RESUMEN
Generally, the major obstacles for efficient gene delivery are cellular internalization and endosomal escape of nucleic acid such as plasmid DNA (pDNA) or small interfering RNA (siRNA). We previously developed Pluronic P123 modified polypropyleneimine (PPI)/pDNA (P123-PPI/pDNA) polyplexes as a gene delivery system. The results showed that P123-PPI/pDNA polyplexes revealed higher transfection efficiency than PPI/pDNA polyplexes in multidrug resistant breast cancer cells. As a continued effort, the present investigation on the factors influencing the transfection efficiency, cellular uptake mechanisms, and intracellular fate of P123-PPI/pDNA polyplexes is reported. The presence of P123 was the main factor influencing the transfection efficiency of P123-PPI/pDNA polyplexes in MCF-7/ADR cells, but other parameters, such as N/P ratio, FBS concentration, incubation time and temperature were important as well. The endocytic inhibitors against clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis (CvME), and macropinocytosis were involved in the internalization to investigate their effects on the cellular uptake and transfection efficiency of P123-PPI/pDNA polyplexes in vitro. The data showed that the internalization of P123-PPI/pDNA polyplexes was obtained from both CME and CvME. Colocalization experiments with TRITC-transferrin (CME indicator), Alexa Fluor 555-CTB (CvME indicator), monoclonal anti-α-tubulin (microtubule indicator), and LysoTracker Green (Endosome/lysosome indicator) were carried out to confirm the internalization routes. The results showed that both CME and CvME played vital roles in the effective transfection of P123-PPI/pDNA polyplexes. Endosome/lysosome system and skeleton, including actin filament and microtubule, were necessary for the transportation after internalization.
Asunto(s)
Plásmidos/genética , Poloxaleno/química , Polipropilenos/química , Transfección/métodos , Citoesqueleto de Actina/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Supervivencia Celular/genética , ADN/química , ADN/genética , ADN/metabolismo , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Endocitosis , Endosomas/metabolismo , Humanos , Lisosomas/metabolismo , Células MCF-7 , Microscopía Confocal , Plásmidos/química , Plásmidos/metabolismoRESUMEN
Multidrug resistance (MDR) remains one of the major reasons for the reductions in efficacy of many chemotherapeutic agents in cancer therapy. As a classical MDR phenotype of human malignancies, the adenosine triphosphate binding cassette (ABC)-transporter P-glycoprotein (MDR1/P-gp) is an efflux protein with aberrant activity that has been linked to multidrug resistance in cancer. For the reversal of MDR by RNA interference (RNAi) technology, an U6-RNA gene promoter-driven expression vector encoding anti-MDR1/P-gp short hairpin RNA (shRNA) molecules was constructed (abbreviated pDNA-iMDR1-shRNA). This study explored the feasibility of using Pluronic P123-conjugated polypropylenimine (PPI) dendrimer (P123-PPI) as a carrier for pDNA-iMDR1-shRNA to overcome tumor drug resistance in breast cancer cells. P123-PPI functionalized with anti-CD44 monoclonal antibody (CD44 receptor targeting ligand) (anti-CD44-P123-PPI) can efficiently condense pDNA into nanocomplexes to achieve efficient delivery of pDNA, tumor specificity and long circulation. The in vitro studies methodically evaluated the effect of P123-PPI and anti-CD44-P123-PPI on pDNA-iMDR1-shRNA delivery and P-gp downregulation. Our in vitro results indicated that the P123-PPI/pDNA and anti-CD44-P123-PPI/pDNA nanocomplexes with low cytotoxicity revealed higher transfection efficiency compared with the PPI/pDNA nanocomplexes and Lipofectamine™ 2000 in the presence of serum. The nanocomplexes loaded with pDNA-iMDR1-shRNA against P-gp could reverse MDR accompanied by the suppression of MDR1/P-gp expression at the mRNA and protein levels and improve the internalization and cytotoxicity of Adriamycin (ADR) in the MCF-7/ADR multidrug-resistant cell line. BALB/c nude mice bearing MCF-7/ADR tumor were utilized as a xenograft model to assess antitumor efficacy in vivo. The results demonstrated that the administration of anti-CD44-P123-PPI/pDNA-iMDR1-shRNA nanocomplexes combined with ADR could inhibit tumor growth more efficiently than ADR alone. The enhanced therapeutic efficacy of ADR may be correlated with increased accumulation of ADR in drug-resistant tumor cells. Consequently, these results suggested that the use of pDNA-iMDR1-shRNA-loaded nanocomplexes may be a promising gene delivery strategy to reverse MDR and improve the effectiveness of chemotherapy.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Receptores de Hialuranos/inmunología , Nanopartículas/química , Plásmidos/metabolismo , ARN Interferente Pequeño/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Animales , Anticuerpos/metabolismo , Muerte Celular/efectos de los fármacos , ADN/metabolismo , Doxorrubicina/farmacología , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Endocitosis/efectos de los fármacos , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Femenino , Silenciador del Gen/efectos de los fármacos , Técnicas de Transferencia de Gen , Genes Reporteros , Células Endoteliales de la Vena Umbilical Humana , Humanos , Espacio Intracelular/metabolismo , Células MCF-7 , Poloxaleno/síntesis química , Poloxaleno/química , Polipropilenos/síntesis química , Polipropilenos/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Distribución Tisular/efectos de los fármacos , TransfecciónRESUMEN
The aim of this study was to clarify the relationship between the concentration of Pluronic P123/F127 block copolymers and P-glycoprotein (P-gp) inhibitory potency. Modulation of multidrug resistance (MDR) by Pluronic P123/F127 was evaluated in P-gp over-expressing human breast cancer cell line MCF-7/ADR and its non-P-gp over-expressing counterpart MCF-7 cells. Four different probes (known as P-gp substrates) including rhodamine 123 (R-123), rhodamine 6G (R-6G), doxorubicin (DOX), and paclitaxel (PTX) were applied to investigate the impact of Pluronic P123/F127 copolymers with different concentrations on the intracellular accumulation of these probes. Additionally, the intracellular ATP and mitochondrial transmembrane potential in MCF-7/ADR cells were determined over a wide concentration range of Pluronic P123/F127. Furthermore, the endocytic mechanisms of Pluronic micelles were performed. It was suggested that P-gp substrate hydrophobicity and the concentration of P123/F127 copolymers had little impact on P-gp inhibitory activity of Pluronic P123/F127 itself. Intracellular ATP depletion was the main mechanism of Pluronic P123/F127 for P-gp inhibition. In vitro cytotoxicity study was also conducted in order to compare cytotoxic effect among different PTX formulations. It indicated that the IC50 of PTX-loaded Pluronic P123/F127 mixed micelles was 6.3-fold lower than free PTX and 2.3-fold lower than Taxol, respectively. Therefore, Pluronic P123/F127 polymeric micelles could be considered a promising drug delivery system to overcome MDR in cancer therapy.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Poloxaleno/administración & dosificación , Polietilenos/administración & dosificación , Polímeros/administración & dosificación , Polipropilenos/administración & dosificación , Adenosina Trifosfato/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Química Farmacéutica/métodos , Doxorrubicina/administración & dosificación , Sistemas de Liberación de Medicamentos , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Células MCF-7 , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Micelas , Paclitaxel/administración & dosificación , Rodamina 123/administración & dosificación , Rodaminas/administración & dosificaciónRESUMEN
Three poly(caprolactone)-modified Pluronic P105 polymers (P105/PCLs) were synthesized using commercially available ε-caprolactone monomers and Pluronic P105 copolymers. The chemical structures, compositions and molecular weights of the P105/PCLs were confirmed by FT-IR, (1)H NMR and GPC measurements. Three paclitaxel (PTX)-loaded P105/PCL polymeric micelles were then prepared, and they showed average diameters in the range of 30-150 nm, drug-loading coefficients of 0.15%-5.43%, and encapsulation ratios of 2.1%-76.53%. The in vitro cytotoxicity assay demonstrated that three PTX-loaded P105/PCL micelles were able to sensitize the resistant SKOV-3/PTX tumor cells. The PTX-loaded P105/PCL(50) micelle was then selected for an in vivo antitumor efficacy study. The tumor volumes in nude mice bearing s.c. resistant SKOV-3/PTX carcinoma treated with this micellar PTX were significantly less than the control group treated with Taxol. It was demonstrated that three PCL-modified P105 monomers and micelles inhibited P-gP efflux activity in the resistant SKOV-3/PTX cells via at least three intracellular events: 1) inhibition of ATPase of P-gP, 2) decrease of membrane microviscosity and 3) a loss of mitochondrial membrane potential and subsequent decrease of ATP levels at the concentration of monomers (0.001%) and/or micelles (0.01-1.0%). Considering other favorable characteristics, such as sustained PTX release in vitro, long-circulating time in vivo and increased PTX concentration in the tissues of ovaries and uterus in mice, the PCL-modified Pluronic P105 polymeric micelle system could have important clinical implications for delivery of paclitaxel and treatment of the resistant ovarian tumors.
Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Portadores de Fármacos/química , Micelas , Paclitaxel/uso terapéutico , Poloxámero/química , Poliésteres/química , Animales , Antineoplásicos Fitogénicos/administración & dosificación , Antineoplásicos Fitogénicos/química , Línea Celular Tumoral , Resistencia a Antineoplásicos , Femenino , Humanos , Ratones , Neoplasias Ováricas/tratamiento farmacológico , Paclitaxel/administración & dosificación , Paclitaxel/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We proposed to develop a polycation lipid nanocarrier (PLN) with higher transfection efficiency than our previously described polycation nanostrucutred lipid nanocarrier (PNLC). PLN was composed of triolein, cetylated low-molecular-weight polyethylenimine, and dioleoyl phosphatidylethanolamine. The physicochemical properties of PLN and the PLN/DNA complexes (PDC) were characterized. The in vitro transfection was performed in human lung adenocarcinoma (SPC-A1) cells, and the intracellular mechanism was investigated as well. The measurements indicated that PLN and PDC are homogenous nanometer-sized particles with a positive charge. The transfection efficiency of PDC significantly increased with the content of triolein and was higher than that of PNLC and commercial Lipofectamine 2000. In particular, the transfection of PLN in the presence of 10% serum was more effective than that in its absence. With the help of specific inhibitors of chlorpromazine and filipin, the clathrin-dependent endocytosis pathway was determined to be the main contributor to the successful transfection mediated by PLN in SPC-A1 cells. The captured images verified that the fluorescent PDC was localized in the lysosomes and nuclei after endocytosis. Thus, PLN represents a novel efficient nonviral gene delivery vector.
Asunto(s)
Portadores de Fármacos/química , Nanopartículas/química , Transfección/métodos , Trioleína/química , Cationes/química , Cationes/farmacocinética , Línea Celular Tumoral , Portadores de Fármacos/farmacocinética , Proteínas Fluorescentes Verdes/química , Humanos , Fosfatidiletanolaminas/química , Polietileneimina/química , Trioleína/farmacocinéticaRESUMEN
The increasing global incidence of malignant melanoma combined with the poor prognosis and low survival rates of patients necessitates the development of new chemotherapeutic strategies. Thus, the objective of this present study was to investigate the therapeutic efficacy of Pluronic polymeric micelles encapsulating paclitaxel (PTX) in both B16F10 melanoma subcutaneous mice model and pulmonary metastatic mice model. Herein, we developed a PTX-loaded polymeric micelles (PF-PTX) consisting of Pluronic P 123 and F127 block copolymers with small particle size (â¼25 nm), high encapsulation efficiency (>90%), good stability in lyophilized form and pH-dependent in vitro release. Furthermore, influence of PF-PTX on in vitro cytotoxicity was determined by MTT assay using B16F10 melanoma cell line, while cellular distribution of PF-PTX was detected by confocal microscopy. Additionally, C57BL/6 mice bearing subcutaneous or pulmonary B16F10 melanoma tumors were treated with Taxol or PF-PTX, and antitumor effect was compared. It was found that antitumor efficacy of PF-PTX in both tumor models showed significant tumor growth delay and increased survival. In summary, the simple Pluronic-based nanocarrier could be harnessed for the delivery of anticancer drug to melanoma, with increased therapeutic index.
Asunto(s)
Antineoplásicos Fitogénicos/administración & dosificación , Neoplasias Pulmonares/secundario , Melanoma Experimental/patología , Micelas , Paclitaxel/administración & dosificación , Poloxámero , Neoplasias Cutáneas/secundario , Animales , Antineoplásicos Fitogénicos/farmacocinética , Rastreo Diferencial de Calorimetría , Ratones , Microscopía Electrónica de Rastreo , Paclitaxel/farmacocinética , Espectroscopía Infrarroja por Transformada de Fourier , Distribución TisularRESUMEN
The aim of this work was to demonstrate the advantage of using paclitaxel (PTX)-loaded Pluronic P123/F127 mixed micelles (PF-PTX) against non-small cell lung cancer (NSCLC) compared to Taxol. Modulation of multidrug resistance (MDR) by Pluronic mixed micelles was evaluated in lung resistance protein (LRP)-overexpressing human lung adenocarcinoma A-549 cell line. Influence of PF-PTX on in vitro cytotoxicity was determined by MTT assay, while cellular apoptosis was detected by cell nuclei staining and Annexin V-FITC apoptosis detection kit. Cell cycle arrest was also confirmed by flow cytometry. Additionally, in vivo fate and antitumor efficacy of PF-PTX were extensively evaluated in comparison with Taxol. It was demonstrated that PF-PTX had superior anti-proliferation activity against A-549 cells compared with Taxol as measured by IC(50). The enhanced anti-cancer efficacy of PF-PTX was associated with PTX-induced apoptosis and cell arrest in the G(2)/M phase. Intracellular ATP depletion and decreased mitochondrial potential caused by Pluronic copolymers were found to be related to modulation of MDR. PF-PTX also exhibited significant advantages in pharmacokinetics and A-549 xenograft tumor model versus Taxol. The PF-PTX formulation achieved 3.0-fold longer mean residence time in circulation, 2.2-fold larger area under the plasma concentration-time curve than Taxol. At 28days, tumor volume in PF-PTX group was only 31.8% that of the Taxol. Therefore, PF-PTX significantly enhanced the anti-cancer activity of PTX and might be considered a promising drug delivery system to overcome MDR in lung cancer.
Asunto(s)
Antineoplásicos Fitogénicos/administración & dosificación , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Micelas , Paclitaxel/administración & dosificación , Poloxaleno/química , Apoptosis , Carcinoma de Pulmón de Células no Pequeñas/patología , Sistemas de Liberación de Medicamentos , Resistencia a Antineoplásicos , Humanos , Neoplasias Pulmonares/patologíaRESUMEN
Third generation cationic dendritic polymeric polypropyleneimine (PPI) was modified by Pluronic P123 and investigated for gene delivery. The cytotoxicity of P123-PPI was evaluated by the MTT assay and shown to be much lower than that of PPI alone. P123-PPI and PPI can both condense plasmid DNA into nanoparticles with a size of approximately 100 nm and a zeta potential of about 15 mV at the N/P ratio 20:1. The nanoparticles can protect plasmid DNA from being digested by DNase I at a concentration of 0.4 U/microg DNA. The nanoparticles were resistant to dissociation induced by 50% fetal bovine serum and 75 microg/mL sodium heparin. The transfection efficiency of SPC-A1 cells using P123-PPI/DNA nanoparticles was much higher than the transfection utilizing PPI/DNA nanoparticles. The addition of free P123 during the preparation of P123-PPI/DNA nanoparticles could significantly enhance the transfection efficiency in the presence of 10% fetal bovine serum. Therefore, P123-PPI/DNA complex nanoparticles may be a safe, efficient and promising cationic conjugate for gene delivery.
Asunto(s)
Núcleo Celular/metabolismo , ADN/metabolismo , Dendrímeros/química , Poloxaleno/química , Polipropilenos/química , Transfección/métodos , Transporte Activo de Núcleo Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , ADN/química , Dendrímeros/toxicidad , Desoxirribonucleasa I/metabolismo , Relación Dosis-Respuesta a Droga , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Heparina/metabolismo , Humanos , Nanopartículas , Conformación de Ácido Nucleico , Poloxaleno/toxicidad , Polipropilenos/toxicidad , Suero/metabolismoRESUMEN
The objective of this study was to optimize and characterize a novel polymeric mixed micelle composed of Pluronic P123 and F127 loaded with paclitaxel (PTX). A Doehlert matrix design was utilized to investigate the effect of four variables, namely P123 mass fraction, amount of water, feeding of PTX and hydration temperature on the responses including drug-loading coefficient (DL %), encapsulation ratio (ER %) and the percentage of PTX precipitated from the drug-loaded mixed micelles after 48 h at 37 (PTX precipitated %) for improvement of drug solubilization efficiency and micelle stability. PTX-loaded P123/F127 mixed micelles were prepared by thin-film hydration method. The optimized formulation showed a particle size of about 25 nm with ER %>90%, and a sustained release behavior compared to Taxol. Micelle formation was confirmed by NMR spectroscopy. The mixed micelles had a low CMC of 0.0059% in water. In addition, micelle stability studies implied that introduction of Pluronic F127 (33 wt%) into P123 micelle system significantly increased the stability of PTX-loaded micelles. More importantly, in vitro cytotoxicity was assessed using human lung adenocarcinoma cell lines SPC-A1 and A-549 and was compared to Taxol and the free drug. The cell viability assay against A-549 cells exhibited the 50% inhibition concentration (IC50) of PTX-loaded P123/F127 mixed micelles (0.1 microg/ml) was much lower than those of Taxol injection (0.4 microg/ml) and the free PTX (1.7 microg/ml). Therefore, PTX-loaded P123/F127 mixed micelles may be considered as an effective anticancer drug delivery system for cancer chemotherapy.