RESUMO
Direct delivery of proteins into cells has been considered an effective approach for treating the protein-related diseases. However, clinical use of proteins has still been limited due to their instability in the blood and poor membrane permeability. To achieve an efficient cellular delivery of the protein to target cells via a systemic administration, a multifunctional carrier system having desirable stability both in the blood stream and the cells, specific cell-targeting property and endosomal escape functions may be required. In this study, we prepared a catalytic nanoparticle containing an active enzyme by cross-tethering multiple superoxide dismutase (SOD) molecules with catechol-derivatized hyaluronic acid (HA). The permeable shell of hydrophilic HA chains effectively protects the enzyme from degradation in the blood after intravenous administration and provides an additional function for targeting hepatocytes expressing HA receptor (CD44). The structure and catalytic activity of the enzyme molecules in the nanoparticle were not significantly compromised in the nanoparticle. In addition, ultra-small calcium phosphate nanoparticles (USCaP, 2-5â¯nm) were crystalized and decorated on the surface of the nanoparticle for the efficient endosomal escape after cellular uptake. The SOD-containing nanoparticle fortified with USCaP was used for the treatment of acetaminophen (APAP)-induced fulminant hepatotoxicity and liver injury. The nanoparticle achieved the efficient hepatic cellular delivery of SOD via a systemic administration and resulted in efficient removal of reactive oxygen species (ROS) in the liver and remarkable improvement of APAP-induced hepatotoxicity and liver injury in animals. STATEMENT OF SIGNIFICANCE: Despite the enormous therapeutic potential, the intracellular delivery of proteins has been limited due to their poor membrane permeability and stability. In this study, we demonstrated an active enzyme-containing nanoparticle functionalized by hyaluronic acid and ultra-small size calcium phosphate nanoparticles (2-5â¯nm) for targeted cellular delivery of superoxide dismutase (SOD). The nanoparticle was designed to integrate all the essential functions, including serum stability, target specificity, and endosomal escape capability, for a systemic delivery of a therapeutic protein to the cells of the liver tissue. The intravenous administration of the nanoparticle efficiently removes reactive oxygen species (ROS) in the liver and remarkably improves the drug-induced hepatotoxicity and the progress of fulminant liver injury in an acetaminophen-overdose animal model.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Sistemas de Liberação de Medicamentos , Nanopartículas , Superóxido Dismutase , Acetaminofen/efeitos adversos , Acetaminofen/farmacologia , Animais , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Enzimas Imobilizadas/química , Enzimas Imobilizadas/farmacologia , Feminino , Células Hep G2 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanopartículas/química , Nanopartículas/uso terapêutico , Superóxido Dismutase/química , Superóxido Dismutase/farmacologiaRESUMO
Paclitaxel (PTX) is used as a major antitumor agent for the treatment of recurrent and metastatic breast cancer. For the clinical application of PTX, it needs to be dissolved in an oil/detergent-based solvent due to its poor solubility in an aqueous medium. However, the formulation often causes undesirable complications including hypersensitivity reactions and limited tumor distribution, resulting in a lower dose-dependent antitumor effect. Herein, we introduce a facile and oil-free method to prepare albumin-based PTX nanoparticles for efficient systemic cancer therapy using a conjugate of human serum albumin (HSA) and poly(ethyleneglycol) (PEG). PTX were efficiently incorporated in the self-assembled HSA-PEG nanoparticles (HSA-PEG/PTX) using a simple film casting and re-hydration procedure without additional processes such as application of high pressure/shear or chemical crosslinking. The spherical HSA-PEG nanoparticle with a hydrodynamic diameter of ca. 280 nm mediates efficient cellular delivery, leading to comparable or even higher cytotoxicity in various breast cancer cells than that of the commercially available Abraxane®. When systemically administered in a mouse xenograft model for human breast cancer, the HSA-PEG-based nanoparticle formulation exhibited an extended systemic circulation for more than 96 h and enhanced intratumoral accumulation, resulting in a remarkable anticancer effect and prolonged survival of the animals.
Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Nanopartículas/administração & dosagem , Paclitaxel/administração & dosagem , Polietilenoglicóis/administração & dosagem , Albumina Sérica/administração & dosagem , Carga Tumoral/efeitos dos fármacos , Animais , Antineoplásicos Fitogênicos/química , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Diagnóstico por Imagem/tendências , Relação Dose-Resposta a Droga , Feminino , Humanos , Células MCF-7 , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanopartículas/química , Paclitaxel/química , Polietilenoglicóis/química , Albumina Sérica/química , Carga Tumoral/fisiologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Conventional cancer treatment strategies have been aimed at eradicating all cancer cells. To this end, standard chemotherapeutic approaches have relied on the maximum tolerated dose (MTD) of cytotoxic drugs with a long off-therapy interval, leading to heavy toxic side effects accompanied by drug resistance. To avoid the problems associated with the traditional MTD chemotherapy, metronomic chemotherapy with relatively low dose continuous treatments of cytotoxic drugs has been proposed as an alternative to the predominant paradigm of directly killing all cancer cells. Low-dose metronomic (LDM) chemotherapy is expected to have not only antitumor effects without toxicity and drug resistance, but also beneficial anti-angiogenic effects by causing selective apoptosis of tumor endothelial cells. In an attempt to keep the drug resistance under control and halt exponential tumor growth, herein, we combined LDM chemotherapy with a second anti-angiogenic strategy. The selective blockade of vascular endothelial growth factor (VEGF) in combination with metronomic doxorubicin (Dox) induced synergistic antitumor effects mainly through an antiangiogenic mechanism. For specific VEGF suppression, VEGF-targeting siRNA was delivered to tumor tissue using polymerized siRNA/thiolated glycol chitosan (poly-siVEGF/tGC) nanoparticles, leading to efficient VEGF gene knockdown in tumor tissue with a sequence-specific manner. Although the single treatment with metronomic Dox and poly-siVEGF/tGC nanoparticles alone showed some antitumor activity, notably, the combination of the two therapies resulted in superb tumor regression without causing systemic toxicity or drug resistance. Thus, these results suggest that the VEGF-targeted RNAi using poly-siRNA/tGC nanoparticles in combination with LDM chemotherapy could be a promising synergistic strategy for controlling tumor growth by enhancing the efficacy of anti-angiogenesis while minimizing toxicity and drug resistance.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Doxorrubicina/administração & dosagem , Nanopartículas/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/genética , Administração Metronômica , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Terapia Combinada , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos Nus , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Interferência de RNARESUMO
Pathological angiogenesis is one of the major symptoms of severe ocular diseases, including corneal neovascularization. The blockade of vascular endothelial growth factor (VEGF) action has been recognized as an efficient strategy for treating corneal neovascularization. In this study, we aimed to investigate whether nanoparticle-based delivery of apatinib, a novel and selective inhibitor of VEGF receptor 2, inhibits VEGF-mediated angiogenesis and suppresses experimental corneal neovascularization. Water-insoluble apatinib was encapsulated in nanoparticles composed of human serum albumin (HSA)-conjugated polyethylene glycol (PEG). In vitro angiogenesis assays showed that apatinib-loaded HSA-PEG (Apa-HSA-PEG) nanoparticles potently inhibited VEGF-induced tube formation, scratch wounding migration, and proliferation of human endothelial cells. In a rat model of alkali burn injury-induced corneal neovascularization, a subconjunctival injection of Apa-HSA-PEG nanoparticles induced a significant decrease in neovascularization compared to that observed with an injection of free apatinib solution or phosphate-buffered saline. An in vivo distribution study using HSA-PEG nanoparticles loaded with fluorescent hydrophobic model drugs revealed the presence of a substantial number of nanoparticles in the corneal stroma within 24 h after injection. These in vitro and in vivo results demonstrate that apatinib-loaded nanoparticles may be promising for the prevention and treatment of corneal neovascularization-related ocular disorders.
Assuntos
Indutores da Angiogênese/administração & dosagem , Neovascularização da Córnea/tratamento farmacológico , Nanopartículas/administração & dosagem , Neovascularização Patológica/tratamento farmacológico , Piridinas/administração & dosagem , Indutores da Angiogênese/farmacologia , Animais , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Nanopartículas/química , Polietilenoglicóis/química , Piridinas/farmacologia , Ratos Sprague-Dawley , Albumina Sérica/química , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
A variety of VEGF inhibitors have been reported to treat cancers by suppressing tumor angiogenesis. Bevacizumab, a monoclonal VEGF antibody, was the first FDA approved anti-angiogenic agent for cancer treatments. However, bevacizumab shows modest therapeutic efficiency and often cause resistant problem in significant populations of cancer patients. To solve these problem, we investigated the therapeutic efficacy of siRNA drugs targeting VEGF and combination of the RNAi drug with bevacizumab for cancer treatments. For efficient VEGF siRNA delivery, chemically polymerized siRNAs were complexed with thiolated-glycol chitosan (psi(VEGF)/tGC). The poly-VEGF siRNA and thiolated-glycol chitosan formed stable nanoparticles via electrostatic interaction and chemical crosslinking, and showed high accumulation in tumor tissues resulting in efficient gene silencing. Both VEGF siRNA nanoparticles and bevacizumab had efficient therapeutic effects in tumor xenograft mouse models. Interestingly, most pronounced therapeutic efficacy was observed when the two distinct VEGF inhibitors were treated in combination revealing synergistic effects. The results showed that the psi(VEGF)/tGC nanoparticle mediated knockdown of VEGF exerts anti-tumor effects and the combination treatments with bevacizumab can extend the treatments options to conventional bevacizumab treatments for cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Bevacizumab/farmacologia , Nanopartículas/química , RNA Interferente Pequeno/farmacologia , Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Bevacizumab/administração & dosagem , Bevacizumab/química , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Inativação Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Polimerização/efeitos dos fármacos , RNA Interferente Pequeno/química , Células Tumorais Cultivadas , Fatores de Crescimento do Endotélio Vascular/genética , Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Cancer is a multifactorial disease which involves complex genetic mutation and dysregulation. Combinatorial RNAi technology and concurrent multiple gene silencing are expected to provide advanced strategies for effective cancer therapy, but a safe and effective carrier system is a prerequisite to successful siRNA delivery in vivo. We previously developed an effective tumor-targeting siRNA delivery system for in vivo application. In response to the success of this development, herein we present a dual-gene targeted siRNA and its delivery system, to achieve synergistic effects in cancer therapy. Two different sequences of siRNA were chemically modified to be randomly copolymerized in a single backbone of siRNA polymer (Dual-poly-siRNA), and the resulting Dual-poly-siRNA was incorporated into tumor-homing glycol chitosan nanoparticles. Based on the stability in serum and delivery in a tumor-targeted manner, intravenously administered Dual-poly-siRNA carrying glycol chitosan nanoparticles (Dual-NP) demonstrated successful dual-gene silencing in tumors. Notably, co-delivery of VEGF and Bcl-2 targeting siRNA led to more effective cancer therapy for convenient application.
Assuntos
Quitosana/química , Neoplasias da Próstata/terapia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Terapêutica com RNAi/métodos , Transfecção/métodos , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Injeções Intravenosas , Masculino , Camundongos Nus , Nanopartículas , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Carga Tumoral , Fator A de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
P-glycoprotein (Pgp) mediated multi-drug resistance (MDR) is a major cause of failure in chemotherapy. In this study, small interfering RNA (siRNA) for Pgp down-regulation was delivered to tumors to overcome MDR in cancer. To achieve an efficient siRNA delivery in vivo, self-polymerized 5'-end thiol-modified siRNA (poly-siRNA) was incorporated in tumor targeting glycol chitosan nanoparticles. Pgp-targeted poly-siRNA (psi-Pgp) and thiolated glycol chitosan polymers (tGC) formed stable nanoparticles (psi-Pgp-tGC NPs), and the resulting nanoparticles protected siRNA molecules from enzymatic degradation. The psi-Pgp-tGC NPs could release functional siRNA molecules after cellular delivery, and they were able to facilitate siRNA delivery to Adriamycin-resistant breast cancer cells (MCF-7/ADR). After intravenous administration, the psi-Pgp-tGC NPs accumulated in MCF-7/ADR tumors and down-regulated P-gp expression to sensitize cancer cells. Consequently, chemo-siRNA combination therapy significantly inhibited tumor growth without systemic toxicity. These psi-Pgp-tGC NPs showed great potential as a supplementary therapeutic agent for drug-resistant cancer.
Assuntos
Quitosana/administração & dosagem , Resistencia a Medicamentos Antineoplásicos , Nanopartículas/administração & dosagem , Neoplasias/tratamento farmacológico , RNA Interferente Pequeno/administração & dosagem , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Inativação Gênica , Humanos , Células MCF-7 , Camundongos Nus , Neoplasias/metabolismo , Neoplasias/patologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cancer chemotherapy is often limited, since more than one molecule is usually involved with the cancer pathogenesis. A combination of therapeutic drugs would be a promising approach to overcome the complexity of tumors. In this study, a conjugate (DA3) of deoxycholic acid and low molecular weight polyethylenimine (PEI 1.8 kDa), which has a property that mimics that of cell penetrating peptides (CPPs), was used for simultaneous delivery of an anticancer drug and siRNA. When complexed with siRNA, DA3 showed significantly higher target gene silencing efficiency than PEI 25 kDa. The gene silencing efficiency of DA3 was maintained even in the presence of endocytosis inhibitors, suggesting that the polymeric carrier can mediate an endocytosis-independent macromolecular transduction similar to CPPs. The capability of forming a micelle-like core-shell structure enables the conjugates to encapsulate and dissolve paclitaxel (PTX), a water-insoluble drug. The drug-loaded cationic micelles can then interact with siRNA to form stable complexes (PTX/DA3/siRNA). The PTX/DA3/siRNA showed significantly enhanced inhibition of cancer cell growth. When administered into tumor-bearing animals, the PTX/DA3/siRNA demonstrated significant suppression of tumor growth, providing potential usefulness in clinical settings.