RESUMO
Toxicity towards non-tumor cells during anticancer therapy can be reduced by using nanoscale systems for anticancer drug delivery. Usually only the loaded drug has anticancer activity. Recently, micellar nanocomplexes (MNCs) comprising green tea catechin derivatives for the delivery of the anticancer proteins, such as Herceptin, have been developed. Herceptin as well as the MNCs without the drug were effective against HER2/neu-overexpressing human tumor cells and had synergistic anticancer effects in vitro and in vivo. It remained unclear which kinds of negative effects the MNCs had on tumor cells exactly, and which of their components mediated them. Also, it was unclear if MNC has any toxicity effects on the normal cells of vital human organ systems. Herein we examined the effects of Herceptin-MNCs and their individual components on human breast cancer cells and on normal primary human endothelial and kidney proximal tubular cells. We applied a novel in vitro model that predicts nephrotoxicity in humans with high accuracy, as well as high-content screening and microfluidic mono- and co-culture models to thoroughly address effects on various cell types. The results showed that MNCs alone were profoundly toxic for breast cancer cells, and induced apoptosis regardless of HER2/neu expression levels. Apoptosis was induced by both green tea catechin derivatives contained within MNCs. In contrast, MNCs were not toxic for normal human cells, and the probability was low that MNCs would be nephrotoxic in humans. Together, the results supported the hypothesis that green tea catechin derivative-based MNCs could improve efficacy and safety of therapies with anticancer proteins.
Assuntos
Neoplasias da Mama , Catequina , Humanos , Feminino , Micelas , Trastuzumab , CháRESUMO
Although a few nanomedicines have been approved for clinical use in cancer treatment, that recognizes improved patient safety through targeted delivery, their improved efficacy over conventional drugs has remained marginal. One of the typical drawbacks of nanocarriers for cancer therapy is a low drug-loading capacity that leads to insufficient efficacy and requires an increase in dosage and/or frequency of administration, which in turn increases carrier toxicity. In contrast, elevating drug-loading would cause the risk of nanocarrier instability, resulting in low efficacy and off-target toxicity. This intractable drug-to-carrier ratio has imposed constraints on the design and development of nanocarriers. However, if the nanocarrier has intrinsic therapeutic effects, the efficacy would be synergistically augmented with less concern for the drug-to-carrier ratio. Sunitinib-loaded micellar nanocomplex (SU-MNC) was formed using poly(ethylene glycol)-conjugated epigallocatechin-3-O-gallate (PEG-EGCG) as such a carrier. SU-MNC specifically inhibited the vascular endothelial growth factor-induced proliferation of endothelial cells, exhibiting minimal cytotoxicity to normal renal cells. SU-MNC showed enhanced anticancer effects and less toxicity than SU administered orally/intravenously on human renal cell carcinoma-xenografted mice, demonstrating more efficient effects on anti-angiogenesis, apoptosis induction, and proliferation inhibition against tumors. In comparison, a conventional nanocarrier, SU-loaded polymeric micelle (SU-PM) comprised of PEG-b-poly(lactic acid) (PEG-PLA) copolymer, only reduced toxicity with no elevated efficacy, despite comparable drug-loading and tumor-targeting efficiency to SU-MNC. Improved efficacy of SU-MNC was ascribed to the carrier-drug synergies with the high-performance carrier of PEG-EGCG besides tumor-targeted delivery.
Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Renais/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Neoplasias Renais/tratamento farmacológico , Nanopartículas/química , Sunitinibe/farmacologia , Chá/química , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Catequina/análogos & derivados , Catequina/química , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Portadores de Fármacos/química , Feminino , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Camundongos , Camundongos Nus , Camundongos Transgênicos , Micelas , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Tamanho da Partícula , Polietilenoglicóis/química , Sunitinibe/administração & dosagem , Sunitinibe/química , Propriedades de Superfície , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Low drug loading and instability in blood circulation are two key challenges that impede the successful clinical translation of nanomedicine, as they result in only marginal therapeutic efficacy and toxic side effects associated with premature drug leakage, respectively. Herein, highly stable and ultrahigh drug loading micellar nanocomplexes (MNCs) based on the self-assembly of the anticancer drug doxorubicin (DOX) and a poly(ethylene glycol)-epigallocatechin-3-O-gallate (EGCG) conjugate are developed. The formation of these MNCs is facilitated by strong favorable intermolecular interactions between the structurally similar aromatic EGCG and DOX molecules, which impart exceptionally high drug-loading capability of up to 88% and excellent thermodynamic and kinetic stability. Unlike two clinical formulations of DOX-free DOX and liposomal DOX, which are not effective below their lethal dosages, these DOX-loaded MNCs demonstrate significant tumor growth inhibition in vivo on a human liver cancer xenograft mouse model with minimal unwanted toxicity. Overall, these MNCs can represent a safe and effective strategy to deliver DOX for cancer therapy.
Assuntos
Nanoestruturas , Animais , Catequina , Linhagem Celular Tumoral , Doxorrubicina , Humanos , Camundongos , Micelas , Neoplasias , Polietilenoglicóis , CháRESUMO
The green tea catechin, (-)-epigallocatechin-3-O-gallate (EGCG), has gained significant attention as a potent adjuvant to enhance the antitumor efficacy of cisplatin while mitigating its harmful side effects. Herein we report the development of a fail-safe cisplatin nanomedicine constructed with hyaluronic acid-EGCG conjugate for ovarian cancer therapy. A simple mixing of this conjugate and cisplatin induces spontaneous self-assembly of micellar nanocomplexes having a spherical core-shell structure. The surface-exposed hyaluronic acid enables efficient delivery of cisplatin into CD44-overexpressing cancer cells via receptor-mediated endocytosis whereas the internally packed EGCG moieties offer an environment favorable for the encapsulation of cisplatin. In addition, the antioxidant effect of EGCG moieties ensures fail-safe protection against off-target organ toxicity originating from cisplatin-evoked oxidative stress. Pharmacokinetic and biodistribution studies reveal the prolonged blood circulation and preferential tumor accumulation of intravenously administered nanocomplexes. Moreover, the nanocomplexes exhibit superior antitumor efficacy over free cisplatin while displaying no toxicity in both a subcutaneous xenograft model and peritoneal metastatic model of human ovarian cancer. Our findings demonstrate proof of concept for the feasibility of green tea catechin-based micellar nanocomplexes as a safe and effective cisplatin nanomedicine for ovarian cancer treatment.
Assuntos
Antineoplásicos/farmacologia , Catequina/análogos & derivados , Cisplatino/química , Ácido Hialurônico/farmacologia , Nanoconjugados/química , Neoplasias Ovarianas/tratamento farmacológico , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Catequina/química , Catequina/metabolismo , Catequina/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Feminino , Humanos , Ácido Hialurônico/química , Camundongos SCID , Micelas , Estresse Oxidativo/efeitos dos fármacos , Tamanho da Partícula , Propriedades de Superfície , Chá/química , Distribuição TecidualRESUMO
Nanosized polyelectrolyte complexes are attractive delivery vehicles for the transfer of therapeutic genes to diseased cells. Here we report the application of self-assembled ternary complexes constructed with plasmid DNA, branched polyethylenimine and hyaluronic acid-green tea catechin conjugates for targeted gene delivery. These conjugates not only stabilize plasmid DNA/polyethylenimine complexes via the strong DNA-binding affinity of green tea catechin, but also facilitate their transport into CD44-overexpressing cells via receptor-mediated endocytosis. The hydrodynamic size, surface charge and physical stability of the complexes are characterized. We demonstrate that the stabilized ternary complexes display enhanced resistance to nuclease attack and polyanion-induced dissociation. Moreover, the ternary complexes can efficiently transfect the difficult-to-transfect HCT-116 colon cancer cell line even in serum-supplemented media due to their enhanced stability and CD44-targeting ability. Confocal microscopic analysis demonstrates that the stabilized ternary complexes are able to promote the nuclear transport of plasmid DNA more effectively than binary complexes and hyaluronic acid-coated ternary complexes. The present study suggests that the ternary complexes stabilized with hyaluronic acid-green tea catechin conjugates can be widely utilized for CD44-targeted delivery of nucleic acid-based therapeutics.
Assuntos
Catequina/análogos & derivados , DNA/administração & dosagem , Ácido Hialurônico/metabolismo , Plasmídeos/administração & dosagem , Transfecção/métodos , Catequina/química , Catequina/metabolismo , DNA/genética , Endocitose , Proteínas de Fluorescência Verde/genética , Células HCT116 , Células HEK293 , Humanos , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/química , Plasmídeos/genética , Polietilenoimina/química , Polietilenoimina/metabolismo , Chá/química , Chá/metabolismoRESUMO
A novel ternary nanogel based on the self-assembly of hyaluronic acid-epigallocatechin gallate conjugates (HA-EGCG), linear polyethylenimine (PEI) and Granzyme B (GzmB) in an aqueous environment was developed for the targeted intracellular delivery of GzmB into cancer cells. Lysozyme-encapsulated HA-EGCG nanogels were first prepared and characterized. HA-EGCG nanogels exhibited smaller particle sizes and a more homogeneous size distribution than the HA counterpart. Fluorescence quenching and lysozyme activity studies revealed that EGCG moieties facilitated protein binding through physical interactions and led to the formation of stable nanogels. When CD44-overexpressing HCT-116 colon cancer cells were treated with GzmB-encapsulated HA-EGCG nanogels in vitro, a significant cytotoxic effect was observed. Caspase assays and intracellular trafficking studies confirmed that cell death was due to apoptosis triggered by the delivery of GzmB to the cytosol of those cells. In comparison, little cytotoxic effect was observed in CD44-deficient cells treated with GzmB-encapsulated HA-EGCG nanogels. This study highlights the potential utility of HA-EGCG as effective intracellular protein carriers for targeted cancer therapy. STATEMENT OF SIGNIFICANCE: Intracellularly activated cytotoxic proteins can be used to kill cancer cells but viable carriers for such proteins are lacking. In this work, we developed novel nanogels based on selfassembly of hyaluronic acid (HA)-(-)-epigallocatechin-3-gallate (EGCG) conjugates, linear polyethylenemine (PEI) and the cytotoxic protein Granzyme B (GzmB) for the intracellular delivery of GzmB for cancer therapy. HA was exploited for its ability to target CD44 which are overexpressed in many types of cancer cells, while EGCG, the main component of green tea catechins, was chosen for its ability to bind to proteins. Characterization studies showed that EGCG facilitated protein complexation through physical interactions and led to the formation of stable nanogels. HA-EGCG nanogels were able to achieve CD44 targeted killing of HCT-116 cancer cells by delivering GzmB into the cytosol of these cells. We believe that the applications of the HA-EGCG nanogels can be expanded to the intracellular delivery of other cytotoxic protein drugs for cancer therapy.
Assuntos
Catequina/análogos & derivados , Sistemas de Liberação de Medicamentos/métodos , Ácido Hialurônico/química , Espaço Intracelular/metabolismo , Muramidase/metabolismo , Polietilenoglicóis/química , Polietilenoimina/química , Chá/química , Animais , Catequina/síntese química , Catequina/química , Sobrevivência Celular/efeitos dos fármacos , Galinhas , Dimerização , Difusão Dinâmica da Luz , Citometria de Fluxo , Granzimas/metabolismo , Células HCT116 , Células Hep G2 , Humanos , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/síntese química , Nanogéis , Espectrometria de FluorescênciaRESUMO
When designing drug carriers, the drug-to-carrier ratio is an important consideration, because the use of high quantities of carriers can result in toxicity as a consequence of poor metabolism and elimination of the carriers. However, these issues would be of less concern if both the drug and carrier had therapeutic effects. (-)-Epigallocatechin-3-O-gallate (EGCG), a major ingredient of green tea, has been shown, for example, to possess anticancer effects, anti-HIV effects, neuroprotective effects and DNA-protective effects. Here, we show that sequential self-assembly of the EGCG derivative with anticancer proteins leads to the formation of stable micellar nanocomplexes, which have greater anticancer effects in vitro and in vivo than the free protein. The micellar nanocomplex is obtained by complexation of oligomerized EGCG with the anticancer protein Herceptin to form the core, followed by complexation of poly(ethylene glycol)-EGCG to form the shell. When injected into mice, the Herceptin-loaded micellar nanocomplex demonstrates better tumour selectivity and growth reduction, as well as longer blood half-life, than free Herceptin.