RESUMO
The potent photodynamic properties of Hypericin (Hyp) elicit a range of light-dose-dependent anti-tumor activities. However, its low water solubility hampers its broad application. Therefore, the administration of Hyp into biological systems requires drug carriers that would enable sufficient bioavailability. Stimuli-triggered nanocarriers, which are sensitive to endogenous or exogenous stimuli, have become an attractive replacement for conventional therapeutic regimens. Herein, we produced optimized Hyp thermosensitive liposomes (Hyp-TSL), self-assembled from DPPC, DSPC, DSPE-PEG2000. Hyp-TSL displayed a hydrodynamic diameter below 100 nm with an adequate encapsulation efficiency of 94.5 % and good colloidal stability. Hyp-TSL exhibited thermal sensitivity over a narrow range with a phase transition temperature of 41.1 °C, in which liposomal destruction was evident in AFM images after elevated temperature above the phase transition temperature. The uptake of TSL-Hyp into MDA-MB-231 cells was significantly increased with hyperthermic treatment of 42 °C when compared to the uptake at a average physiological temperature of 37 °C. Consequent enhancement of cellular reactive oxygen species was observed after hyperthermic treatment at 42 °C. The half-maximal inhibitory concentration of Hyp TSL was reduced by 3.8 fold after hyperthermic treatment at 42 °C in comparison to treatment at 37 °C. Hyp-TSL were considered safe for intravenous applications as compared by hemocompatibility studies, where coagulation time was <50 s and hemolytic potential was <10%. Conclusively, the enhancement in tumor drug availability correlated with improved therapeutic outcomes.
Assuntos
Hipertermia Induzida , Perileno , Antracenos , Lipossomos , Perileno/análogos & derivados , SolubilidadeRESUMO
Lung cancer is one of the most common causes for a high number of cancer related mortalities worldwide. Therefore, it is important to improve the therapy by finding new targets and developing convenient therapies. One of these novel non-invasive strategies is the combination of pulmonary delivered tetraether liposomes and photodynamic therapy. In this study, liposomal model formulations containing the photosensitiser curcumin were nebulised via two different technologies, vibrating-mesh nebulisation and air-jet nebulisation, and compared with each other. Particle size and ζ-potential of the liposomes were investigated using dynamic light scattering and laser Doppler anemometry, respectively. Furthermore, atomic force microscopy and transmission electron microscopy were used to determine the morphological characteristics. Using a twin glass impinger, suitable aerodynamic properties were observed, with the fine particle fraction of the aerosols being ≤62.7 ± 1.6%. In vitro irradiation experiments on lung carcinoma cells (A549) revealed an excellent cytotoxic response of the nebulised liposomes in which the stabilisation of the lipid bilayer was the determining factor. Internalisation of nebulised curcumin-loaded liposomes was visualised utilising confocal laser scanning microscopy. Based on these results, the pulmonary application of curcumin-loaded tetraether liposomes can be considered as a promising approach for the photodynamic therapy against lung cancer.
RESUMO
Metastatic breast cancer is one of the most common causes of cancer-related death in women worldwide. The transmembrane metalloprotease-disintegrin (ADAM8) protein is highly overexpressed in triple-negative breast cancer (TNBC) cells and potentiates tumor cell invasion and extracellular matrix remodeling. Exploiting the high expression levels of ADAM8 in TNBC cells by delivering anti-ADAM8 antibodies efficiently to the targeted site can be a promising strategy for therapy of TNBC. For instance, a targeted approach with the aid of ultra-high field magnetic resonance imaging (UHF-MRI) activatable thermosensitive liposomes (LipTS-GD) could specifically increase the intracellular accumulation of cytotoxic drugs. The surface of doxorubicin-loaded LipTS-GD was modified by covalent coupling of MAB1031 antibody (LipTS-GD-MAB) in order to target the overexpressed ADAM8 in ADAM8 positive MDA-MB-231 cells. Physicochemical characterization of these liposomes was performed using size, surface morphology and UHF-MRI imaging analysis. In vitro cell targeting was investigated by the washing and circulation method. Intracellular trafficking and lysosomal colocalization were assessed by fluorescence microscopy. Cell viability, biocompatibility and in-ovo CAM assays were performed to determine the effectiveness and safety profiles of liposome formulations. Our results show specific binding and induction of doxorubicin release after LipTS-GD-MAB treatment caused a higher cytotoxic effect at the cellular target site.
Assuntos
Proteínas ADAM/metabolismo , Antibióticos Antineoplásicos/administração & dosagem , Anticorpos Monoclonais/farmacologia , Imagem por Ressonância Magnética Intervencionista , Proteínas de Membrana/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Antibióticos Antineoplásicos/farmacocinética , Disponibilidade Biológica , Mama/diagnóstico por imagem , Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Embrião de Galinha , Membrana Corioalantoide , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacocinética , Liberação Controlada de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Lipossomos , Neoplasias de Mama Triplo Negativas/diagnóstico , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Photodynamic therapy (PDT) is a minimally invasive therapeutic approach used in the treatment of various medical conditions and cancerous diseases, involving light, a photosensitizing substance, and oxygen. Curcumin, a naturally occurring compound, carries antitumor activities and potentially could be exploited as a photosensitizer in PDT. Only little is known about liposomal-encapsulated curcumin that could help in increasing the efficacy, stability, and bioavailability of this compound. This study investigates the in vitro effects of curcumin-loaded liposomes in combination with PDT. Three papilloma virus-associated cell lines were treated with curcumin-loaded liposomes corresponding to a curcumin concentration of 0-100 µmol/L for 4 h followed by illumination at 457 nm (blue) for 45, 136, and 227 s at a fluence of 220.2 W/m2 (100 mA) corresponding to 1, 3 and 5 J·cm-2. After 24 h, the biological outcome of the treatment was assessed with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), SYTO9/PI (propidium iodide), Annexin V-FITC (fluorescein isothiocyanate)/PI, clonogenic survival, and scratch (wound closure) assays. Photoactivation of curcumin-loaded liposomes led to a significant reduction in colony formation and migratory abilities, as well as to an increase in tumor cell death. The results point to the combination of curcumin-loaded liposomes with PDT as a potentially useful tool for the treatment of papillomavirus-associated malignancies.
RESUMO
To limit the massive cytotoxicity of chemotherapeutic agents, it is desirable to establish an appropriate subtle blend of formulation design based on a dual-responsive strategy. In this study, a combined therapeutic platform based on magnetic thermosensitive liposomes (LipTS-GD) was developed. The incorporation of chelated-gadolinium imparted magnetic properties to thermosensitive liposomes (LipTS). The application of an ultra high field magnetic resonance imaging (UHF-MRI) induced hyperthermia, thus provided an improved chemotherapeutic effect of Doxorubicin (DOX). The paramagnetic platform demonstrated thermal sensitivity over a narrow temperature range starting at 37.8 °C, hence the release of DOX from LipTS-GD can be well triggered by inducing hyperthermia using UHF-MRI application. The prepared LipTS-GD were below 200 nm in diameter and an adequate release of DOX reaching 68% was obtained after 1 h UHF-MRI exposure. Profoundly, triple-negative breast cancer (TNBC) cells that were treated with LipTS-GD and subjected thereafter to UHF-MRI exposure for 60 min showed 36% viability. Hemocompatibility studies of LipTS-GD showed a physiological coagulation time and minimal hemolytic potential. Conclusively, LipTS-GD guided local delivery of DOX to solid tumors will potentially raise the therapeutic index, thus reducing the required dose and frequency of DOX administered systemically without influencing the adjacent tissues.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Hipertermia Induzida/métodos , Antibióticos Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Terapia Combinada , Doxorrubicina/química , Composição de Medicamentos , Feminino , Humanos , Lipossomos , Imageamento por Ressonância MagnéticaRESUMO
5,10,15,20-Tetrakis(3-hydroxyphenyl)chlorin (mTHPC; temoporfin) is one of the most potent second-generation photosensitizers available today for the treatment of a variety of clinical disorders and has a unique capability of being activated at different wavelengths. However, due to its highly lipophilic nature, poor solubility in the aqueous media and poor bioavailability limits its application in anticancer therapies. To overcome these potential issues, we developed three different liposomal formulations with mTHPC encapsulated in hydrophobic milieu thus increasing the bioavailability of the drug. The prepared formulations were characterized in terms of hydrodynamic diameter, surface charge, encapsulation efficiency, and stability studies. The mean size of the liposomes was found to be in the nanoscale range (about 100 nm) with zeta potential ranging from -6.0 to -13.7 mV. mTHPC loaded liposomes were also evaluated for morphology using atomic force microscopy (AFM) and cryo-transmission electron microscopy (cryo-TEM). Data obtained from the hemocompatibility experiments showed that these formulations were compatible with blood showing less than 10% hemolysis and coagulation time lower than 40 s. The results obtained from the single-cell gel electrophoresis assay also demonstrated no incidence of genotoxicity. Photodynamic destruction of SK-OV-3 cells using mTHPC loaded liposomes showed a dose-response relationship upon irradiation with two different wavelength lights (blue λ = 457 nm & red λ = 652 nm). A 10-fold pronounced effect was produced when liposomal formulations were irradiated at 652 nm as compared to 457 nm. This was also evaluated by the quantitative assessment of reactive oxygen production (ROS) using fluorescence microscopy. The qualitative assessment of PDT pre- and post-irradiation was visualized using confocal laser scanning microscopy (CLSM) which demonstrated an intense localization of mTHPC liposomes in the perinuclear region. Chick chorioallantoic membrane assay (CAM) was used as an alternative in-ovo model to demonstrate the localized destruction of tumor microvasculature. Overall, the prepared nanoformulation is a biocompatible, efficient and well characterized delivery system for mTHPC for the safe and effective PDT.
Assuntos
Carcinoma/tratamento farmacológico , Membrana Corioalantoide/irrigação sanguínea , Lipídeos/química , Mesoporfirinas/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Animais , Carcinoma/patologia , Linhagem Celular Tumoral , Embrião de Galinha , Relação Dose-Resposta a Droga , Composição de Medicamentos , Feminino , Humanos , Lipossomos , Mesoporfirinas/química , Densidade Microvascular/efeitos dos fármacos , Nanopartículas , Neoplasias Ovarianas/patologia , Fármacos Fotossensibilizantes/química , SolubilidadeRESUMO
Surface modification of nanoparticles with aptamer is gaining popularity lately due to its selective targeting and low immunogenicity. In this study, sorafenib tosylate (SFB) was loaded in biodegradable PLGA nanoparticles prepared by solvent evaporation method. The surfaces of drug deprived and drug-loaded particles (PN and PNS, respectively) were coupled with aptamer to target ErbB3 using EDC/NHS chemical modification. Nanoparticles were characterized with regard to their size, shape and chemical composition by dynamic light scattering, atomic force microscopy, FTIR and elemental analysis respectively. To evaluate the particles in vitro cell culture studies were performed. Cell viability assay, pathway analysis and apoptosis assay showed cellular toxicity in the presence of aptamer in PNS-Apt (p < 0.001). Metastatic progression assay showed decreased cell migration in the presence of aptamer and SFB. Confocal laser scanning microscopy was used to visualize the receptor-mediated time-dependent intracellular uptake and distribution of the nanoparticles throughout the cytoplasm. The findings of the current study demonstrated the potential efficacy of the surface modified SFB-loaded particles against ErbB3.
Assuntos
Aptâmeros de Nucleotídeos/farmacologia , Portadores de Fármacos/química , Nanopartículas/química , Receptor ErbB-3/antagonistas & inibidores , Sorafenibe/farmacologia , Apoptose/efeitos dos fármacos , Aptâmeros de Nucleotídeos/administração & dosagem , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Tamanho da Partícula , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Sorafenibe/administração & dosagem , Tecnologia Farmacêutica/métodosRESUMO
The naturally occurring anthraquinone derivative hypericin is a highly potent photosensitiser. Several in vitro studies show high phototoxicity of the pigment towards gram-positive bacteria. Nevertheless, the highly lipophilic nature and poor bioavailability prevent its application in daily clinical practice thus leading to a limited therapeutic value of hypericin. Liposomal encapsulation could help overcome these limitations and would make hypericin available for daily clinical practice. The use of liposomes as carriers for hypericin in antimicrobial photodynamic therapy (aPDT) is quite new. The aim of this work was to improve the photodynamic efficiency of the previously mentioned carriers by entrapping hypericin in the aqueous compartment of the liposomes. Therefore, a water-soluble inclusion complex of hypericin and (2-hydroxypropyl)-beta-cyclodextrin (Hyp-HPßCD) was prepared. After encapsulation of the inclusion complex into DSPC and DSPC/DPPC/DSPE-PEG liposomes with the dehydration-rehydration vesicle (DRV) method, the formulations were physicochemical characterised. The photodynamic efficiency towards the gram-positive model strain Staphylococcus saprophyticus subsp. bovis. was tested on planktonic cells as well as on biofilms. DSPC liposomes achieved a 4.1log reduction and the DSPC/DPPC/DSPE-PEG liposomes a 2.6log reduction in growth of planktonic bacteria, while Hyp-HPßCD showed total eradication. Even bacterial cells growing in a biofilm could be treated effectively in vitro.
Assuntos
Antibacterianos/química , Lipossomos/química , Perileno/análogos & derivados , Antracenos , Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Composição de Medicamentos/métodos , Perileno/química , Fosfatidiletanolaminas/química , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/química , Polietilenoglicóis/química , Staphylococcus saprophyticus/efeitos dos fármacos , beta-Ciclodextrinas/químicaRESUMO
Catheter related infections are causing one third of all blood stream infections. The mortality of those infections is very high and the gold standard for catheter related blood stream infections (CR-BSI) is still the removal of the catheter and systemic antibiotic therapy. There already exist some approaches to prevent the biofilm formation on catheter material, which are far from ideal. A new strategy to prevent bacterial colonization on catheter surfaces is the application of photodynamic therapy (PDT). Therefor the surface has to be modified with substances that can be activated by light, leading to the production of cell toxic reactive oxygen species (ROS). Only small concentrations of the so called photosensitizer (PS) are necessary, avoiding side effects in human therapy. Furthermore, there is no resistance development in PDT. In this study polyurethane (PUR) surfaces were coated with hypericin nanoformulations, leading to 4.3 log10 reduction in bacterial growth in vitro. The effect could be enhanced by the application of ultrasound. The combination of PDT with ultrasound therapy led to a synergistic effect resulting in a 6.8 log10 reduction of viable counts. This minimal invasive method requires only an optical fibre inserted in the catheter lumen and an ultrasound device. Thus the implementation in daily clinical practice is very simple.
Assuntos
Infecções Relacionadas a Cateter/microbiologia , Cateteres Venosos Centrais/microbiologia , Perileno/análogos & derivados , Fotoquimioterapia/métodos , Ultrassom/métodos , Antracenos , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Infecções Relacionadas a Cateter/terapia , Linhagem Celular , Humanos , Teste de Materiais , Nanoestruturas/química , Perileno/química , Perileno/farmacologia , Poliuretanos , Espécies Reativas de Oxigênio/metabolismo , Staphylococcus/efeitos dos fármacos , Staphylococcus/patogenicidade , Staphylococcus/fisiologia , Propriedades de SuperfícieRESUMO
Accumulation of photosensitisers in photodynamic therapy in healthy tissues is often the cause of unwanted side effects. Using nanoparticles, improved bioavailability and site-specific drug uptake can be achieved. In this study, curcumin, a natural product with anticancer properties, albeit with poor aqueous solubility, was encapsulated in biodegradable polymeric poly(lactic-co-glycolic acid) (PLGA) nanoparticles (CUR-NP). Dynamic light scattering, laser Doppler anemometry and atomic force microscopy were used to characterise the formulations. Using haemolysis, serum stability and activated partial thromboplastin time tests, the biocompatibility of CUR-NP was assessed. Particle uptake and accumulation were determined by confocal laser scanning microscopy. Therapeutic efficacy of the formulation was tested in SK-OV-3 human ovarian adenocarcinoma cells post low level LED irradiation by determining the generation of reactive oxygen species and cytotoxicity. Pharmacologic inhibitors of cellular uptake pathways were used to identify the particle uptake mechanism. CUR-NP exhibited better physicochemical properties such as stability in the presence of light and improved serum stability compared to free curcumin. In addition, the novel nanoformulation facilitated the use of higher amounts of curcumin and showed strong apoptotic effects on tumour cells.
RESUMO
Photodynamic therapy is amongst the most rapidly developing therapeutic strategies against cancer. However, most photosensitizers are administered intravenously with very few reports about pulmonary applications. To address this issue, an inhalable formulation consisting of nanoparticles loaded with photosensitizer (i.e. curcumin) was developed. The nanoparticles were prepared using nanoprecipitation method. Dynamic light scattering measurements of the curcumin loaded nanoparticles revealed a hydrodynamic diameter of 181.20⯱â¯11.52â¯nm. In vitro irradiation experiments with human lung epithelial carcinoma cells (A549) showed a selective cellular toxicity of the nanoparticles upon activation using LED irradiating device. Moreover, curcumin nanoparticles exhibited a dose-dependent photocytotoxicity and the IC50 values of curcumin were directly dependent on the radiation fluence used. The nanoparticles were subsequently spray dried using mannitol as a stabilizer to produce Nano-in-Microparticles with appropriate aerodynamic properties for a sufficient deposition in the lungs. This was confirmed using the next generation impactor, which revealed a large fine particle fraction (64.94⯱â¯3.47%) and a mass median aerodynamic diameter of 3.02⯱â¯0.07⯵m. Nano-in-Microparticles exhibited a good redispersibility and disintegrated into the original nanoparticles upon redispersion in aqueous medium. The Langmuir monolayer experiments revealed an excellent compatibility of the nanoparticles with the lung surfactant. Results from this study showed that the Nano-in-Microparticles are promising drug carriers for the photodynamic therapy of lung cancer.
Assuntos
Curcumina/administração & dosagem , Portadores de Fármacos/administração & dosagem , Nanopartículas/administração & dosagem , Neoplasias/terapia , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/administração & dosagem , Células A549 , Administração por Inalação , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Curcumina/farmacologia , Composição de Medicamentos , Humanos , Luz , Fármacos Fotossensibilizantes/farmacologiaRESUMO
Photodynamic therapy (PDT) is an established noninvasive tumor treatment. The hydrophobic natural occurring pigment hypericin shows a lot of attractive properties for the application in PDT. Hence, the administration to biological systems or patients requires the formulation in drug carriers enabling sufficient bioavailability. Therefore, free hypericin was encapsulated by the thin film hydration method or a hypericin-hydroxypropyl-ß-cyclodextrin inclusion complex (Hyp-HPßCD) was incorporated by dehydration-rehydration vesicle method in either conventional or ultra-stable tetraether lipid (TEL) liposomes. The hydrodynamic diameter of the prepared nanoformulations ranged between 127 and 212 nm. These results were confirmed by atomic force microscopy. All liposomes showed a good stability under physiological conditions. TEL liposomes which tend to build more rigid bilayers, generate higher encapsulation efficiencies than their conventional counterparts. Furthermore, the suitability for intravenous application was confirmed by hemocompatibility studies resulting in a hemolytic potential less than 20% and a coagulation time less than 50 sec. The uptake of liposomal hypericin into human ovarian carcinoma cells (SK-OV-3) was confirmed using confocal microscopy and further characterized by pathway studies. It was demonstrated that the lipid composition and intraliposomal hypericin localization influenced the anti-vascular effect in the chorioallantoic membrane (CAM). While hypericin TEL liposomes exhibit substantial destruction of the microvasculature drug-in-cyclodextrin TEL liposomes showed no effect. Nevertheless, both formulations yielded severe photocytotoxicity in SK-OV-3 cells in a therapeutic dosage range. Conclusively, hypericin TEL liposomes would be perfectly suited for anti-vascular targeting while Hyp-HPßCD TEL liposomes could deliver the photosensitizer to the tumor site in a more protected manner.
Assuntos
Inibidores da Angiogênese/administração & dosagem , Antineoplásicos/administração & dosagem , Perileno/análogos & derivados , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/administração & dosagem , Inibidores da Angiogênese/metabolismo , Animais , Antracenos , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Galinhas , Membrana Corioalantoide/efeitos dos fármacos , Membrana Corioalantoide/metabolismo , Relação Dose-Resposta a Droga , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Feminino , Humanos , Lipossomos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Perileno/administração & dosagem , Perileno/metabolismo , Fármacos Fotossensibilizantes/metabolismoRESUMO
Photodynamic therapy is one of the most promising non-invasive strategies employed for the treatment of several kinds of bacterial infections. Though the vast majority of clinically approved photosensitisers are administered intravenously, most of the in vitro experiments are performed under static conditions which do not represent the physiological environment of the venous bloodstream. To address this issue, a dynamic circulation model was developed to facilitate in situ antibacterial photodynamic therapy under flow conditions to mimic blood stream infections.
Assuntos
Bacteriemia/tratamento farmacológico , Fotoquimioterapia/métodos , Humanos , Teste de Materiais , Nanopartículas/efeitos adversos , Nanopartículas/químicaRESUMO
Non-viral vectors are a safe, efficient and non-toxic alternative to viral vectors for gene therapy against many diseases ranging from genetic disorders to cancers. Polyamidoamine (PAMAM), a positively charged dendrimer has a tendency to complex with nucleic acids (to form dendriplexes) like plasmid DNA (pDNA) and small interfering RNA (siRNA) and can shield them from enzymatic degradation, thereby facilitating endocytosis and endosomal release. In this study, we developed an advanced variant of the dendriplexes by encapsulating them within liposomes to enhance their gene delivery efficiency. This liposome encapsulated dendriplex system can further reduce unwanted cytotoxicity and enhance cellular uptake of nucleic acids. A broad range of lipid combinations were used to optimize the lipodendriplexes in terms of their physicochemical characteristics including size, shape and zeta potential. The optimized lipodendriplexes were tested for pDNA transfection, in vitro cell viability, cellular uptake, siRNA mediated knockdown, hemocompatibility, metastatic progression and in ovo in chorioallantoic membrane model (CAM). The optimized system has shown significant improvement in pDNA transfection (pâ¯<â¯0.01) with higher GFP expression and gene silencing and has shown improved cell viability (pâ¯<â¯0.05) compared to the parent dendriplex system. The hemocompatibility and CAM analysis, revealed an efficient yet biocompatible gene delivery system in the form of lipodendriplexes.
Assuntos
Dendrímeros/química , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos , Animais , Linhagem Celular , Sobrevivência Celular/genética , Galinhas , Membrana Corioalantoide/metabolismo , DNA/administração & dosagem , Técnicas de Silenciamento de Genes , Inativação Gênica , Vetores Genéticos/efeitos adversos , Proteínas de Fluorescência Verde/genética , Humanos , Lipídeos/química , Lipossomos , Nanoestruturas , Plasmídeos/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , TransfecçãoRESUMO
Oncological use of photodynamic therapy is an evolving field in cancer therapeutics. Photosensitisers are prone to accumulation inside healthy tissues causing undesirable effects. To avoid this, we have developed tetraether lipid liposomal formulations containing curcumin which is a naturally occurring anti-cancer substance and deemed to be safe towards healthy cells. Upon excitation with light at a specific wavelength, curcumin produces reactive oxygen species (ROS) in presence of oxygen, thereby exhibiting a cytotoxic effect towards the surrounding tissues, giving a total control on the onset of therapy. In our study, we examined two different liposomal formulations wherein curcumin is encapsulated within the hydrophobic milieu with the intent to increase its bioavailability. Hydrodynamic diameter, surface charge, stability, morphology and haemocompatibility of the liposomes were studied. The results confirmed the formation of stable nanometre range liposomal vesicles (200-220 nm) containing curcumin which were haemocompatible with coagulation time less than 50 s and a haemolytic potential below 40%. Increased ROS generation post irradiation (>50% compared to un-irradiated samples) was confirmed using fluorescence spectroscopy. The efficiency and selectivity of the PDT was demonstrated by assessing their viability post irradiation and by qualitative analysis using confocal microscopy showing nuclear perforation induced by PDT. Photo-destructive effects of PDT on the microvasculature were studied in vivo using chick chorioallantoic membrane model (CAM). Considerable phototoxicity could be observed in the irradiated area of the CAM 30 min post irradiation. Phototoxic effects in vitro (in SK-OV-3 and PCS-100-020™) and in vivo (in chorioallantoic membrane model) in combination with a novel custom manufactured LED irradiating device showed a formulation dependant selective photodynamic effect of the curcumin liposomes.
Assuntos
Curcumina/administração & dosagem , Portadores de Fármacos/administração & dosagem , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/administração & dosagem , Animais , Linhagem Celular Tumoral , Células Cultivadas , Embrião de Galinha , Membrana Corioalantoide/efeitos dos fármacos , Membrana Corioalantoide/metabolismo , Curcumina/metabolismo , Portadores de Fármacos/metabolismo , Humanos , Lipossomos , Fármacos Fotossensibilizantes/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Cytotoxicity is a major drawback impeding the therapeutic use of gene delivery and gene down-regulation vehicles. Apart from cytotoxicity, rapid degradation and low cellular uptake are other major factors affecting therapeutic use. Considering the above factors, formulation and development of PEI (Polyethylenimine) based, liposome encapsulated delivery vehicles with improved transfection efficiency and low cytotoxicity which can be used for gene delivery and gene knockdown. DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine), DPPC (Dipalmitoylphosphatidylcholine) and cholesterol have been considered as lipids of choice bearing in mind various factors such as rigidness and surface charge which greatly influence the formation of liposomes, polyplex encapsulation and transfection efficiency. For the condensation of plasmid DNA (pDNA) and short interfering RNA (siRNA), branched PEI 25kDa (bPEI) and deacylated linear PEI 22kDa (lPEI) were employed. lPEI and siRNA polyplexes encapsulated within DOPE/DPPC/Cholesterol (DDC) liposomes exhibited higher luc (luciferase) gene knockdown in vitro compared to the controls. They also showed superior transfection efficiencies compared to polyplexes in experiments using pCMV-luc (luciferase reporter plasmid) and pEGFP-N1 (Green Fluorescence protein reporter plasmid). This can partly be attributed to the improved integrity imparted by the liposomal layer which was confirmed by complex stability and integrity assays. Cytotoxicity and coagulation time assays of DDC-lPEI based lipopolyplexes showed decreased cytotoxic potential and negligible influence on coagulation respectively for compared to polyplexes, thus rendering them suitable for gene therapy.