Publicação
Study of the interactions of surface-modified particles with membrane model systems
| Resumo: | The complexity of cell membranes and the development of nano/micro drug delivery systems make the topic of interactions between these two structures challenging. Studies point that the surface properties of these carriers like size, surface charge, shape and hydrophobicity largely influence such interactions. This project aims to study the interactions of surface modified Poly(lactic-co-glycolic acid) (PLGA) Microparticles (MPs) with Giant Unilamellar Vesicles (GUVs). MPs were formulated by the double emulsion solvent evaporation method and surface modified using chitosan (CH) and alginate (ALG) in order to manipulate the surface charge. Five concentrations of coumarin-6 (0.04, 0.10, 0.20, 0.40 and 1.00 g/mg) were entrapped into the PLGA matrix, after formulation optimization without probe. Phisichochemical characterization of MPs was made in terms of size, surface charge, coumarin-6 loading and morphology. Spectral properties of coumarin-6 were analyzed by fluorescence spectroscopy with the five different probe concentrations and five concentrations of MPs in suspension (0.10, 0.20, 0.25, 0.40, and 0.50 mg/mL). GUVs with different lipid composition and membrane properties, namely, homogeneously fluid GUVs and GUVs displaying gel-fluid and lo-ld phase separation were produced. Confocal microscopy was used to monitor the possible interactions between coumarin-6-loaded PLGA MPs and GUVs. MPs size (D50%) of non-fluorescent and fluorescent coumarin-6-loaded PLGA MPs ranged between 1.4±0.3 to 3.8±0.2 μm. The span values ranged between 1.1±0.2 to 4.8±1.1, demonstrating polidisperse populations. The surface modification did not demonstrate a statistically significant impact on the D50% values. In addition, the presence of coumarin-6 did not demonstrate a statistically significant impact on the D50% values, except for PLGA ALG MPs loaded with 0.4 and 1.0 μg/mL of coumarin-6 (p < 0.01). As expected, the surface modification produced a statistically significant impact on the surface charge (p < 0.001). Negative surface charges were displayed for non-fluorescent and fluorescent PLGA PVA MPs (-17.8±0.9 to -19.9±0.2 mV) and for non-fluorescent and fluorescent PLG ALG MPs (-30.8±2.3 to -36.8±4.4 mV). PLGA MPs containing CH PLGA showed a highly positive surface charge (50.8±2.7 and 58.7±3.4 mV). The presence of coumarin-6 did not demonstrate statistically significant impact on surface charge. Spectral studies showed that once increasing the MPs concentration in suspension no spectral changes were detected, suggesting that coumarin-6 did not change the partition environment to the surface and so remains on the hydrophobic matrix. In addition, this observation was further supported by the mainly linear variation of fluorescence intensity maximum upon increasing the concentration of the coumarin-6-loaded PLGA-C6 MPs, independently of their surface modification. At coumarin-6 concentrations above 0.2 µg/mg, the increase of the non-encapsulated molecules localized in the surface of the MPs or even aqueous medium was mainly evidenced by the decrease of anisotropy values. Regardless of MP composition, the red shift in probe emition of an aproximately 15 nm once increasing probe concentration from 0.04 to 1.00 The complexity of cell membranes and the development of nano/micro drug delivery systems make the topic of interactions between these two structures challenging. Studies point that the surface properties of these carriers like size, surface charge, shape and hydrophobicity largely influence such interactions. This project aims to study the interactions of surface modified Poly(lactic-co-glycolic acid) (PLGA) Microparticles (MPs) with Giant Unilamellar Vesicles (GUVs). MPs were formulated by the double emulsion solvent evaporation method and surface modified using chitosan (CH) and alginate (ALG) in order to manipulate the surface charge. Five concentrations of coumarin-6 (0.04, 0.10, 0.20, 0.40 and 1.00 g/mg) were entrapped into the PLGA matrix, after formulation optimization without probe. Phisichochemical characterization of MPs was made in terms of size, surface charge, coumarin-6 loading and morphology. Spectral properties of coumarin-6 were analyzed by fluorescence spectroscopy with the five different probe concentrations and five concentrations of MPs in suspension (0.10, 0.20, 0.25, 0.40, and 0.50 mg/mL). GUVs with different lipid composition and membrane properties, namely, homogeneously fluid GUVs and GUVs displaying gel-fluid and lo-ld phase separation were produced. Confocal microscopy was used to monitor the possible interactions between coumarin-6-loaded PLGA MPs and GUVs. MPs size (D50%) of non-fluorescent and fluorescent coumarin-6-loaded PLGA MPs ranged between 1.4±0.3 to 3.8±0.2 μm. The span values ranged between 1.1±0.2 to 4.8±1.1, demonstrating polidisperse populations. The surface modification did not demonstrate a statistically significant impact on the D50% values. In addition, the presence of coumarin-6 did not demonstrate a statistically significant impact on the D50% values, except for PLGA ALG MPs loaded with 0.4 and 1.0 μg/mL of coumarin-6 (p < 0.01). As expected, the surface modification produced a statistically significant impact on the surface charge (p < 0.001). Negative surface charges were displayed for non-fluorescent and fluorescent PLGA PVA MPs (-17.8±0.9 to -19.9±0.2 mV) and for non-fluorescent and fluorescent PLG ALG MPs (-30.8±2.3 to -36.8±4.4 mV). PLGA MPs containing CH PLGA showed a highly positive surface charge (50.8±2.7 and 58.7±3.4 mV). The presence of coumarin-6 did not demonstrate statistically significant impact on surface charge. Spectral studies showed that once increasing the MPs concentration in suspension no spectral changes were detected, suggesting that coumarin-6 did not change the partition environment to the surface and so remains on the hydrophobic matrix. In addition, this observation was further supported by the mainly linear variation of fluorescence intensity maximum upon increasing the concentration of the coumarin-6-loaded PLGA-C6 MPs, independently of their surface modification. At coumarin-6 concentrations above 0.2 µg/mg, the increase of the non-encapsulated molecules localized in the surface of the MPs or even aqueous medium was mainly evidenced by the decrease of anisotropy values. Regardless of MP composition, the red shift in probe emition of an aproximately 15 nm once increasing probe concentration from 0.04 to 1.00 µg/mg also account that observation. Studies on the MPs-membranes interaction studies were performed under the following experimental conditions: i) concentration of coumarin-6 0.2 µg/mg and ii) MPs concentration 0.25 mg/mL. Data showed that highly positive PLGA CH-C6-3 MPs and highly negative PLGA ALG-C6-3 MPs interacted with GUVs and were mainly directed to the gel phase and interface of fluid-gel phases. Results show that the MP surface modification plays an important role on surface charge. Entrapment of the fluorescent dye coumarin-6 conducts to a probe/MPs stable system. It was demonstrated that surface charge and biophysical membrane behaviour are important on interactions of surface modified MPs and GUVs. g/mg also account that observation. Studies on the MPs-membranes interaction studies were performed under the following experimental conditions: i) concentration of coumarin-6 0.2 µg/mg and ii) MPs concentration 0.25 mg/mL. Data showed that highly positive PLGA CH-C6-3 MPs and highly negative PLGA ALG-C6-3 MPs interacted with GUVs and were mainly directed to the gel phase and interface of fluid-gel phases. Results show that the MP surface modification plays an important role on surface charge. Entrapment of the fluorescent dye coumarin-6 conducts to a probe/MPs stable system. It was demonstrated that surface charge and biophysical membrane behaviour are important on interactions of surface modified MPs and GUVs. |
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| Autores principais: | Sirage, Melissa Margarete Jessen |
| Assunto: | Micropartículas ácido poli(láctico-co-glicólico) Cumarina-6 Carga superficial Vesículas unilamelares gigantes Interacções Teses de mestrado - 2016 |
| Ano: | 2016 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | The complexity of cell membranes and the development of nano/micro drug delivery systems make the topic of interactions between these two structures challenging. Studies point that the surface properties of these carriers like size, surface charge, shape and hydrophobicity largely influence such interactions. This project aims to study the interactions of surface modified Poly(lactic-co-glycolic acid) (PLGA) Microparticles (MPs) with Giant Unilamellar Vesicles (GUVs). MPs were formulated by the double emulsion solvent evaporation method and surface modified using chitosan (CH) and alginate (ALG) in order to manipulate the surface charge. Five concentrations of coumarin-6 (0.04, 0.10, 0.20, 0.40 and 1.00 g/mg) were entrapped into the PLGA matrix, after formulation optimization without probe. Phisichochemical characterization of MPs was made in terms of size, surface charge, coumarin-6 loading and morphology. Spectral properties of coumarin-6 were analyzed by fluorescence spectroscopy with the five different probe concentrations and five concentrations of MPs in suspension (0.10, 0.20, 0.25, 0.40, and 0.50 mg/mL). GUVs with different lipid composition and membrane properties, namely, homogeneously fluid GUVs and GUVs displaying gel-fluid and lo-ld phase separation were produced. Confocal microscopy was used to monitor the possible interactions between coumarin-6-loaded PLGA MPs and GUVs. MPs size (D50%) of non-fluorescent and fluorescent coumarin-6-loaded PLGA MPs ranged between 1.4±0.3 to 3.8±0.2 μm. The span values ranged between 1.1±0.2 to 4.8±1.1, demonstrating polidisperse populations. The surface modification did not demonstrate a statistically significant impact on the D50% values. In addition, the presence of coumarin-6 did not demonstrate a statistically significant impact on the D50% values, except for PLGA ALG MPs loaded with 0.4 and 1.0 μg/mL of coumarin-6 (p < 0.01). As expected, the surface modification produced a statistically significant impact on the surface charge (p < 0.001). Negative surface charges were displayed for non-fluorescent and fluorescent PLGA PVA MPs (-17.8±0.9 to -19.9±0.2 mV) and for non-fluorescent and fluorescent PLG ALG MPs (-30.8±2.3 to -36.8±4.4 mV). PLGA MPs containing CH PLGA showed a highly positive surface charge (50.8±2.7 and 58.7±3.4 mV). The presence of coumarin-6 did not demonstrate statistically significant impact on surface charge. Spectral studies showed that once increasing the MPs concentration in suspension no spectral changes were detected, suggesting that coumarin-6 did not change the partition environment to the surface and so remains on the hydrophobic matrix. In addition, this observation was further supported by the mainly linear variation of fluorescence intensity maximum upon increasing the concentration of the coumarin-6-loaded PLGA-C6 MPs, independently of their surface modification. At coumarin-6 concentrations above 0.2 µg/mg, the increase of the non-encapsulated molecules localized in the surface of the MPs or even aqueous medium was mainly evidenced by the decrease of anisotropy values. Regardless of MP composition, the red shift in probe emition of an aproximately 15 nm once increasing probe concentration from 0.04 to 1.00 The complexity of cell membranes and the development of nano/micro drug delivery systems make the topic of interactions between these two structures challenging. Studies point that the surface properties of these carriers like size, surface charge, shape and hydrophobicity largely influence such interactions. This project aims to study the interactions of surface modified Poly(lactic-co-glycolic acid) (PLGA) Microparticles (MPs) with Giant Unilamellar Vesicles (GUVs). MPs were formulated by the double emulsion solvent evaporation method and surface modified using chitosan (CH) and alginate (ALG) in order to manipulate the surface charge. Five concentrations of coumarin-6 (0.04, 0.10, 0.20, 0.40 and 1.00 g/mg) were entrapped into the PLGA matrix, after formulation optimization without probe. Phisichochemical characterization of MPs was made in terms of size, surface charge, coumarin-6 loading and morphology. Spectral properties of coumarin-6 were analyzed by fluorescence spectroscopy with the five different probe concentrations and five concentrations of MPs in suspension (0.10, 0.20, 0.25, 0.40, and 0.50 mg/mL). GUVs with different lipid composition and membrane properties, namely, homogeneously fluid GUVs and GUVs displaying gel-fluid and lo-ld phase separation were produced. Confocal microscopy was used to monitor the possible interactions between coumarin-6-loaded PLGA MPs and GUVs. MPs size (D50%) of non-fluorescent and fluorescent coumarin-6-loaded PLGA MPs ranged between 1.4±0.3 to 3.8±0.2 μm. The span values ranged between 1.1±0.2 to 4.8±1.1, demonstrating polidisperse populations. The surface modification did not demonstrate a statistically significant impact on the D50% values. In addition, the presence of coumarin-6 did not demonstrate a statistically significant impact on the D50% values, except for PLGA ALG MPs loaded with 0.4 and 1.0 μg/mL of coumarin-6 (p < 0.01). As expected, the surface modification produced a statistically significant impact on the surface charge (p < 0.001). Negative surface charges were displayed for non-fluorescent and fluorescent PLGA PVA MPs (-17.8±0.9 to -19.9±0.2 mV) and for non-fluorescent and fluorescent PLG ALG MPs (-30.8±2.3 to -36.8±4.4 mV). PLGA MPs containing CH PLGA showed a highly positive surface charge (50.8±2.7 and 58.7±3.4 mV). The presence of coumarin-6 did not demonstrate statistically significant impact on surface charge. Spectral studies showed that once increasing the MPs concentration in suspension no spectral changes were detected, suggesting that coumarin-6 did not change the partition environment to the surface and so remains on the hydrophobic matrix. In addition, this observation was further supported by the mainly linear variation of fluorescence intensity maximum upon increasing the concentration of the coumarin-6-loaded PLGA-C6 MPs, independently of their surface modification. At coumarin-6 concentrations above 0.2 µg/mg, the increase of the non-encapsulated molecules localized in the surface of the MPs or even aqueous medium was mainly evidenced by the decrease of anisotropy values. Regardless of MP composition, the red shift in probe emition of an aproximately 15 nm once increasing probe concentration from 0.04 to 1.00 µg/mg also account that observation. Studies on the MPs-membranes interaction studies were performed under the following experimental conditions: i) concentration of coumarin-6 0.2 µg/mg and ii) MPs concentration 0.25 mg/mL. Data showed that highly positive PLGA CH-C6-3 MPs and highly negative PLGA ALG-C6-3 MPs interacted with GUVs and were mainly directed to the gel phase and interface of fluid-gel phases. Results show that the MP surface modification plays an important role on surface charge. Entrapment of the fluorescent dye coumarin-6 conducts to a probe/MPs stable system. It was demonstrated that surface charge and biophysical membrane behaviour are important on interactions of surface modified MPs and GUVs. g/mg also account that observation. Studies on the MPs-membranes interaction studies were performed under the following experimental conditions: i) concentration of coumarin-6 0.2 µg/mg and ii) MPs concentration 0.25 mg/mL. Data showed that highly positive PLGA CH-C6-3 MPs and highly negative PLGA ALG-C6-3 MPs interacted with GUVs and were mainly directed to the gel phase and interface of fluid-gel phases. Results show that the MP surface modification plays an important role on surface charge. Entrapment of the fluorescent dye coumarin-6 conducts to a probe/MPs stable system. It was demonstrated that surface charge and biophysical membrane behaviour are important on interactions of surface modified MPs and GUVs. |
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