Browsing by Author "Oliveira, Maria Alice de"

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    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.
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    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.
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    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.
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    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.
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    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.