RESUMO
Chlamydia trachomatis is the most common sexually transmitted human pathogen. Infection results in minimal to no symptoms in approximately two-thirds of women and therefore often goes undiagnosed. C. trachomatis infections are a major public health concern because of the potential severe long-term consequences, including an increased risk of ectopic pregnancy, chronic pelvic pain, and infertility. To date, several point-of-care tests have been developed for C. trachomatis diagnostics. Although many of them are fast and specific, they lack the required sensitivity for large-scale application. We describe a rapid and sensitive form of detection directly from urine samples. The assay uses recombinase polymerase amplification and has a minimum detection limit of 5 to 12 pathogens per test. Furthermore, it enables detection within 20 minutes directly from urine samples without DNA purification before the amplification reaction. Initial analysis of the assay from clinical patient samples had a specificity of 100% (95% CI, 92%-100%) and a sensitivity of 83% (95% CI, 51%-97%). The whole procedure is fairly simple and does not require specific machinery, making it potentially applicable in point-of-care settings.
Assuntos
Infecções por Chlamydia/diagnóstico , Infecções por Chlamydia/urina , Chlamydia trachomatis/genética , Doenças Bacterianas Sexualmente Transmissíveis/diagnóstico , Doenças Bacterianas Sexualmente Transmissíveis/urina , Infecções por Chlamydia/genética , Chlamydia trachomatis/enzimologia , DNA Bacteriano/análise , DNA Polimerase Dirigida por DNA/química , Diacilglicerol Colinofosfotransferase/genética , Feminino , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da Polimerase , Recombinases/química , Sensibilidade e Especificidade , Doenças Bacterianas Sexualmente Transmissíveis/genéticaRESUMO
STUDY QUESTION: Do cell penetrating peptides (CPPs) translocate into spermatozoa and, if so, could they be utilized to deliver a much larger protein cargo? SUMMARY ANSWER: Chemically diverse polycationic CPPs rapidly and efficiently translocate into spermatozoa. They exhibit differential accumulation within intracellular compartments without detrimental influences upon cellular viability or motility but they are relatively ineffective in transporting larger proteins. WHAT IS ALREADY KNOWN: Endocytosis, the prevalent route of protein internalization into eukaryotic cells, is severely compromised in mature spermatozoa. Thus, the translocation of many bioactive agents into sperm is relatively inefficient. However, the delivery of bioactive moieties into mature spermatozoa could be significantly improved by the identification and utility of an efficient and inert vectorial delivery technology. STUDY DESIGN: CPP translocation efficacies, their subsequent differential intracellular distribution and the influence of peptides upon viability were determined in bovine spermatozoa. Temporal analyses of sperm motility in the presence of exogenously CPPs utilized normozoospermic human donor samples. MATERIALS AND METHODS: CPPs were prepared by manual, automated and microwave-enhanced solid phase synthesis. Confocal fluorescence microscopy determined the intracellular distribution of rhodamine-conjugated CPPs in spermatozoa. Quantitative uptake and kinetic analyses compared the translocation efficacies of chemically diverse CPPs and conjugates of biotinylated CPPs and avidin. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) conversion assays were employed to analyse the influence of CPPs upon sperm cell viability and sperm class assays determined the impact of CPPs on motility in capacitated and non-capacitated human samples. MAIN RESULTS: Chemically heterogeneous CPPs readily translocated into sperm to accumulate within discrete intracellular compartments. Mitoparan (INLKKLAKL(Aib)KKIL), for example, specifically accumulated within the mitochondria located in the sperm midpiece. The unique plasma membrane composition of sperm is a critical factor that directly influences the uptake efficacy of structurally diverse CPPs. No correlations in efficacies were observed when comparing CPP uptake into sperm with either uptake into fibroblasts or direct translocation across a phosphatidylcholine membrane. These comparative investigations identified C105Y (CSIPPEVKFNKPFVYLI) as a most efficient pharmacokinetic modifier for general applications in sperm biology. Significantly, CPP uptake induced no detrimental influence upon either bovine sperm viability or the motility of human sperm. As a consequence of the lack of endocytotic machinery, the CPP-mediated delivery of much larger protein complexes into sperm is relatively inefficient when compared with the similar process in fibroblasts. LIMITATIONS, REASONS FOR CAUTION: It is possible that some CPPs could directly influence aspects of sperm biology and physiology that were not analysed in this study. WIDER IMPLICATIONS OF THE FINDINGS: CPP technologies have significant potential to deliver selected bioactive moieties and so could modulate the biology and physiology of human sperm biology both prior- and post-fertilization.
Assuntos
Peptídeos Penetradores de Células/metabolismo , Motilidade dos Espermatozoides , Espermatozoides/metabolismo , Animais , Bovinos , Permeabilidade da Membrana Celular , Endocitose , Humanos , Masculino , Camundongos , Células NIH 3T3 , Transporte ProteicoRESUMO
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
The successful applicability of gene therapy approaches will heavily rely on the development of efficient and safe nonviral gene delivery vectors, for example, cell-penetrating peptides (CPPs). CPPs can condense oligonucleotides and plasmid DNA (pDNA) into nanoparticles, thus allowing the transfection of genetic material into cells. However, despite few promising attempts, CPP-mediated pDNA delivery has been relatively inefficient due to the unfavorable nanoparticle characteristics or the nanoparticle entrapment to endocytic compartments. In many cases, both of these drawbacks could be alleviated by modifying CPPs with a stearic acid residue, as demonstrated in the delivery of both the pDNA and the short oligonucleotides. In this study, PepFect14 (PF14) peptide, previously used for the transport of shorter oligonucleotides, is demonstrated to be suited also for the delivery of pDNA. It is shown that PF14 forms stable nanoparticles with pDNA with a negative surface charge and size of around 130-170 nm. These nanoparticles facilitate efficient gene delivery and expression in a variety of regular adherent cell lines and also in difficult-to-transfect primary cells. Uptake studies indicate that PF14/pDNA nanoparticles are utilizing class A scavenger receptors (SCARA) and caveolae-mediated endocytosis as the main route for cellular internalization. Conclusively, PF14 is an efficient nonviral vector for gene delivery.
Assuntos
Peptídeos Penetradores de Células/administração & dosagem , Peptídeos Penetradores de Células/genética , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Lipopeptídeos/administração & dosagem , Lipopeptídeos/genética , Animais , Células CHO , Técnicas de Cultura de Células , Peptídeos Penetradores de Células/metabolismo , Cricetinae , DNA/genética , Endocitose/genética , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Lipopeptídeos/metabolismo , Nanopartículas/administração & dosagem , Oligonucleotídeos/administração & dosagem , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Tamanho da Partícula , Plasmídeos/genética , Plasmídeos/metabolismo , Transfecção/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
Finding suitable nonviral delivery vehicles for nucleic acid-based therapeutics is a landmark goal in gene therapy. Cell-penetrating peptides (CPPs) are one class of delivery vectors that has been exploited for this purpose. However, since CPPs use endocytosis to enter cells, a large fraction of peptides remain trapped in endosomes. We have previously reported that stearylation of amphipathic CPPs, such as transportan 10 (TP10), dramatically increases transfection of oligonucleotides in vitro partially by promoting endosomal escape. Therefore, we aimed to evaluate whether stearyl-TP10 could be used for the delivery of plasmids as well. Our results demonstrate that stearyl-TP10 forms stable nanoparticles with plasmids that efficiently enter different cell-types in a ubiquitous manner, including primary cells, resulting in significantly higher gene expression levels than when using stearyl-Arg9 or unmodified CPPs. In fact, the transfection efficacy of stearyl-TP10 almost reached the levels of Lipofectamine 2000 (LF2000), however, without any of the observed lipofection-associated toxicities. Most importantly, stearyl-TP10/plasmid nanoparticles are nonimmunogenic, mediate efficient gene delivery in vivo, when administrated intramuscularly (i.m.) or intradermally (i.d.) without any associated toxicity in mice.
Assuntos
Peptídeos Penetradores de Células/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos , Plasmídeos/metabolismo , Transfecção/métodos , Animais , Transporte Biológico , Linhagem Celular , Cricetinae , Cricetulus , Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Endossomos/metabolismo , Terapia Genética/métodos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Ácidos Nucleicos/metabolismoRESUMO
While small interfering RNAs (siRNAs) have been rapidly appreciated to silence genes, efficient and non-toxic vectors for primary cells and for systemic in vivo delivery are lacking. Several siRNA-delivery vehicles, including cell-penetrating peptides (CPPs), have been developed but their utility is often restricted by entrapment following endocytosis. Hence, developing CPPs that promote endosomal escape is a prerequisite for successful siRNA implementation. We here present a novel CPP, PepFect 6 (PF6), comprising the previously reported stearyl-TP10 peptide, having pH titratable trifluoromethylquinoline moieties covalently incorporated to facilitate endosomal release. Stable PF6/siRNA nanoparticles enter entire cell populations and rapidly promote endosomal escape, resulting in robust RNAi responses in various cell types (including primary cells), with minimal associated transcriptomic or proteomic changes. Furthermore, PF6-mediated delivery is independent of cell confluence and, in most cases, not significantly hampered by serum proteins. Finally, these nanoparticles promote strong RNAi responses in different organs following systemic delivery in mice without any associated toxicity. Strikingly, similar knockdown in liver is achieved by PF6/siRNA nanoparticles and siRNA injected by hydrodynamic infusion, a golden standard technique for liver transfection. These results imply that the peptide, in addition to having utility for RNAi screens in vitro, displays therapeutic potential.
Assuntos
Peptídeos Penetradores de Células/química , Lipopeptídeos/química , Quinolinas/química , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Animais , Peptídeos Penetradores de Células/metabolismo , Peptídeos Penetradores de Células/toxicidade , Células Cultivadas , Endossomos/metabolismo , Humanos , Indicadores e Reagentes , Mediadores da Inflamação/metabolismo , Lipídeos , Lipopeptídeos/metabolismo , Camundongos , Nanopartículas/química , Nanopartículas/toxicidade , Quinolinas/metabolismoRESUMO
BACKGROUND: Although plasma fibrinogen levels are related to cardiovascular risk, data regarding the role of fibrinogen genetic variation in myocardial infarction (MI) or coronary artery disease (CAD) etiology remain inconsistent. The purpose of the present study was to investigate the effect of fibrinogen A (FGA), fibrinogen B (FGB) and fibrinogen G (FGG) gene SNPs and haplotypes on susceptibility to CAD in a homogeneous Greek population. METHODS: We genotyped for rs2070022, rs2070016, rs2070006 in FGA gene, the rs7673587, rs1800789, rs1800790, rs1800788, rs1800787, rs4681 and rs4220 in FGB gene and for the rs1118823, rs1800792 and rs2066865 SNPs in FGG gene applying an arrayed primer extension-based genotyping method (APEX-2) in a sample of CAD patients (n = 305) and controls (n = 305). Logistic regression analysis was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs), before and after adjustment for potential confounders. RESULTS: None of the FGA and FGG SNPs and FGA, FGB, FGG and FGA-FGG haplotypes was associated with disease occurrence after adjustment. Nevertheless, rs1800787 and rs1800789 SNPs in FGB gene seem to decrease the risk of CAD, even after adjustment for potential confounders (OR = 0.42, 95%CI: 0.19-0.90, p = 0.026 and OR = 0.44, 95%CI:0.21-0.94, p = 0.039, respectively). CONCLUSIONS: FGA and FGG SNPs as well as FGA, FGB, FGG and FGA-FGG haplotypes do not seem to be important contributors to CAD occurrence in our sample. On the contrary, FGB rs1800787 and rs1800789 SNPs seem to confer protection to disease onset lowering the risk by about 50% in homozygotes for the minor alleles.
Assuntos
Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/prevenção & controle , Fibrinogênio/genética , Variação Genética , Polimorfismo de Nucleotídeo Único , Síndrome Coronariana Aguda/sangue , Síndrome Coronariana Aguda/genética , Síndrome Coronariana Aguda/prevenção & controle , Idoso , Estudos de Casos e Controles , Doença da Artéria Coronariana/sangue , Feminino , Frequência do Gene , Genes Recessivos , Grécia , Haplótipos , Homozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Genéticos , Razão de Chances , Fatores de RiscoRESUMO
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.