RESUMEN
In this study, transferrin (Tf)-modified poly(ethylene glycol)-phosphatidylethanolamine (mPEG-PE) micelles loaded with the poorly water-soluble drug, R547 (a potent and selective ATP-competitive cyclin-dependent kinase (CDK) inhibitor), were prepared and evaluated for their targeting efficiency and cytotoxicity in vitro and in vivo to A2780 ovarian carcinoma cells, which overexpress transferrin receptors (TfR). At 10 mM lipid concentration, both Tf-modified and plain micelles solubilized 800 µg of R547. Tf-modified micelles showed enhanced interaction with A2780 ovarian carcinoma cells in vitro. The involvement of TfR in endocytosis of Tf-modified micelles was confirmed by colocalization studies of micelle-treated cells with the endosomal marker Tf-Alexa488. We confirmed endocytosis of micelles in an intact form with micelles loaded with a fluorescent dye and additionally labeled with fluorescent lipid. The in vitro cytotoxicity and in vivo tumor growth inhibition studies in A2780-tumor bearing mice confirmed the enhanced efficacy of Tf-modified R547-loaded micelles compared to free drug solution and to nonmodified micelles. The results of this study demonstrate the potential application of Tf-conjugated polymeric micelles in the treatment of tumors overexpressing TfR.
Asunto(s)
Sistemas de Liberación de Medicamentos , Micelas , Neoplasias Glandulares y Epiteliales/tratamiento farmacológico , Neoplasias Ováricas/tratamiento farmacológico , Pirimidinas/química , Pirimidinas/farmacología , Transferrina/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Carcinoma Epitelial de Ovario , Línea Celular Tumoral , Femenino , Citometría de Flujo , Humanos , Ratones , Microscopía Confocal , Solubilidad , Agua/químicaRESUMEN
The use of lysosome-targeted liposomes may significantly improve the delivery of therapeutic enzymes and chaperones into lysosomes for the treatment of lysosomal storage disorders. The aim of this research was to synthesize new potentially lysosomotropic ligands on a base of Neutral Red and rhodamine B and to study their ability to enhance specific lysosomal delivery of surface-modified liposomes loaded with a model compound, fluorescein isothiocyanate-dextran (FD). The delivery of these liposomes and their content to lysosomes in HeLa cells was investigated by confocal immunofluorescent microscopy, subcellular fractionation, and flow cytometry. Confocal microscopy demonstrated that liposomes modified with derivatives of rhodamine B provide a good rate of colocalization with the specific lysosomal markers. The comparison of fluorescence of FD in lysosomes isolated by subcellular fractionation also showed that the efficiency of lysosomal delivery of the liposomal load by liposomes modified with some of synthesized ligands was significantly higher compared to that with plain liposomes. These results were additionally confirmed by flow cytometry of the intact cells treated with liposomes loaded with 5-dodecanoylaminofluorescein di-ß-d-galactopyranoside, a specific substrate for the intralysosomal ß-galactosidase, using a number of cell lines, including macrophages with induced phenotype of lysosomal enzyme deficiency; two of the synthesized ligands-rhodamine B DSPE-PEG(2k)-amide and 6-(3-(DSPE-PEG(2k))-thioureido) rhodamine B-demonstrated enhanced lysosomal delivery, in some cases, higher than that for commercially available rhodamine B octadecyl ester, with the best results (the enhancement of the lysosomal delivery up to 75% greater in comparison to plain liposomes) shown for the cells with induced lysosomal enzyme deficiency phenotype. Use of liposomes modified with rhodamine B derivatives may be advantageous for the development of drug delivery systems for the treatment of lysosome-associated disorders.
Asunto(s)
Ligandos , Lisosomas/metabolismo , Fraccionamiento Celular , Dextranos/metabolismo , Citometría de Flujo , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Fluoresceínas/química , Fluoresceínas/metabolismo , Galactosa/química , Galactosa/metabolismo , Células HeLa , Humanos , Inmunohistoquímica , Liposomas/química , Liposomas/metabolismo , Lisosomas/ultraestructura , Microscopía Confocal , Estructura Molecular , Rodaminas/química , Rodaminas/metabolismoRESUMEN
Aging of red blood cells (RBCs) is associated with alteration in a wide range of RBC features, occurring each on its own timescale. A number of these changes are interrelated and initiate a cascade of biochemical and structural transformations, including band-3 clustering and phosphatidylserine (PS) externalization. Using specific band-3 clustering agents (acridine orange (AO) and ZnCl2), we examined whether treatment of RBCs with these agents may affects PS externalization and whether this process is Ca2+-dependent. RBCs were isolated from the blood of eight healthy donors upon obtaining their informed consent. The suspension was supplemented with increasing concentrations of AO or ZnCl2 (from 0.5 to 2.0 mM) and incubated at 25 °C for 60 min. To detect PS at the RBC surface, we used allophycocyanin-conjugated recombinant human Annexin V. We demonstrated, that treatment of RBCs with both clustering agents caused an elevation in the percent of cells positively labeled by Annexin-V (RBCPS), and that this value was not dependent on the presence of calcium in the buffer: RBCs treated with AO in the presence of either EDTA, EGTA or calcium exhibited similar percentage of RBCPS. Moreover, the active influx of Zn2+ into RBCs induced by their co-incubation with both ZnCl2 and A23187 did not increase the percent of RBCPS as compared to RBCs incubated with ZnCl2 alone. Taken together, these results demonstrate that the band-3 clustering agents (AO or ZnCl2) induce PS externalization in a Ca2+ independent manner, and we hereby suggest a possible scenario for this phenomenon.
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Proteína 1 de Intercambio de Anión de Eritrocito/metabolismo , Análisis por Conglomerados , Eritrocitos/citología , Fosfatidilserinas/metabolismo , Naranja de Acridina/farmacología , Anexina A5/metabolismo , Calcio/farmacología , Senescencia Celular , Cloruros/farmacología , Humanos , Compuestos de Zinc/farmacologíaRESUMEN
Combinations of chemotherapy with immunotherapy have seen recent clinical success, including two approvals of anti-PD-1/L1 agents in combination with taxane-based chemotherapy in non-small cell lung cancer and triple-negative breast cancer. Here, we present a study on the combination activity and mechanistic rationale of a novel EphA2-targeted liposomal taxane (EphA2-ILs-DTXp) and anti-PD-1. This combination was highly active in mouse syngeneic tumor models, with complete responses observed in 3 of 5 models. In the EMT-6 tumor model, combination of EphA2-ILs-DTXp with anti-PD-1 resulted in a 60% complete response rate, with durable responses that were resistant to rechallenge. These responses were not observed in the absence of CD8+ T cells. Characterization of the immune infiltrates in EMT-6 tumors reveals increased CD8+ T cells, increased CD8+ IFNγ+ CTLs, and an increased CD8/regulatory T-cell (Treg) ratio. These immunomodulatory effects were not observed in mice treated with a combination of docetaxel and anti-PD-1. Pharmacokinetic analysis revealed that the AUC of docetaxel was increased 15 times, from 52.1 to 785 ng/mL/hour, when delivered by EphA2-ILs-DTXp. A dose reduction study of EphA2-ILs-DTXp showed a dose-response relationship for both tumor growth inhibition and the CD8/Treg ratio. Our data indicate that synergism between docetaxel and anti-PD-1 is achievable with nanoliposomal delivery.
Asunto(s)
Hidrocarburos Aromáticos con Puentes/uso terapéutico , Neoplasias/tratamiento farmacológico , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor EphA2/metabolismo , Taxoides/uso terapéutico , Animales , Hidrocarburos Aromáticos con Puentes/farmacología , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Neoplasias/patología , Taxoides/farmacologíaRESUMEN
Ephrin receptor A2 (EphA2) is a member of the Ephrin/Eph receptor cell-to-cell signaling family of molecules, and it plays a key role in cell proliferation, differentiation, and migration. EphA2 is overexpressed in a broad range of cancers, and its expression is in many cases associated with poor prognosis. We recently developed a novel EphA2-targeting antibody-directed nanotherapeutic encapsulating a labile prodrug of docetaxel (EphA2-ILs-DTXp) for the treatment of EphA2-expressing malignancies. Here, we characterized the expression of EphA2 in bladder cancer using immunohistochemistry in 177 human bladder cancer samples and determined the preclinical efficacy of EphA2-ILs-DTXp in four EphA2-positive patient-derived xenograft (PDX) models of the disease, either as a monotherapy, or in combination with gemcitabine. EphA2 expression was detected in 80-100% of bladder cancer samples and correlated with shorter patient survival. EphA2 was found to be expressed in tumor cells and/or tumor-associated blood vessels in both primary and metastatic lesions with a concordance rate of approximately 90%. The EphA2-targeted antibody-directed nanotherapeutic EphA2-ILs-DTXp controlled tumor growth, mediated greater regression, and was more active than free docetaxel at equitoxic dosing in all four EphA2-positive bladder cancer PDX models. Combination of EphA2-ILs-DTXp and gemcitabine in one PDX model led to improved tumor growth control compared to monotherapies or the combination of free docetaxel and gemcitabine. These data demonstrating the prevalence of EphA2 in bladder cancers and efficacy of EphA2-ILs-DTXp in PDX models support the clinical exploration of EphA2 targeting in bladder cancer.
RESUMEN
BACKGROUND: Blood banking procedures are associated with elevated adherence of red blood cells (RBCs) to blood vessel wall endothelial cells (ECs), which can introduce a circulatory risk to recipients. This study was undertaken to examine the possibility of repairing this damage by a poststorage "rejuvenation" procedure before transfusion. STUDY DESIGN AND METHODS: Stored RBCs were treated with rejuvenation solution (Rejuvesol, enCyte Systems, Inc.), and their adhesion to cultured human microvascular ECs was determined as a function of shear stress using a cell flow properties analyzer. The adherence of rejuvenation-treated stored RBCs (stRBCs) was compared to that of untreated stRBCs and of freshly donated RBCs. RESULTS: Strong elevation of stRBC/EC adhesion was induced by cold storage and it correlated with translocation of phosphatidylserine (PS) to the RBC surface, a known mediator of RBC/EC adhesion. The role of RBC surface PS in stRBC/EC interaction was confirmed by the suppression of adhesion after the blocking of the stRBC surface PS with annexin V. Concomitantly, RBC storage elevated intracellular levels of reactive oxygen species (ROS) and Ca(2+), the latter known to facilitate PS externalization. Poststorage rejuvenation treatment of stRBCs reversed all the above changes (ROS, Ca(2+), PS), along with complete suppression of the enhanced RBC/EC adhesion, restoring it to that of normal, freshly collected RBCs. CONCLUSION: Poststorage RBC rejuvenation treatment is effective in reversing the storage-induced RBC/EC interaction. This provides further documentation for the potential clinical benefit of poststorage rejuvenation.
Asunto(s)
Conservación de la Sangre/efectos adversos , Adhesión Celular/efectos de los fármacos , Células Endoteliales/citología , Eritrocitos/citología , Calcimicina/farmacología , Calcio/metabolismo , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Ionóforos/farmacología , Fosfatidilserinas/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Ephrin A2 targeted immunoliposomes incorporating pH-sensitive taxane prodrugs were developed for sustained delivery of active drug to solid tumors. Here we describe the systematic formulation development and characterization of these immunoliposomes. We synthesized both paclitaxel and docetaxel prodrugs to formulate as ephrin A2-targeted liposomes stabilized in the aqueous core with sucroseoctasulfate (SOS). The optimized lipid formulation was comprised of egg-sphingomyelin, cholesterol, and polyethylene glycol distearoyl glycerol (PEG-DSG). The formulations examined had a high efficiency of prodrug encapsulation (as high as 114â¯mol% taxane per mole phospholipid) and subsequent stability (>3â¯years at 2-8⯰C). The taxane prodrug was stabilized with extraliposomal citric acid and subsequently loaded into liposomes containing a gradient of SOS, resulting in highly stable SOS-drug complexes being formed inside the liposome. The internal prodrug and SOS concentrations were optimized for their impact on in vivo drug release and drug degradation. Cryo-electron microscope images revealed dense prodrug-SOS complex in the aqueous core of the immunoliposomes. Ephrin A2-targeted taxane liposomes exhibited sub-nanomolar (0.69â¯nM) apparent equilibrium dissociation constant toward the extracellular domain of the ephrin A2 receptor, long circulation half-life (8-12â¯h) in mouse plasma, a release rate dependent on intraliposomal drug concentration and stable long-term storage. At an equitoxic dose of 50â¯mg taxane/kg, ephrin A2-targeted liposomal prodrug showed greater antitumor activity than 10â¯mg/kg of docetaxel in A549 non-small cell lung, as well as MDA-MB-436 and SUM149 triple negative breast cancer xenograft models. The lead molecule entered a Phase I clinical trial in patients with solid tumors (NCT03076372).
Asunto(s)
Antineoplásicos/administración & dosificación , Hidrocarburos Aromáticos con Puentes/administración & dosificación , Portadores de Fármacos/química , Efrina-A2/metabolismo , Nanopartículas/química , Profármacos/administración & dosificación , Taxoides/administración & dosificación , Células A549 , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Hidrocarburos Aromáticos con Puentes/química , Hidrocarburos Aromáticos con Puentes/farmacocinética , Hidrocarburos Aromáticos con Puentes/farmacología , Línea Celular Tumoral , Composición de Medicamentos , Liberación de Fármacos , Femenino , Humanos , Liposomas , Ratones Desnudos , Tamaño de la Partícula , Profármacos/química , Profármacos/farmacocinética , Profármacos/farmacología , Unión Proteica , Taxoides/química , Taxoides/farmacocinética , Taxoides/farmacología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Antibody-mediated tumour targeting and nanoparticle-mediated encapsulation can reduce the toxicity of antitumour drugs and improve their efficacy. Here, we describe the performance of a nanotherapeutic encapsulating a hydrolytically sensitive docetaxel prodrug and conjugated to an antibody specific for EphA2-a receptor overexpressed in many tumours. Administration of the nanotherapeutic in mice led to slow and sustained release of the prodrug, reduced exposure of active docetaxel in the circulation (compared with administration of the free drug) and maintenance of optimal exposure of the drug in tumour tissue. We also show that administration of the nanotherapeutic in rats and dogs resulted in minimal haematological toxicity, as well as the absence of neutropenia and improved overall tolerability in multiple rodent models. Targeting of the nanotherapeutic to EphA2 improved tumour penetration and resulted in markedly enhanced antitumour activity (compared with administration of free docetaxel and non-targeted nanotherapeutic controls) in multiple tumour-xenografted mice. This nanomedicine could become a potent and safe therapeutic alternative for cancer patients undergoing chemotherapy.
Asunto(s)
Antineoplásicos/uso terapéutico , Nanopartículas/uso terapéutico , Receptor EphA2/metabolismo , Animales , Antineoplásicos/farmacología , Hidrocarburos Aromáticos con Puentes/farmacología , Hidrocarburos Aromáticos con Puentes/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Docetaxel/sangre , Docetaxel/química , Docetaxel/farmacocinética , Docetaxel/uso terapéutico , Humanos , Liposomas , Ratones Endogámicos NOD , Ratones SCID , Taxoides/farmacología , Taxoides/uso terapéutico , Distribución Tisular/efectos de los fármacos , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tumor necrosis factor receptor 2 (TNFR2) is the alternate receptor for TNF and can mediate both pro- and anti-inflammatory activities of T cells. Although TNFR2 has been linked to enhanced suppressive activity of regulatory T cells (Tregs) in autoimmune diseases, the viability of TNFR2 as a target for cancer immunotherapy has been underappreciated. Here, we show that new murine monoclonal anti-TNFR2 antibodies yield robust antitumor activity and durable protective memory in multiple mouse cancer cell line models. The antibodies mediate potent Fc-dependent T cell costimulation and do not result in significant depletion of Tregs Corresponding human agonistic monoclonal anti-TNFR2 antibodies were identified and also had antitumor effects in humanized mouse models. Anti-TNFR2 antibodies could be developed as a novel treatment option for patients with cancer.
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Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/antagonistas & inhibidores , Receptores Tipo II del Factor de Necrosis Tumoral/inmunología , Animales , Neoplasias del Colon/inmunología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/terapia , Modelos Animales de Enfermedad , Femenino , Humanos , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
beta-Thalassaemia is a congenital haemoglobinopathy, associated with red blood cells (RBC) anomalies, leading to impairment of their flow-affecting properties, namely, RBC deformability, self-aggregability, and adherence to endothelial cells (EC). Treatment of normal RBC with phenylhydrazine (PHZ) causes selective association of oxidized alpha-globin chains with the membrane skeleton, leading to reduced RBC deformability, characteristic of beta-thalassaemia. PHZ has thus been used to mimic phenotypes of beta-thalassaemia RBC. The present study was undertaken to further elucidate the suitability of PHZ-treated RBC as a model for beta-thalassemic RBC, by comparing the aggregability and adhesiveness of PHZ-treated RBC to those of RBC from thalassaemia intermedia (TI) patients, using image analysis of RBC under flow. In addition, the externalization of phosphatidylserine (PS), a mediator of RBC/EC interaction, was determined. It was found that PHZ caused enhanced RBC adhesiveness to extracellular matrix, similar to TI-RBC. Furthermore, in both conditions, the enhanced adhesiveness was mediated by PS translocated to the RBC surface. In contrast, PHZ treatment completely abolished RBC aggregability, while TI-RBC aggregability was slightly elevated. It is proposed that PHZ-treated RBC resemble beta-thalassaemia RBC in their deformability and adhesiveness, but not in their aggregability, and thus can be used as a limited model for beta-thalassaemia RBC phenotypes.
Asunto(s)
Eritrocitos/efectos de los fármacos , Modelos Cardiovasculares , Fenilhidrazinas/farmacología , Talasemia beta/sangre , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Agregación Eritrocitaria/efectos de los fármacos , Deformación Eritrocítica/efectos de los fármacos , Eritrocitos/fisiología , Hemorreología/efectos de los fármacos , HumanosRESUMEN
Cells respond to DNA damage by activating complex signaling networks that decide cell fate, promoting not only DNA damage repair and survival but also cell death. We have developed a multiscale computational model that quantitatively links chemotherapy-induced DNA damage response signaling to cell fate. The computational model was trained and calibrated on extensive data from U2OS osteosarcoma cells, including the cell cycle distribution of the initial cell population, signaling data measured by Western blotting, and cell fate data in response to chemotherapy treatment measured by time-lapse microscopy. The resulting mechanistic model predicted the cellular responses to chemotherapy alone and in combination with targeted inhibitors of the DNA damage response pathway, which we confirmed experimentally. Computational models such as the one presented here can be used to understand the molecular basis that defines the complex interplay between cell survival and cell death and to rationally identify chemotherapy-potentiating drug combinations.
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Antineoplásicos/farmacología , Neoplasias Óseas/patología , Daño del ADN , Osteosarcoma/patología , Neoplasias Ováricas/patología , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Apoptosis , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/genética , Ciclo Celular , Proliferación Celular , Reparación del ADN , Quimioterapia Combinada , Femenino , Humanos , Ratones , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/genética , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Transducción de Señal , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Multidrug resistance (MDR) is a hallmark of cancer cells and a crucial factor in chemotherapy failure, cancer reappearance, and patient deterioration. We have previously described the physicochemical characteristics and the in vitro anticancer properties of a multifunctional doxorubicin-loaded liposomal formulation. Lipodox(®), a commercially available PEGylated liposomal doxorubicin, was made multifunctional by surface-decorating with a cell-penetrating peptide, TATp, conjugated to PEG 1000-PE, to enhance liposomal cell uptake. A pH-sensitive polymer, PEG 2000-Hz-PE, with a pH-sensitive hydrazone (Hz) bond to shield the peptide in the body and expose it only at the acidic tumor cell surface, was used as well. In addition, an anti-nucleosome monoclonal antibody 2C5 attached to a long-chain polymer to target nucleosomes overexpressed on the tumor cell surface was also present. Here, we report the in vitro cell uptake and cytotoxicity of the modified multifunctional immunoliposomes as well as the in vivo studies on tumor xenografts developed subcutaneously in nude mice with MDR and drug-sensitive human ovarian cancer cells (SKOV-3). Our results show the ability of multifunctional immunoliposomes to overcome MDR by enhancing cytotoxicity in drug-resistant cells, compared with non-modified liposomes. Furthermore, in comparison with the non-modified liposomes, upon intravenous injection of these multifunctional immunoliposomes into mice with tumor xenografts, a significant reduction in tumor growth and enhanced therapeutic efficacy of the drug in both drug-resistant and drug-sensitive mice was obtained. The use of "smart" multifunctional delivery systems may provide the basis for an effective strategy to develop, improve, and overcome MDR cancers in the future.
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Antibióticos Antineoplásicos/administración & dosificación , Anticuerpos Monoclonales/química , Doxorrubicina/análogos & derivados , Neoplasias Ováricas/tratamiento farmacológico , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/química , Animales , Antibióticos Antineoplásicos/química , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/administración & dosificación , Doxorrubicina/química , Resistencia a Antineoplásicos , Femenino , Xenoinjertos , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Nucleosomas/inmunología , Nucleosomas/metabolismo , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/patología , Polietilenglicoles/administración & dosificación , Polietilenglicoles/químicaRESUMEN
AIM: We hypothesized that liposomes modified with lysosomotropic octadecyl-rhodamine B (Rh) and loaded with therapeutic glucocerebroside velaglucerase alfa (VPRIV™) will improve lysosomal delivery of the enzyme into Gaucher's cells. MATERIALS & METHODS: Confocal microscopy and flow cytometry were used to evaluate the ability of Rh-modified liposomes loaded with VPRIV to improve the lysosomal targeting in monocyte-derived macrophages and Gaucher's fibroblasts. RESULTS: Confocal microscopy demonstrated that Rh-modified liposomes localized primarily in the lysosomes. As confirmed by flow cytometry using specific substrate 5-(pentafluorobenzoylamino)fluorescein diglucoside, intralysosomal accumulation of VPRIV in the cells treated with Rh-modified liposomes was significantly increased (up to 68%) relative to the cells treated with plain liposomes or free VPRIV. CONCLUSION: Rh-modified lysosomotropic liposomes can improve lysosomal accumulation of liposomal enzymes both in nonphagocytic Gaucher's fibroblasts and phagocytic monocyte-derived macrophages.
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Fibroblastos/metabolismo , Glucosilceramidasa/metabolismo , Liposomas/química , Liposomas/metabolismo , Lisosomas/química , Rodaminas/química , Línea Celular , Citometría de Flujo , Enfermedad de Gaucher/enzimología , Enfermedad de Gaucher/metabolismo , Humanos , Microscopía ConfocalRESUMEN
Bleomycin (BLM) is an example of an anticancer drug that should be delivered into cytosol for its efficient therapeutic action. With this in mind, we developed octaarginine (R8)-modified fusogenic DOPE-liposomes (R8-DOPE-BLM). R8-modification dramatically increased (up to 50-fold) the cell-liposome interaction. R8-DOPE-liposomes were internalized via macropinocytosis and did not end up in the lysosomes. R8-DOPE-BLM led to a significantly stronger cell death and DNA damage in vitro relative to all controls. R8-DOPE-BLM demonstrated a prominent anticancer effect in the BALB/c mice bearing 4T1 tumors. Thus, R8-DOPE-BLM provided efficient intracellular delivery of BLM leading to strong tumor growth inhibition in vivo.
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Bleomicina/administración & dosificación , Liposomas/administración & dosificación , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Oligopéptidos/administración & dosificación , Animales , Apoptosis/efectos de los fármacos , Bleomicina/química , Procesos de Crecimiento Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Células HeLa , Humanos , Liposomas/química , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Oligopéptidos/química , Fosfatidiletanolaminas/administración & dosificación , Fosfatidiletanolaminas/química , Distribución Aleatoria , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Significant progress has been made recently in the area of immunoconjugated drugs and drug delivery systems (DDS). The immuno-modification of either the drug or DDS has proven to be a very promising approach that has significantly improved the targeted accumulation in pathological sites while decreasing its undesirable side effects in healthy tissues. The arrangement for both prolonged life in the circulation and specific target recognition represents another potent strategy in the development of immuno-targeted systems. The longevity of immuno-targeted DDS such as immunoliposomes and immunomicelles improves their targetability even in the presence of the additional passive accumulation in areas with a compromised vasculature. The added use of the immuno-targeted systems takes advantage of the specific microenvironment of pathological sites including lowered pH, increased temperature, and variation in the enzymatic activity. "Smart" stimulus-responsive systems combine different valuable functionalities including PEG-protection, targeting antibody, cell-penetration, and stimulus-sensitive functions. In this review we examined the evolution, current status and future directions in the area of therapeutical immunoconjugates and long-circulating immuno-targeted DDS.
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Sistemas de Liberación de Medicamentos/métodos , Inmunoconjugados/administración & dosificación , Inmunoconjugados/química , Preparaciones Farmacéuticas/administración & dosificación , Preparaciones Farmacéuticas/química , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos/tendencias , Predicción , Humanos , Inmunoconjugados/farmacocinética , Liposomas/administración & dosificación , Liposomas/química , Liposomas/farmacocinética , Micelas , FarmacocinéticaRESUMEN
We prepared and evaluated transferrin (Tf) and monoclonal antibody (mAb) 2C5-modified dual ligand-targeted poly(ethylene glycol)-phosphatidylethanolamine micelles loaded with a poorly soluble drug, R547 (a selective adenosine triphosphate-competitive cyclin-dependent kinase inhibitor) for enhancement of targeting efficiency and cytotoxicity in vitro and in vivo to A2780 ovarian carcinoma compared to single ligand-targeted micelles. Micellar solubilization significantly improved the solubility of R547 from 1 to 800 µg/mL. The size of modified and non-modified micelles was 13-16 nm. Flow cytometry indicated significantly enhanced cellular association of dual ligand-targeted micelles compared to single ligand-targeted micelles. Confocal microscopy confirmed the Tf receptor-mediated endocytosis of rhodamine-labeled Tf-modified micelles after staining the micelle-treated cells with the endosomal marker Tf-Alexa488. The optimized dual-targeted micelles enhanced cytotoxicity in vitro against A2780 ovarian cancer cells compared to plain and single ligand-targeted micelles. Interestingly, in vivo anti-tumor efficacy was more pronounced for the preparation with a single-targeting ligand (Tf). The specific combination Tf and mAb 2C5 did not yield the expected increase in efficacy as was observed in vitro. This observation suggests that the relationships between targeting ligands in vivo could be more complex than in simplified in vitro systems, and the results of the optimization process should always be verified in vivo.
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Antineoplásicos/administración & dosificación , Antineoplásicos/uso terapéutico , Micelas , Neoplasias/tratamiento farmacológico , Antineoplásicos/química , Células Cultivadas , Ensayos de Selección de Medicamentos Antitumorales , Citometría de Flujo , Humanos , Ligandos , Microscopía Confocal , SolubilidadRESUMEN
The completion of human genome project has increased our knowledge of the molecular mechanisms of many diseases, including cancer, thus providing new opportunities for gene therapy. Antisense oligodeoxynucleotides (AsODN) possess great potential as sequence-specific therapeutic agents, which in contrast to classic treatments provide more efficient and target-specific approach to modulate disease-related genes. To be therapeutically effective, sufficient concentrations of intact AsODN must bypass membrane barriers and access the site of action. In this study, a dendrosome delivery strategy was designed to improve the encapsulation of AsODN in non-cationic liposomes to target PKC-α in lung cancer cells in vitro. Subcellular trafficking of fluorescently labeled AsODN was visualized using confocal microscopy. Uptake and expression of mRNA and target protein after AsODN delivery was measured by flow cytometry, qRT-PCR and Western blot analysis, respectively. Dendrosomes showed favorable physicochemical parameters: high encapsulation efficiency and uptake in serum-containing medium with no apparent cytotoxicity. AsODN encapsulated in dendrosome efficiently and specifically suppress the target gene at both mRNA and protein levels. Additional in vivo studies on the application of dendrosome as a delivery system for nucleic acid molecules may lead to improvement of this technology and facilitate the development of therapeutic antisense techniques.
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Regulación hacia Abajo , Neoplasias Pulmonares/genética , Oligodesoxirribonucleótidos Antisentido/administración & dosificación , Proteína Quinasa C-alfa/genética , Western Blotting , Línea Celular Tumoral , Dendrímeros/administración & dosificación , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Humanos , Liposomas , Neoplasias Pulmonares/terapia , Microscopía Confocal , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Lysosomes are a promising therapeutic target for induction apoptosis in cancer cells due to lysosomal membrane permeabilization (LMP) leading to leakage of hydrolytic enzymes, especially the cathepsins, into the cytoplasm. We hypothesized that with the modification of the ceramide-loaded liposomes with transferrin (Tf), we would achieve both tumor targeting and increased delivery of lysosome-destabilizing agents, such as ceramides to lysosomes, to initiate LMP-induced apoptosis. We prepared Tf-modified (TL) and plain (PL) liposomes and loaded with short (C6)- or long (C16) N-acyl chain ceramides. Uptake, intracellular localization of liposomes, stability of the lysosomal membrane and release of cathepsin D were investigated on Hela cells by fluorescence microscopy and flow cytometry. Apoptosis was evaluated by binding of fluorescently-labeled Annexin V. Antitumor and pro-apoptotic effects of C6Cer-loaded Tf-liposomes were demonstrated in vivo in an A2780-ovarian carcinoma xenograft mouse model. TL were internalized specifically via the TfR-dependent endocytic pathway and localized within the endosome-lysosomal compartment. Ceramide-loaded Tf-liposomes significantly increased apoptosis compared with ceramide-free and ceramide-loaded non-modified liposomes. The treatment of cancer cells with TL led to increased LMP and cytoplasmic relocation of the intralysosomal cathepsin D. A strong antitumor and pro-apoptotic effect of C6Cer-loaded TL was also demonstrated in vivo in an A2780-ovarian carcinoma xenograft mouse model. The lysosomal accumulation of ceramides delivered by Tf-liposomes initiates the permeabilization of the lysosomal membranes required for the release of lysosomal cathepsins into the cytoplasm and initiation of the cancer cell apoptosis both in vitro and in vivo.
Asunto(s)
Ceramidas/administración & dosificación , Liposomas/administración & dosificación , Transferrina/administración & dosificación , Animales , Apoptosis/efectos de los fármacos , Ceramidas/química , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Células HeLa , Humanos , Liposomas/química , Lisosomas/química , Lisosomas/metabolismo , Ratones , Ratones Desnudos , Microscopía Fluorescente , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Transferrina/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The effectiveness of many promising drug candidates (e.g. anticancer agents) awaits the development of drug forms capable of delivery of their drug load specifically to particular sites of an organism or a cell. To make universal and efficient drug carriers, administered drug-loaded vehicles should be able to reach the pathologic zone, recognize and bind their targets at a therapeutic concentration before clearance from the organism. Numerous methods have been developed to couple drug vehicles with active targeting substances - including monoclonal antibodies and substrates or ligands for pathologic cell receptors. Other approaches have included the use of such factors as decreased pH and elevated activity of enzymes in tumor tissues and the hypoxic environment inside the tumor core. This review makes an attempt to analyze the main factors that influence targeting on the kinetic or thermodynamic level that may provide the basis for a strategy to develop and improve drug delivery systems.
Asunto(s)
Sistemas de Liberación de Medicamentos , Diseño de Fármacos , Termodinámica , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Portadores de Fármacos/química , Humanos , Concentración de Iones de Hidrógeno , Ligandos , Modelos Biológicos , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Distribución TisularRESUMEN
The use of lysosome-targeted liposomes may significantly improve a delivery of therapeutic enzymes into lysosomes for the treatment of lysosome-associated diseases. The aim of this research was to achieve a specific intracellular targeting of lysosomes, by using liposomes modified with the lysosomotropic octadecyl-rhodamine B (RhB) and loaded with a model compound, fluorescein isothiocyanate (FITC)-dextran (FD). Plain and RhB-modified liposomes were prepared by hydration of lipid films and loaded with FD or with 5-dodecanoylaminofluorescein di-ß-d-galactopyranoside (C(12)FDG), a specific substrate for the intralysosomal ß-galactosidase. The delivery of these liposomes and their content to lysosomes in HeLa cells was investigated by confocal microscopy, flow cytometry, and subcellular fractionation. Confocal microscopy demonstrated that RhB-liposomes co-localize well with the specific lysosomal markers, unlike plain liposomes. The comparison of the FITC fluorescence of the lysosomes isolated by subcellular fractionation also showed that the efficiency of FD delivery into lysosomes by RhB-modified liposomes was significantly higher compared with plain liposomes. These results were additionally confirmed by the flow cytometry of the intact cells treated with C(12)FDG-loaded liposomes that also demonstrated increased lysosomal targeting by RhB-modified liposomes. The modification of the liposomal surface with a lysosomotropic ligand, such as octadecyl-RhB, can significantly increase the delivery of liposomal loads to lysosomes.