-NC. Treatment with TMX-NC reduced parasite-DNA quantified in liver and spleen compared to free-TMX; and provided a similar reduction of parasite burden compared with meglumine antimoniate in mice and hamster models. Image-guided biodistribution showed accumulation of NC in liver and spleen after 30 min post-administration. TMX-NC reduced the number of liver granulomas and restored the aspect of capsules and trabeculae in the spleen of infected animals. TMX-NC was tested for the first time against VL models, indicating a promising formulation for oral treatment.
Síntese de polilactídeo com fluorescência no infravermelho próximo para futuras aplicações nanoteranósticas.
(2019) Oliveira, Maria Alice de; Mosqueira, Vanessa Carla Furtado; Lana, Gwenaelle Elza Nathalie Pound; Barros, André Luís Branco de; Santos, Claudio Gouvea dos; Mosqueira, Vanessa Carla Furtado
Os marcadores fluorescentes que absorvem na região espectral do infravermelho próximo (NIR) podem ser empregados como fotossensitizadores nas terapias fotodinâmica e fototérmica para tratamento de câncer. Além disso, essas sondas são úteis na detecção de nanocarreadores, pois permitem o rastreio do destino dessas partículas in vivo por meio de técnicas de imagem pouco invasivas. No entanto, são necessárias estratégias para garantir a estabilidade de ligação e a concentração adequada do marcador nas nanopartículas. Diante disso, o presente trabalho teve como objetivo a conjugação de um derivado carbocianina, o marcador IR780, com polilactídeos (PLA) para obtenção de um polímero que possa ser empregado em estudos pré-clínicos para avaliação da biodistribuição de nanopartículas. PLA reativos foram sintetizados via polimerização por abertura de anel (ROP) do D,L-lactídeo com um iniciador contendo um grupo ciclooctila, via catalise por octanoato de estanho ou triazabiciclodeceno (TBD). Análise da reatividade dos PLA demonstrou que apenas os polímeros obtidos por reação catalisada pelo TBD eram reativos frente a azidas orgânicas. O IR780 foi derivatizado e conjugado à PLA via reação multicomponente de cicloadição azida-alcino promovida por tensão de ciclo (SPAAC). O sucesso da reação foi confirmado por cromatografia líquida de alta eficiência (CLAE). Nanopartículas contendo o IR780 conjugado ao PLA (IR-PLA) associado a diferentes poliésteres (PCL, PLGA e PEG-PLA) foram preparadas por nanoprecipitação e caracterizadas pela técnica de fracionamento em campo de fluxo assimétrico (AF4) associada a detecção por espalhamento de luz e fluorescência (AF4-MALS-FLD). Análises por AF4-MALS-FLD demonstraram que as nanopartículas apresentam fluorescência estável, que não foi transferida para proteínas séricas quando incubadas a 37°C em meio DMEM contendo 10% de soro fetal bovino, além disso observou-se que o IR-PLA foi capaz de se associar a diferentes poliésteres, formando NP estáveis. Sendo assim, o IR-PLA mostrou ser um marcador fluorescente polimérico versátil, que pode ser associado a diferentes poliésteres biodegradáveis para a marcação dos nanocarreadores mais seguros e mais utilizados na área farmacêutica.