Development, physico-chemical and toxicological characterisation of solid lipid nanoparticles for application in breast cancer therapy
Ficheiros
Data
2013
Autores
Título da revista
ISSN da revista
Título do Volume
Editora
Resumo
As nanopartículas de lípidos sólidos (SLN, do inglês “solid lipid nanoparticles”) são
transportadores coloidais de fármacos, constituídos por uma matriz de lípidos sólidos à
temperatura corporal e à temperatura ambiente, estabilizados por agentes tensioactivos
apropriados. No âmbito da presente tese, estes sistemas foram desenvolvidos com objectivo
de administração de fármacos pouco solúveis em água, e para facilitar a administração
direccionada a células de cancro.
O objectivo deste trabalho consistiu em explorar o potencial das SLN no tratamento da
invasão celular de cancro de mama, nomeadamente das células HER2/neu positivas. Foram
desenvolvida partículas cuja composição consistiu no Imwitor 900K ou Compritol 888 ATO
(como lípidos sólidos), no cetyltrimethylammonium bromide (CTAB) como lípido
catiónico/tensioactivo e no Lutrol F68 ou Miranol C32 Ultra como agente tensioactivo. Como
método de produção, foi utilizada a homogenização a alta pressão ou a alta velocidade,
seguindo-se a optimização das respectivas formulações, as quais foram utilizadas para os
estudos posteriores. As SLN foram caracterizadas em termos de distribuição de tamanho
médio das partículas, cristalinidade de matriz lipídica sólida e estabilidade durante
armazenamento. Foram obtidas SLN com tamanho médio das partículas entre 115 nm e 334
nm e d0.90 inferior a 1 μm. O estado sólido das mesmas foi confirmado por calorimetria
diferencial de varrimento e por difracção de raios X. Todas as formulações apresentaram
estabilidade adequada ao longo de 5 semanas, quer à temperatura ambiente, quer a 4 °C.
Apesar da liofilização com o crioprotector trealose, a estabilidade das SLN não liofilizadas
revelou-se bastante superior. A formulação designada como cSLN-C manteve-se estável
durante um período mínimo de 12 semanas.
As SLN são, em geral, consideradas como transportadores coloidais com baixa
toxicidade. Mesmo assim, o efeito das SLN per si tem importância na interpretação da
interacção de formulações que contém um fármaco ou um anticorpo com as células. As SLN
desenvolvidas neste trabalho não apresentaram toxicidade na concentração de 0,01 mg/ml.
Utilizando a concentração de 0,1 mg/ml a viabilidade celular diminuiu dependendo da linha
celular utilizada e tempo de exposição. A dose 1,0 mg/ml foi tóxica nas linhas celulares
seleccionadas para este trabalho. Dentro destas, MCF-7 (carcinoma de mama, receptor de
estrogénio positivo, HER2-neu negativo) foram as mais susceptíveis aos danos causados pelas
SLN, as BT-474 (carcinoma mamário, HER2-neu positivo), HepG2 (hepatocarcinoma) e
Caco-2 (cólon adenocarcinoma) foram menos susceptíveis em ordem decrescente. A
toxicidade das SLN foi causada por disrupção de integridade das membranas celulares. Danos
em ácido deoxiribonucléico (ADN) foram detectados por ensaio cometa. Foram reportados
poucos danos – quando comparado com controlo sem tratamento (não significativo nas
concentrações não tóxicas). Também foram detectados danos em purinas, que não causaram
quebras de ADN. Alguns sinais de stress oxidativo foram detectados em células HepG2: a
fluorescência de diacetato de diclorofluoresceina (DCFDA) encontrou-se aumentada
relativamente aos controlos sem tratamento e aos positivos, verificou-se um aumento da
actividade da enzima superóxido dismutase e uma diminuição da actividade de glutationa
reductase. Apesar destes sinais de existência de stress oxidativo, os lípidos membranares não
foram afectados (determinação como substâncias reactivas ao ácido tiobarbitúrico, TBARS).
Estes resultados estão em concordância com poucos danos detectados em ADN (relativamente
ao controlo sem tratamento). Os danos causados por stress oxidativo podem ocorrer em
células com capacidade antioxidante inferior à das células HepG2. A capacidade de indução
de stress oxidativo pode, hipoteticamente, ser vantajosa em veiculação de fármacos
quimioterapêuticos, cujo mecanismo de acção exige existência de radicais livres, e pode,
parcialmente, contribuir para a melhoria de eficácia destes medicamentos, quando veiculadas
em SLN in vitro e in vivo.
A Curcumina foi seleccionada como fármaco-modelo com potencial actividade
antineoplásica. A baixa solubilidade aquosa, instabilidade em pH alcalino e fotossensibilidade
são propriedades que fazem da curcumina um fármaco ideal para a encapsulação em SLN.
Contudo, a solubilidade em vários lípidos foi igual ou inferior a 1 %. A baixa solubilidade em
lípidos influenciou a capacidade de carga. Em combinação com as limitações atribuídas à
toxicidade das SLN, apenas pode ser administrada 10 μg/ml (27 μM) no máximo, uma dose
que é insuficiente para observar os efeitos anticancerígenos da curcumina. Um anticorpo anti-
HER2/neu foi colocado na superfície das SLN utilizando a interacção streptavidina-biotina. O
efeito de complexo anticorpo-SLN foi governado pela toxicidade das próprias SLN. A
conjugação com o anticorpo melhorou significativamente a internalização de complexos nas
células de cancro mamário. O efeito foi mais marcado em células BT474, HER2/neu
positivas. O tratamento com complexo SLN-anticorpo causou uma diminuição de viabilidade
celular das linhas de cancro de mama superior ao efeito das partículas isoladas ou do anticorpo isolado.
Solid lipid nanoparticles (SLN) are colloidal carriers consisting of lipid cores that are solid at body and room temperature dispersed in aqueous phase and stabilized by suitable surfactant. They were developed to improve drug delivery of drugs that are poorly soluble in water and to enable targeted delivery to cancer cells. The aim of this work was to explore the potential of SLN in treatment of breast cancer cell invasion, namely HER2/neu positive breast cancer cells. A series of SLN composed of Imwitor 900K or Compritol 888 ATO as solid lipid, cetyltrimethylammonium bromide (CTAB) as cationic lipid/surfactant and Lutrol F68 or Miranol C32 Ultra as surfactants was developed. Optimized high shear homogenisation of high pressure homogenisation were used as preparation methods. SLN were characterized in terms of particle size distibution, lipid core crystalinity and storage stability. SLN with mean particle size between 115 nm and 334 nm and d0.50 below 0.5 μm were obtained; the crystalline state of lipid cores was confirmed by differential scanning calorimetry and X-ray diffraction. All SLN were stable for at least 4 weeks at room temperature and 4 °C, which was superior to stability of the same SLN freezedryed with trehalose. Compritol-composed SLN were stable over 12 weeks. SLN are in general considered as safe colloidal carriers, their effect on living cells however cannot be neglected when interpreting the studies of interaction of drug-loaded and/or targeted SLN with cells. SLN developed in this work were non-toxic to living cells at a dose 0.01 mg/ml; cell viability was reduced to various extent at 0.1 mg/ml depending on cell line and time of exposure and at 1.0 mg/ml the SLN were toxic to the selected cell lines. Among the used cell lines, MCF-7 cells (breast carcinoma, estrogen receptor positive, Her2/neu negative) were the most susceptible to our SLN, followed by BT-474 (breast carcinoma, HER2/neu positive), HepG2 (hepatocarcinoma) and Caco-2 (colorectal carcinoma) cells. Toxicity of SLN was caused mostly by disruption of membrane integrity. DNA damage was examined by comet assay and was detected in a limited extent, compared to untreated controls (not significant at non-toxic concentrations). Damage to purine bases that did not directly lead to DNA strand breaks was also detected. Some signs of oxidative stress was detected in HepG2 cells: dichlorofluorescein-diacetate (DCFDA) assay revealed increase in free radicals content compared to untreated and positive controls, activity of superoxid dismutase was found increased and activity of glutathion reductase was drastically decreased. Despite these signs of oxidative stress, membrane lipids were not affected – as determined by thiobarbituric acid reactive species (TBARS) determination. This finding is in line with only slightly increased DNA strand breaks (compared to untreated control). Damage caused by oxidative stress after SLN exposure may however occur in cells with lower antioxidant capacity than HepG2 cells. The capacity to induce oxidative stress can hypotethically be beneficial for delivery of chemotherapeutic drugs – that require some free radical increase for their action – and may partly explain many reports on SLN improving efficiency of chemotherapeutics in vitro and in vivo. Curcumin was selected as model drug with potential chemotherapeutic effect. Its low solubility, instability at alkaline pH and light make it an ideal candidate for encapsulation into SLN. Unfortunately, its solubility in solid lipids was limited to 1% (w/w) and to lipid mixtures containing either monoacylglycerides or polyethylenglycol. This affected the resulting drug loading, which together with limitations by SLN toxicity only enabled use of dose equal or lower than 10 μg/ml (27μM) of curcumin – i.e. doses lower than those at which anticancer effects were observed. An antiHER2/neu antibody was attached to SLN surface via streptavidin-biotin binding. The effect of targeted complex was influenced mostly by the toxicity of SLN alone, but at non-toxic dose of SLN a synergistic effect between SLN and the antibody was observed. The antibody improved significantly cell internalization into breast cancer cells, mostly in HER2/neu positive BT-474 cells but to some extent also in MCF-7 cells. Exposure to targeted SLN leads to cell viabilities lower than when exposed to antibody alone or SLN alone.
Solid lipid nanoparticles (SLN) are colloidal carriers consisting of lipid cores that are solid at body and room temperature dispersed in aqueous phase and stabilized by suitable surfactant. They were developed to improve drug delivery of drugs that are poorly soluble in water and to enable targeted delivery to cancer cells. The aim of this work was to explore the potential of SLN in treatment of breast cancer cell invasion, namely HER2/neu positive breast cancer cells. A series of SLN composed of Imwitor 900K or Compritol 888 ATO as solid lipid, cetyltrimethylammonium bromide (CTAB) as cationic lipid/surfactant and Lutrol F68 or Miranol C32 Ultra as surfactants was developed. Optimized high shear homogenisation of high pressure homogenisation were used as preparation methods. SLN were characterized in terms of particle size distibution, lipid core crystalinity and storage stability. SLN with mean particle size between 115 nm and 334 nm and d0.50 below 0.5 μm were obtained; the crystalline state of lipid cores was confirmed by differential scanning calorimetry and X-ray diffraction. All SLN were stable for at least 4 weeks at room temperature and 4 °C, which was superior to stability of the same SLN freezedryed with trehalose. Compritol-composed SLN were stable over 12 weeks. SLN are in general considered as safe colloidal carriers, their effect on living cells however cannot be neglected when interpreting the studies of interaction of drug-loaded and/or targeted SLN with cells. SLN developed in this work were non-toxic to living cells at a dose 0.01 mg/ml; cell viability was reduced to various extent at 0.1 mg/ml depending on cell line and time of exposure and at 1.0 mg/ml the SLN were toxic to the selected cell lines. Among the used cell lines, MCF-7 cells (breast carcinoma, estrogen receptor positive, Her2/neu negative) were the most susceptible to our SLN, followed by BT-474 (breast carcinoma, HER2/neu positive), HepG2 (hepatocarcinoma) and Caco-2 (colorectal carcinoma) cells. Toxicity of SLN was caused mostly by disruption of membrane integrity. DNA damage was examined by comet assay and was detected in a limited extent, compared to untreated controls (not significant at non-toxic concentrations). Damage to purine bases that did not directly lead to DNA strand breaks was also detected. Some signs of oxidative stress was detected in HepG2 cells: dichlorofluorescein-diacetate (DCFDA) assay revealed increase in free radicals content compared to untreated and positive controls, activity of superoxid dismutase was found increased and activity of glutathion reductase was drastically decreased. Despite these signs of oxidative stress, membrane lipids were not affected – as determined by thiobarbituric acid reactive species (TBARS) determination. This finding is in line with only slightly increased DNA strand breaks (compared to untreated control). Damage caused by oxidative stress after SLN exposure may however occur in cells with lower antioxidant capacity than HepG2 cells. The capacity to induce oxidative stress can hypotethically be beneficial for delivery of chemotherapeutic drugs – that require some free radical increase for their action – and may partly explain many reports on SLN improving efficiency of chemotherapeutics in vitro and in vivo. Curcumin was selected as model drug with potential chemotherapeutic effect. Its low solubility, instability at alkaline pH and light make it an ideal candidate for encapsulation into SLN. Unfortunately, its solubility in solid lipids was limited to 1% (w/w) and to lipid mixtures containing either monoacylglycerides or polyethylenglycol. This affected the resulting drug loading, which together with limitations by SLN toxicity only enabled use of dose equal or lower than 10 μg/ml (27μM) of curcumin – i.e. doses lower than those at which anticancer effects were observed. An antiHER2/neu antibody was attached to SLN surface via streptavidin-biotin binding. The effect of targeted complex was influenced mostly by the toxicity of SLN alone, but at non-toxic dose of SLN a synergistic effect between SLN and the antibody was observed. The antibody improved significantly cell internalization into breast cancer cells, mostly in HER2/neu positive BT-474 cells but to some extent also in MCF-7 cells. Exposure to targeted SLN leads to cell viabilities lower than when exposed to antibody alone or SLN alone.
Descrição
Tese de Doutoramento em Genética Molecular Comparativa
Palavras-chave
Nanopartículas , Curcumina , Terapia génica , Neoplasias mamárias , Citotoxicidade imunológica , Genotoxicidade