RESUMEN
Dendrimer-based nanoparticles have shown promising applications in delivery of small interference RNA (siRNA) to downregulate proteins that contribute to multidrug resistance (MDR). Various types of modification can further enhance the anti-tumor efficacy of dendrimer-based nanoparticles. In this study, generation 4 polyamodoamine (PAMAM) was conjugated with PEG2k-DOPE. The PAMAM-PEG2k-DOPE co-polymer, together with mPEG2k-DOPE, was formulated into mixed dendrimer micelles (MDMs) that can complex siRNA through the cationic PAMAM moieties and encapsulate hydrophobic drug in the micellar lipid cores. DOPE-conjugated hyaluronic acid (HA) was coated on the surface of MDMs to shield the exposed positive charge on PAMAM and to increase the cellular association with CD44+ cancer cells. The HA-modified MDMs could form stable complexes with siRNA, prevent RNase-mediated siRNA degradation and maintain its integrity. Cellular association and cytotoxicity of HA-modified MDMs were investigated in A2780 ADR, MDA-MB-231 and HCT 116 cell lines. The HA-modified MDMs alleviated the toxicity from cationic charge, increase the cancer cell specificity and enhance the cancer cell killing effect in CD44+ cell line.
Asunto(s)
Dendrímeros/administración & dosificación , Doxorrubicina/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Ácido Hialurónico/administración & dosificación , Nanopartículas/administración & dosificación , Antibióticos Antineoplásicos/administración & dosificación , Antibióticos Antineoplásicos/síntesis química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Dendrímeros/síntesis química , Relación Dosis-Respuesta a Droga , Doxorrubicina/síntesis química , Resistencia a Múltiples Medicamentos/fisiología , Células HCT116 , Humanos , Ácido Hialurónico/síntesis química , Nanopartículas/químicaRESUMEN
PURPOSE: Since the last decade, it is established that nonspecific delivery of chemotherapeutics fails to effectively treat cancer due to systemic cytotoxicity, poor biodistribution at tumor site and most importantly the development of drug resistance (MDR). Stimuli-sensitive drug delivery systems gained significant attention in recent years for effective tumor therapy and reversal of MDR. The aim of this study was developing a redox sensitive micellar prodrug system, by taking the advantage of the significant difference in GSH levels between extracellular and intracellular environments, but more importantly in healthy and tumor tissues. METHODS: Redox sensitive PEG2000-S-S-PTX micelles were developed for intracellular paclitaxel delivery and characterized in vitro. In vitro release studies were carried out and followed by cytotoxicity studies in chemo-resistant ovarian and breast cancer cells in various reducing environments for different time periods to confirm their potential. RESULTS: PEG2000-S-S-PTX, was synthesized and characterized as a redox sensitive micellar prodrug system. The reduction sensitivity and in vitro PTX release properties were confirmed in reducing environments comparatively with physiological conditions. Cytotoxicity studies suggested that ovarian (SK-OV-3) cells could be better candidates for treatment with redox-sensitive drug delivery systems than breast (MCF-7) cancer cells. CONCLUSIONS: The results of this study highlights the importance of personalized therapy since no fits-for-all system can be developed for different cancer with significantly different metabolic activities. Graphical Abstract Schematic representation of self-assembly of reduction-sensitive PEG2000-S-S-PTX micelles and GSH dependent release of PTX.
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Antineoplásicos Fitogénicos/administración & dosificación , Neoplasias de la Mama/tratamiento farmacológico , Preparaciones de Acción Retardada/química , Disulfuros/química , Neoplasias Ováricas/tratamiento farmacológico , Paclitaxel/administración & dosificación , Antineoplásicos Fitogénicos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Femenino , Humanos , Micelas , Oxidación-Reducción , Paclitaxel/farmacología , Polietilenglicoles/químicaRESUMEN
The effectiveness of existing anti-cancer therapies is based mainly on the stimulation of apoptosis of cancer cells. Most of the existing therapies are somewhat toxic to normal cells. Therefore, the quest for nontoxic, cancer-specific therapies remains. We have demonstrated the ability of liposomes containing anacardic acid, mitoxantrone and ammonium ascorbate to induce the mitochondrial pathway of apoptosis via reactive oxygen species (ROS) production by the killing of cancer cells in monolayer culture and shown its specificity towards melanoma cells. Liposomes were prepared by a lipid hydration, freeze-and-thaw (FAT) procedure and extrusion through polycarbonate filters, a remote loading method was used for dug encapsulation. Following characterization, hemolytic activity, cytotoxicity and apoptosis inducing effects of loaded nanoparticles were investigated. To identify the anticancer activity mechanism of these liposomes, ROS level and caspase 9 activity were measured by fluorescence and by chemiluminescence respectively. We have demonstrated that the developed liposomal formulations produced a high ROS level, enhanced apoptosis and cell death in melanoma cells, but not in normal cells. The proposed mechanism of the cytotoxic action of these liposomes involved specific generation of free radicals by the iron ions mechanism.
RESUMEN
Recent advances in combination therapy by using chemotherapeutic drugs and small noncoding RNAs have highlighted the need for optimization of such agents to allow their carriage in a single delivery system. This protocol details the synthesis of a doxorubicin prodrug, where a NHS coupling reaction was used to sensitize the drug to the proteolytic activity of tumor microenvironments. The design of a lipid-modified miRNA by an S-S coupling reaction is also described. Modification of both, doxorubicin and miRNA, facilitated their simultaneous incorporation into mixed micelles for use in combination therapy against tumor cells.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , MicroARNs/genética , Neoplasias/tratamiento farmacológico , Profármacos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Terapia Combinada , Doxorrubicina/química , Doxorrubicina/farmacología , Humanos , MicroARNs/farmacología , Neoplasias/genética , Polímeros/química , Profármacos/químicaRESUMEN
Modification of nanoparticle surfaces with PEG has been widely considered the gold standard for many years. However, PEGylation presents controversial and serious challenges including lack of functionality, hindered cellular interaction, allergic reactions, and stimulation of IgM production after repetitive dosing that accelerates blood clearance of the nanoparticles. We report the development of novel liposomal formulations surface-modified with a low molecular weight, branched polyethyleneimine (bPEI)-lipid conjugate for use as an alternative to PEG. The formulations had very good stability characteristics in ion- and protein-rich mediums. Protein adsorption onto the liposomal surface did not interfere with the cellular interaction. bPEI-modified liposomes (PEIPOS) showed enhanced association with three different cell lines by up to 75 times compared to plain or PEGylated liposomes and were without carrier toxicity. They also penetrated the deeper layers of 3D spheroids. Encapsulating paclitaxel (PTX) into PEIPOS did not change its main mechanism of action. PEIPOS complexed and intracellularly delivered siRNAs and downregulated resistance-associated proteins. Finally, tumor growth inhibition was observed in a mouse ovarian xenograft tumor model, without signs of toxicity, in animals treated with the siRNA/PTX co-loaded formulation. These complex-in-nature but simple-in-design novel liposomal formulations constitute viable and promising alternatives with added functionality to their PEGylated counterparts.
Asunto(s)
Nanopartículas , Paclitaxel/administración & dosificación , Polietileneimina/química , ARN Interferente Pequeño/administración & dosificación , Animales , Antineoplásicos Fitogénicos/administración & dosificación , Antineoplásicos Fitogénicos/farmacología , Línea Celular Tumoral , Portadores de Fármacos/química , Resistencia a Antineoplásicos , Femenino , Humanos , Lípidos/química , Liposomas , Ratones , Ratones Desnudos , Neoplasias Ováricas/terapia , Paclitaxel/farmacología , Polietilenglicoles/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Multidrug resistance (MDR) significantly decreases the therapeutic efficiency of anti-cancer drugs. Its reversal could serve as a potential method to restore the chemotherapeutic efficiency. Downregulation of MDR-related proteins with a small interfering RNA (siRNA) is a promising way to reverse the MDR effect. Additionally, delivery of small molecule therapeutics simultaneously with siRNA can enhance the efficiency of chemotherapy by dual action in MDR cell lines. Here, we conjugated the dendrimer, generation 4 polyamidoamine (G4 PAMAM), with a polyethylene glycol (PEG)-phospholipid copolymer. The amphiphilic conjugates obtained spontaneously self-assembled into a micellar nano-preparation, which can be co-loaded with siRNA onto PAMAM moieties and sparingly water-soluble chemotherapeutics into the lipid hydrophobic core. This system was co-loaded with doxorubicin (DOX) and therapeutic siRNA (siMDR-1) and tested for cytotoxicity against MDR cancer cells: human ovarian carcinoma (A2780 ADR) and breast cancer (MCF7 ADR). The combination nanopreparation effectively downregulated P-gp in MDR cancer cells and reversed the resistance towards DOX.
Asunto(s)
Dendrímeros/administración & dosificación , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Nanomedicina/métodos , Poliaminas/administración & dosificación , ARN Interferente Pequeño/administración & dosificación , Dendrímeros/metabolismo , Resistencia a Múltiples Medicamentos/fisiología , Resistencia a Antineoplásicos/fisiología , Quimioterapia Combinada , Humanos , Células MCF-7 , Nanomedicina/tendencias , Poliaminas/metabolismo , ARN Interferente Pequeño/metabolismoRESUMEN
Gene silencing using small interfering RNA (siRNA) is a promising strategy for the treatment of multiple diseases. However, the low in vivo stability of siRNA, its poor pharmacokinetics and inability to penetrate inside cells limit its employment in the clinic. Here, we present a novel redox-sensitive micellar nanopreparation based on a triple conjugate of polyethylene glycol, polyethyleneimine and phosphatidylethanolamine, PEG-SS-PEI-PE (PSSPD). This non-toxic system efficiently condenses siRNA and specifically downregulates target green fluorescent protein (GFP) only under reducing conditions via intracellular siRNA release after de-shielding of PEG due to increased glutathione (GSH) levels characteristic of cancer cells.
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Portadores de Fármacos/química , Nanopartículas/química , ARN Interferente Pequeño/química , Animales , Línea Celular , Disulfuros/síntesis química , Disulfuros/química , Disulfuros/toxicidad , Portadores de Fármacos/síntesis química , Portadores de Fármacos/toxicidad , Glutatión/química , Ratones , Micelas , Nanopartículas/toxicidad , Oxidación-Reducción , Tamaño de la Partícula , Fosfatidiletanolaminas/síntesis química , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/toxicidad , Polietilenglicoles/síntesis química , Polietilenglicoles/química , Polietilenglicoles/toxicidad , Polietileneimina/síntesis química , Polietileneimina/química , Polietileneimina/toxicidadRESUMEN
OBJECTIVES: We aimed to develop lipid-polyethylene glycol (PEG)-polymer hybrid nanoparticles, which have high affinity to tumour tissue with active ingredient, a new generation antineoplastic drug, farnesylthiosalicylic acid (FTA) for treatment of glioblastoma. METHOD: Farnesylthiosalicylic acid-loaded poly(lactic-co-glycolic acid)-1,2 distearoyl-glycerol-3-phospho-ethanolamine-N [methoxy (PEG)-2000] ammonium salt (PLGA-DSPE-PEG) with or without 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) hybrid nanoparticles has been prepared and evaluated for in-vitro characterization. Cytotoxicity of FTA-loaded nanoparticles along with its efficacy on rat glioma-2 (RG2) cells was also evaluated both in vitro (in comparison with non-malignant cell line, L929) and in vivo. KEY FINDINGS: Scanning electron microscopy studies showed that all formulations prepared had smooth surface and spherical in shape. FTA and FTA-loaded nanoparticles have cytotoxic activity against RG2 glioma cell lines in cell culture studies, which further increases with addition of DOTAP. Magnetic resonance imaging and histopathologic evaluation on RG2 tumour cells in rat glioma model (49 female Wistar rats, 250-300 g) comparing intravenous and intratumoral injections of the drug have been performed and FTA-loaded nanoparticles reduced tumour size significantly in in-vivo studies, with higher efficiency of intratumoral administration than intravenous route. CONCLUSION: Farnesylthiosalicylic acid-loaded PLGA-DSPE-PEG-DOTAP hybrid nanoparticles are proven to be effective against glioblastoma in both in-vitro and in-vivo experiments.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Farnesol/análogos & derivados , Glioblastoma/tratamiento farmacológico , Nanopartículas/administración & dosificación , Polietilenglicoles/administración & dosificación , Polímeros/administración & dosificación , Salicilatos/administración & dosificación , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Neoplasias Encefálicas/diagnóstico por imagen , Farnesol/administración & dosificación , Farnesol/química , Femenino , Glioblastoma/diagnóstico por imagen , Lípidos/administración & dosificación , Lípidos/química , Nanopartículas/química , Polietilenglicoles/química , Polímeros/química , Ratas , Ratas Wistar , Salicilatos/química , Resultado del Tratamiento , Carga TumoralRESUMEN
A novel drug delivery system for doxorubicin (DOX), based on organic-inorganic composites was developed. DOX was incorporated in micelles (M-DOX) of polyethylene glycol-phosphatidylethanolamine (PEG-PE) which in turn were adsorbed by the clay, montmorillonite (MMT). The nano-structures of the PEG-PE/MMT composites of LOW and HIGH polymer loadings were characterized by XRD, TGA, FTIR, size (DLS) and zeta measurements. These measurements suggest that for the LOW composite a single layer of polymer intercalates in the clay platelets and the polymer only partially covers the external surface, while for the HIGH composite two layers of polymer intercalate and a bilayer may form on the external surface. These nanostructures have a direct effect on formulation stability and on the rate of DOX release. The release rate was reversely correlated with the degree of DOX interaction with the clay and followed the sequence: M-DOX>HIGH formulation>LOW formulation>DOX/MMT. Despite the slower release from the HIGH formulation, its cytotoxicity effect on sensitive cells was as high as the "free" DOX. Surprisingly, the LOW formulation, with the slowest release, demonstrated the highest cytotoxicity in the case of Adriamycin (ADR) resistant cells. Confocal microscopy images and association tests provided an insight into the contribution of formulation-cell interactions vs. the contribution of DOX release rate. Internalization of the formulations was suggested as a mechanism that increases DOX efficiency, particularly in the ADR resistant cell line. The employment of organic-inorganic hybrid materials as drug delivery systems, has not reached its full potential, however, its functionality as an efficient tunable release system was demonstrated. STATEMENT OF SIGNIFICANCE: DOX PEG-PE/clay formulations were design as an efficient drug delivery system. The main aim was to develop PEG-PE/clay formulations of different structures based on various PEG-PE/clay ratios in order to achieve tunable release rates, to control the external surface characteristics and formulation stability. The formulations showed significantly higher toxicity in comparison to "free" DOX, explained by formulation internalization. For each cell line tested, sensitive and ADR resistant, a different formulation structure was found most efficient. The potential of PEG-PE/clay-DOX formulations to improve DOX administration efficacy was demonstrated and should be further explored and implemented for other cancer drugs and cells.
Asunto(s)
Silicatos de Aluminio , Citotoxinas , Doxorrubicina , Portadores de Fármacos , Neoplasias/tratamiento farmacológico , Polietilenglicoles , Silicatos de Aluminio/química , Silicatos de Aluminio/farmacocinética , Silicatos de Aluminio/farmacología , Bentonita/química , Bentonita/farmacocinética , Bentonita/farmacología , Línea Celular Tumoral , Arcilla , Citotoxinas/química , Citotoxinas/farmacocinética , Citotoxinas/farmacología , Doxorrubicina/química , Doxorrubicina/farmacocinética , Doxorrubicina/farmacología , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/farmacología , Humanos , Neoplasias/metabolismo , Neoplasias/patología , Polietilenglicoles/química , Polietilenglicoles/farmacocinética , Polietilenglicoles/farmacologíaRESUMEN
PURPOSE: The effect of existing anti-cancer therapies is based mainly on the stimulation of apoptosis in cancer cells. Here, we have demonstrated the ability of a catalytically-reactive nanoparticle-based complex of cytochrome c with cardiolipin (Cyt-CL) to induce the apoptosis and killing of cancer cells in a monolayer cell culture. METHODS: Cyt-CL nanoparticles were prepared by complexing CytC with different molar excesses of CL. Following characterization, cytotoxicity and apoptosis inducing effects of nanoparticles were investigated. In an attempt to identify the anticancer activity mechanism of Cyt-CL, pseudo-lipoxygenase and lipoperoxidase reaction kinetics were measured by chemiluminescence. RESULTS: Using chemiluminescence, we have demonstrated that the Cyt-CL complex produces lipoperoxide radicals in two reactions: by decomposition of lipid hydroperoxides, and by lipid peroxidation under the action of H2O2. Antioxidants inhibited the formation of lipid radicals. Cyt-CL nanoparticles, but not the CytC alone, dramatically enhanced the level of apoptosis and cell death in two cell lines: drug-sensitive (A2780) and doxorubicin-resistant (A2780-Adr). The proposed mechanism of the cytotoxic action of Cyt-CL involves either penetration through the cytoplasm and outer mitochondrial membrane and catalysis of lipid peroxidation reactions at the inner mitochondrial membrane, or/and activation of lipid peroxidation within the cytoplasmic membrane. CONCLUSIONS: Here we propose a new type of anticancer nano-formulation, with an action based on the catalytic action of Cyt-CL nanoparticles on the cell membrane and and/or mitochondrial membranes that results in lipid peroxidation reactions, which give rise to activation of apoptosis in cancer cells, including multidrug resistant cells.
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Antineoplásicos/farmacología , Cardiolipinas/farmacología , Citocromos c/farmacología , Nanopartículas/química , Antineoplásicos/química , Apoptosis , Cardiolipinas/química , Catálisis , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Membrana Celular/metabolismo , Supervivencia Celular , Citocromos c/química , Doxorrubicina/química , Doxorrubicina/farmacología , Composición de Medicamentos , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Humanos , Peróxido de Hidrógeno/química , Peróxido de Hidrógeno/metabolismo , Cinética , Peroxidación de LípidoRESUMEN
In this study, a transferrin (Tf)-modified polyethylene glycol-phosphatidyl ethanolamine (PEG-PE)-based micellar delivery system containing paclitaxel (PTX) and tariquidar (TRQ), a potent third generation P-gp inhibitor, was prepared. The nanoformulation was evaluated by targeting efficiency, cellular association, cellular internalization pathway and cytotoxicity for reversal of PTX resistance on two multidrug resistant (MDR) ovarian carcinoma cell lines, SKOV-3TR and A2780-Adr. PTX and TRQ are both hydrophobic compounds. They were successfully encapsulated into the micellar structure containing vitamin E as the encapsulation enhancer. The Tf-targeted micelles were internalized mainly via clathrin-dependent endocytosis by both cell lines. For SKOV-3TR, additional mechanisms including caveolin-dependent endocytosis and macropinocytosis were found to play a significant role. The PTX cytotoxicity against the SKOV-3TR and A2780-Adr MDR cells was increased significantly in the presence of micellar encapsulation. However, unlike the A2780-Adr cell line, the Tf-targeting effect was significant on SKOV-3TR cells when co-administrated with TRQ. Penetration of the Tf-targeted micelles in a cancer cell spheroid culture was also investigated.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Micelas , Neoplasias Ováricas/tratamiento farmacológico , Femenino , Humanos , Paclitaxel/administración & dosificación , Polímeros/química , Quinolinas/administración & dosificaciónRESUMEN
Many of the newly developed drugs for cancer, and some of those for cardiovascular disease, are poorly soluble in water and cannot be taken orally. This can be overcome by employing a new and effective delivery system utilizing nanotechnology. We present a new method for oral preparation of poorly soluble drugs that entails assembling (printing) drug-loaded polymeric micelles into sub-100 nm orally acceptable nanorods (NRs). Due to their small size, these NRs will have a high permeability through cells and thus should transport through the intestine to allow for drug delivery in the blood. These NRs drugs are expected to penetrate tumors more efficiently and much faster than individual nanoparticles and may also be useful for drug delivery to atherosclerotic plaque. This should lead to better bioavailability of the drug with reduced toxicity and side effects. Currently used micellar formulations are administered intravenously, which is invasive and could be toxic due to high doses and interaction with normal healthy tissues. Oral drug administration is the easiest and most desirable way to deliver most drugs, including those that are poorly soluble.
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Enfermedades Cardiovasculares/tratamiento farmacológico , Química Farmacéutica/métodos , Sistemas de Liberación de Medicamentos , Micelas , Nanopartículas , Nanotecnología/métodos , Neoplasias/tratamiento farmacológico , Administración Oral , Animales , Humanos , Tamaño de la PartículaRESUMEN
NF-κB is strongly associated with poor prognosis of different cancer types and an important factor responsible for the malignant phenotype of glioblastoma. Overcoming chemotherapy-induced resistance caused by activation of PI3K/Akt and NF-κB pathways is crucial for successful glioblastoma therapy. We developed an all-in-one nanomedicine formulation for co-delivery of a chemotherapeutic agent (topoisomerase II inhibitor, doxorubicin) and a multidrug resistance modulator (NF-κB inhibitor, curcumin) for treatment of glioblastoma due to their synergism. Both agents were incorporated into PEG-PE-based polymeric micelles. The glucose transporter-1 (GLUT1) is overexpressed in many tumors including glioblastoma. The micellar system was decorated with GLUT1 antibody single chain fragment variable (scFv) as the ligand to promote blood brain barrier transport and glioblastoma targeting. The combination treatment was synergistic (combination index, CI of 0.73) against U87MG glioblastoma cells. This synergism was improved by micellar encapsulation (CI: 0.63) and further so with GLUT1 targeting (CI: 0.46). Compared to non-targeted micelles, GLUT1 scFv surface modification increased the association of micelles (>20%, P<0.01) and the nuclear localization of doxorubicin (â¼3-fold) in U87MGcells, which also translated into enhanced cytotoxicity. The increased caspase 3/7 activation by targeted micelles indicates successful apoptosis enhancement by combinatory treatment. Moreover, GLUT1 targeted micelles resulted in deeper penetration into the 3D spheroid model. The increased efficacy of combination nanoformulations on the spheroids compared to a single agent loaded, or to non-targeted formulations, reinforces the rationale for selection of this combination and successful utilization of GLUT1 scFv as a targeting agent for glioblastoma treatment.
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Curcumina/administración & dosificación , Doxorrubicina/administración & dosificación , Glioblastoma/metabolismo , Nanomedicina/métodos , Neoplasias/patología , Anticuerpos de Cadena Única/química , Antineoplásicos/administración & dosificación , Biotinilación , Barrera Hematoencefálica , Línea Celular Tumoral/efectos de los fármacos , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos/métodos , Evaluación Preclínica de Medicamentos , Resistencia a Antineoplásicos/efectos de los fármacos , Citometría de Flujo , Transportador de Glucosa de Tipo 1/metabolismo , Humanos , Ligandos , Micelas , FN-kappa B/antagonistas & inhibidores , Fenotipo , Polímeros , Pronóstico , Esferoides CelularesRESUMEN
Dual stimuli-sensitive mixed polymeric micelles (MM) are developed for co-delivery of the endogenous tumor suppressor miRNA-34a and the chemotherapeutic agent doxorubicin (Dox) into cancer cells. The novelty of the system resides in two stimuli-sensitive prodrugs, a matrix metalloproteinase 2 (MMP2)-sensitive Dox conjugate and a reducing agent (glutathione, GSH)-sensitive miRNA-34a conjugate, self-assembled in a single particle decorated with a polyethylene glycol corona for longevity, and a cell-penetrating peptide (TATp) for enhanced intracellular delivery. The MMP2-sensitivity of the system results in threefold higher cytotoxicity in MMP2-overexpressing HT1080 cells compared to low MMP2-expressing MCF7 cells. Cellular internalization of Dox increases by more than 70% after inclusion of TATp to the formulation. MMP2-sensitive MM also inhibits proliferation and migration of HT1080 cells. Moreover, GSH-sensitive MM allows for an efficient downregulation of Bcl2, survivin, and notch1 (65%, 55%, and 46%, respectively) in HT1080 cells. Combination of both conjugates in dual sensitive MM reduces HT1080 cell viability to 40% and expression of Bcl2 and survivin. Finally, 50% cell death is observed in 3D models of tumor mass. The results confirm the potential of the MM to codeliver miRNA-34a and doxorubicin triggered by dual stimuli inherent of tumor tissues.
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Doxorrubicina/uso terapéutico , Sistemas de Liberación de Medicamentos , Técnicas de Transferencia de Gen , Micelas , MicroARNs/administración & dosificación , Neoplasias/tratamiento farmacológico , Tamaño de la Partícula , Polímeros/química , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glutatión/metabolismo , Humanos , Metaloproteinasa 2 de la Matriz/metabolismo , MicroARNs/metabolismo , Neoplasias/genética , Neoplasias/patología , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/metabolismoRESUMEN
Cancer-specific drug delivery represents an attractive approach to prevent undesirable side-effects and increase the accumulation of the drug in the tumor. Surface modification of nanoparticles such as liposomes with targeting moieties specific to the up-regulated receptors on the surface of tumor cells thus represents an effective strategy. Furthermore, since this receptor expression can be heterogeneous, using a dual-combination of targeting moieties may prove advantageous. With this in mind, the anti-cancer activity of PEGylated doxorubicin-loaded liposomes targeted with folic acid (F), transferrin (Tf) or both (F+Tf) was evaluated. The dual-targeted liposomes showed a 7-fold increase in cell association compared to either of the single-ligand targeted ones in human cervical carcinoma (HeLa) cell monolayers. The increased penetration and cell association of the dual-targeted liposomes were also demonstrated using HeLa cell spheroids. The in vitro cytotoxicity of the doxorubicin liposomes (LD) was then evaluated using HeLa and A2780-ADR ovarian carcinoma cell monolayers. In both these cell lines, the (F+Tf) LD showed significantly higher cytotoxic effects than the untargeted, or single-ligand targeted liposomes. In a HeLa xenograft model in nude mice, compared to the untreated group, though the untargeted LD showed 42% tumor growth inhibition, both the (F) LD and (F+Tf) LD showed 75% and 79% tumor growth inhibition respectively. These results thus highlight that though the dual-targeted liposomes represent an effective cytotoxic formulation in the in vitro setting, they were equally effective as the folic acid-targeted liposomes in reducing tumor burden in the more complex in vivo setting in this particular model.
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Antibióticos Antineoplásicos/farmacología , Doxorrubicina/farmacología , Ácido Fólico/química , Liposomas , Transferrina/química , Antibióticos Antineoplásicos/administración & dosificación , Antibióticos Antineoplásicos/química , Doxorrubicina/administración & dosificación , Doxorrubicina/química , Células HeLa , HumanosRESUMEN
RNA interference (RNAi) is an evolutionary conserved highly specific gene-silencing mechanism initiated by small interfering RNA (siRNA) molecules. Fast-paced preclinical and clinical studies helped the siRNA technology become an efficient tool for undruggable targets in different diseases including genetic diseases, viral diseases and cancer. Despite great feature of siRNAs that can down-regulate any protein in the cells, the full potential and the success of the preclinical studies could not be translated into largely successful clinical outcomes. It has become clear that the possibility of overcoming the pitfalls for in vivo siRNA therapy fully depends on delivery systems. In this review, we start with the challenges and barriers for in vivo siRNA delivery. Then we briefly discuss the recent developments in siRNA modification technology. We specifically focused on siRNA lipidation and delivery approaches with special emphasis on the lipid based hybrid systems. Here we summarize the journey of lipid-based micelle-like nanoparticle systems that combine longevity in blood, effective cellular uptake and endosomal escape for successful siRNA delivery and discuss the multifunctional stimuli-sensitive systems based on lipids as the next generation smart systems.
Asunto(s)
Lípidos/química , Nanopartículas/química , Neoplasias/tratamiento farmacológico , ARN Interferente Pequeño/administración & dosificación , Endosomas/metabolismo , Silenciador del Gen/fisiología , HumanosRESUMEN
Current research in cancer therapy is beginning to shift toward the use of combinational drug treatment regimens. However, the efficient delivery of drug combinations is governed by a number of complex factors in the clinical setting. Therefore, the ability to synchronize the pharmacokinetics of the individual therapeutic agents present in combination not only to allow for simultaneous tumor accumulation but also to allow for a synergistic relationship at the intracellular level could prove to be advantageous. In this work, we report the development of a novel folic acid-targeted liposomal formulation simultaneously co-loaded with C6 ceramide and doxorubicin [FA-(C6+Dox)-LP]. In vitro cytotoxicity assays showed that the FA-(C6+Dox)-LP was able to significantly reduce the IC50 of Dox when compared to that after the treatment with the doxorubicin-loaded liposomes (Dox-LP) as well as the untargeted drug co-loaded (C6+Dox)-LP on HeLa, A2780-ADR, and H69-AR cells. The analysis of the cell cycle distribution showed that while the C6 liposomes (C6-LP) did not cause cell cycle arrest, all the Dox-containing liposomes mediated cell cycle arrest in HeLa cells in the G2 phase at Dox concentrations of 0.3 and 1 µM and in the S phase at the higher concentrations. It was also found that this arrest in the S phase precedes the progression of the cells to apoptosis. The targeted FA-(C6+Dox)-LP were able to significantly enhance the induction of apoptotic events in HeLa cell monolayers as compared to the other treatment groups. Next, using time-lapse phase holographic imaging microscopy, it was found that upon treatment with the FA-(C6+Dox)-LP, the HeLa cells underwent rapid progression to apoptosis after 21 h as evidenced by a drastic drop in the average area of the cells after loss of cell membrane integrity. Finally, upon evaluation in a HeLa spheroid cell model, treatment with the FA-(C6+Dox)-LP showed significantly higher levels of cell death compared to those with C6-LP and Dox-LP. Overall, this study clearly shows that the co-delivery of C6 ceramide and Dox using a liposomal platform significantly correlates with an antiproliferative effect due to cell cycle regulation and subsequent induction of apoptosis and thus warrants its further evaluation in preclinical animal models.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Ceramidas/química , Doxorrubicina/farmacología , Sistemas de Liberación de Medicamentos , Ácido Fólico/química , Liposomas/administración & dosificación , Antibióticos Antineoplásicos/química , Ciclo Celular/efectos de los fármacos , Doxorrubicina/química , Portadores de Fármacos , Femenino , Humanos , Técnicas In Vitro , Liposomas/química , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Células Tumorales Cultivadas , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/patologíaRESUMEN
In the 1990s an unexpected gene-silencing phenomena in plants, the later called RNA interference (RNAi), perplexed scientists. Following the proof of activity in mammalian cells, small interfering RNAs (siRNAs) have quickly crept into biomedical research as a new powerful tool for the potential treatment of different human diseases based on altered gene expression. In the past decades, several promising data from ongoing clinical trials have been reported. However, despite surprising successes in many pre-clinical studies, concrete obstacles still need to be overcome to translate therapeutic siRNAs into clinical reality. Here, we provide an update on the recent advances of RNAi-based therapeutics and highlight novel synthetic platforms for the intracellular delivery of siRNAs.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , Tratamiento con ARN de Interferencia/métodos , Animales , Aptámeros de Nucleótidos/química , Dendrímeros/química , Humanos , Lípidos/química , Nanopartículas/química , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , Tratamiento con ARN de Interferencia/tendenciasRESUMEN
Multicellular 3D cancer cell culture (spheroids) resemble to in vivo tumors in terms of shape, cell morphology, growth kinetics, gene expression and drug response. However, these characteristics cause very limited drug penetration into deeper parts of the spheroids. In this study, we used multi drug resistant (MDR) ovarian cancer cell spheroid and in vivo tumor models to evaluate the co-delivery of paclitaxel (PCL) and a potent NF-κB inhibitor curcumin (CUR). PCL and CUR were co-loaded into the polyethylene glycol-phosphatidyl ethanolamine (PEG-PE) based polymeric micelles modified with transferrin (TF) as the targeting ligand. Cytotoxicity, cellular association and accumulation into the deeper layers were investigated in the spheroids and compared with the monolayer cell culture. Comparing to non-targeted micelles, flow cytometry and confocal imaging proved significantly deeper and higher micelle penetration into the spheroids with TF-targeting. Both in monolayers and in spheroids, PCL cytotoxicity was significantly increased when co-delivered with CUR in non-targeted micelles or as single agent in TF-targeted micelles, whereas TF-modification of co-loaded micelles did not further enhance the cytotoxicity. In vivo tumor inhibition studies showed good correlation with the 3D cell culture experiments, which suggests the current spheroid model can be used as an intermediate model for the evaluation of co-delivery of anticancer compounds in targeted micelles.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Curcumina/administración & dosificación , Micelas , Neoplasias Ováricas/patología , Paclitaxel/administración & dosificación , Fosfatidiletanolaminas/química , Polietilenglicoles/química , Esferoides Celulares , Línea Celular Tumoral , Resistencia a Antineoplásicos , Femenino , HumanosRESUMEN
PURPOSE: The ability to successfully treat advanced forms of cancer remains a challenge due to chemotherapy resistance. Numerous studies indicate that NF-κB, a protein complex that controls the expression of numerous genes, as being a key factor in producing chemo-resistant tumors. In this study, the therapeutic potential of transferrin (TF)-targeted mixed micelles, made of PEG-PE and vitamin E co-loaded with curcumin (CUR), a potent NF-κB inhibitor, and paclitaxel (PCL), was examined. METHODS: The cytotoxicity of non-targeted and TF-targeted CUR and PCL micelles as a single agent or in combination was investigated against SK-OV-3 human ovarian adenocarcinoma along with its multi-drug resistant (MDR) version SK-OV-3-PCL-resistant (SK-OV-3TR) cells in vitro. RESULTS: Our results indicated that the TF-targeted combination micelles were able to improve the net cytotoxic effect of CUR and PCL to clear synergistic one against the SK-OV-3 cells. In addition, even though the non-targeted combination treatment demonstrated a synergistic effect against the SK-OV-3TR cells, the addition of the TF-targeting moiety significantly increased this cytotoxic effect. While keeping CUR constant at 5 and 10 µM and varying the PCL concentration, the PCL IC50 decreased from ~1.78 to 0.68 µM for the non-targeted formulations to ~0.74 and 0.1 µM for the TF-targeted ones, respectively. CONCLUSION: Our results indicate that such co-loaded targeted mixed micelles could have significant clinical advantages for the treatment of resistant ovarian cancer and provide a clear rational for further in vivo investigation.