DEFAR - Departamento de Farmácia
URI permanente desta comunidadehttp://www.hml.repositorio.ufop.br/handle/123456789/530
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11 resultados
Resultados da Pesquisa
Item 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 FurtadoIR780 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.Item 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 FurtadoFluorescent 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.Item 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 depH-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.Item 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 FurtadoNear-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.Item 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 AmaralSimple 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.Item 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 FurtadoNear-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.Item 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 AmaralA 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.Item 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 FurtadoFor 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.Item 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 FurtadoLychnopholide, 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.Item 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 FurtadoA 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.