RESUMO
Gliomas are therapeutically challenging cancers with poor patient prognosis. New drug delivery strategies are needed to achieve a more efficient chemotherapy-based approach against brain tumors. The current paper demonstrates development of a tumor-targeted delivery vector that is based on a cell-penetrating peptide pVEC and a novel glioma-targeting peptide sequence gHo. The unique tumor-homing peptide gHo was identified using in vitro phage display technology. The novel delivery vector, which we designated as gHoPe2, was constructed by a covalent conjugation of pVEC, gHo, and a cargo; the latter could be either a labeling moiety (such as a fluorescent marker) or a cytostatic entity. Using a fluorescent marker, we demonstrate efficient uptake of the vector in glioma cells and selective labeling of glioma xenograft tumors in a mouse model. This is the first time that we know where in vitro phage display has yielded an efficient, in vivo working vector. We also demonstrate antitumor efficacy of the delivery vector gHoPe2 using a well-characterized chemotherapeutic drug doxorubicin. Vectorized doxorubicin proved to be more efficient than the free drug in a mouse glioma xenograft model after systemic administration of the drugs. In conclusion, we have characterized a novel glioma-homing peptide gHo, demonstrated development of a new and potential glioma-targeted drug delivery vector gHoPe2, and demonstrated the general feasibility of the current approach for constructing cell-penetrating peptide-based targeted delivery systems.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Peptídeos Penetradores de Células/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Vetores Genéticos/administração & dosagem , Glioma/tratamento farmacológico , Sequência de Aminoácidos , Animais , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/genética , Feminino , Vetores Genéticos/genética , Glioma/genética , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Cell-penetrating peptide based vehicles have been developed for the delivery of different payloads into the cells in culture and in animals. However, several biological features, among which is the tendency to trigger innate immune response, limit the development of highly efficient peptide-based drug delivery vectors. This study aims to evaluate the influence of transportan 10 (TP10) and its chemically modified derivatives, PepFects (PFs), on the innate immune response of the host system. PFs have shown high efficiency in nucleic acid delivery in vitro and in vivo; hence, the estimation of their possible toxic side effects would be of particular interest. In this study, we analyzed cytotoxic and immunogenic response of PF3, PF4, and PF6 peptides in monocytic leukemia and peripheral blood mononuclear cell lines. In comparison with amphipathic PFs, TP10, TAT, stearyl-(RxR)(4) peptides, and the most widely used transfection reagents Lipofectamine 2000 and Lipofectamine RNAiMAX were also analyzed in this study. IL-1ß, IL-18, and TNF-α cytokine release was detected using highly sensitive enzyme-linked immunosorbent assay (ELISA). Cell viability was detected by measuring the activity of cellular enzymes that reduce water-soluble tetrazolium salts to formazan dyes and apoptosis was evaluated by measuring the levels of caspase-1 and caspase-3/7 over untreated cells. All peptides were found to be nontoxic and nonimmunogenic in vitro at the concentrations of 10 µM and 5 µM, respectively, and at a dose of 5 mg/kg in vivo, suggesting that these CPPs exhibit a promising potential in the delivery of therapeutic molecules into the cell without risks of toxicity and inflammatory reactions.
Assuntos
Peptídeos Penetradores de Células/imunologia , Peptídeos Penetradores de Células/toxicidade , Portadores de Fármacos/toxicidade , Galanina/imunologia , Galanina/toxicidade , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/toxicidade , Venenos de Vespas/imunologia , Venenos de Vespas/toxicidade , Animais , Caspases/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/genética , Células Cultivadas , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Galanina/química , Humanos , Interleucina-18/imunologia , Interleucina-1beta/imunologia , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Lipopeptídeos/química , Lipopeptídeos/imunologia , Lipopeptídeos/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de Fusão/química , Transfecção , Fator de Necrose Tumoral alfa/imunologia , Venenos de Vespas/químicaRESUMO
In recent years, oligonucleotide-based molecules have been intensely used to modulate gene expression. All these molecules share the common feature of being essentially impermeable over cellular membranes and they therefore require efficient delivery vectors. Cell-penetrating peptides are a group of delivery peptides that has been readily used for nucleic acid delivery. In particular, polyarginine and derivates thereof, i.e. the (RxR)(4) peptide, have been applied with success both in vitro and in vivo. A major problem, however, with these arginine-rich peptides is that they frequently remain trapped in endosomal compartments following internalization. The activity of polyarginine has previously been improved by conjugation to a stearyl moiety. Therefore, we sought to investigate what impact such modification would have on the pre-clinically used (RxR)(4) peptide for non-covalent delivery of plasmids and splice-correcting oligonucleotides (SCOs) and compare it with stearylated Arg9 and Lipofectamine 2000. We show that stearyl-(RxR)(4) mediates efficient plasmid transfections in several cell lines and the expression levels are significantly higher than when using unmodified (RxR)(4) or stearylated Arg9. Although the transfection efficiency is lower than with Lipofectamine 2000, we show that stearyl-(RxR)(4) is substantially less toxic. Furthermore, using a functional splice-correction assay, we show that stearyl-(RxR)(4) complexed with 2'-OMe SCOs promotes significant splice correction whereas stearyl-Arg9 fails to do so. Moreover, stearyl-(RxR)(4) promotes dose-dependent splice correction in parity with (RxR)(4)-PMO covalent conjugates, but at least 10-times lower concentration. These features make this stearic acid modified analog of (RxR)(4) an intriguing vector for future in vivo experiments.
Assuntos
Portadores de Fármacos/química , Ácidos Nucleicos/administração & dosagem , Peptídeos/química , Oligonucleotídeos Fosforotioatos/administração & dosagem , Ácidos Esteáricos/química , Processamento Alternativo/efeitos dos fármacos , Processamento Alternativo/genética , Animais , Células CHO , Permeabilidade da Membrana Celular , Proliferação de Células , Cricetinae , Cricetulus , Citometria de Fluxo , Expressão Gênica , Células HeLa , Humanos , Lipídeos , Luciferases/genética , Ácidos Nucleicos/química , Ácidos Nucleicos/genética , Oligonucleotídeos Fosforotioatos/química , Oligonucleotídeos Fosforotioatos/genética , Plasmídeos , TransfecçãoRESUMO
BACKGROUND: Rs619203 (Cys2229Ser) and rs529038 (Asp2213Asn) polymorphisms in the ROS1 gene have been studied in relation to myocardial infarction (MI) yielding inconsistent results. We investigated the role of ROS1 rs529038 polymorphism in coronary artery disease (CAD) in Greeks using a case-control study. METHODS: Genotyping for rs529038 polymorphism was performed using a multiplex PCR technique in patients with CAD (n=294) and controls (n=311). Logistic regression analysis was used to calculate crude and adjusted odds ratios (ORs). RESULTS: Logistic regression analysis did not show any statistically significant effect of ROS1 polymorphism in the occurrence of CAD (AG vs. AA, OR: 1.08, p=0.635; GG vs. AA, OR: 0.62, p=0.220). Adjustment for confounding factors gave similar results, irrespective of the type of disease (i.e., stable coronary artery disease vs. acute coronary syndrome). CONCLUSIONS: Our findings do not support the hypothesis that ROS1 rs529038 polymorphism is an important contributing factor in the etiology of CAD in the Greek population.