Matrix stiffness affects endocytic uptake of MK2-inhibitor peptides.

In this study, the role of substrate stiffness on the endocytic uptake of a cell-penetrating peptide was investigated. The cell-penetrating peptide, an inhibitor of mitogen-activated protein kinase activated protein kinase II (MK2), enters a primary mesothelial cell line predominantly through caveolae. Using tissue culture polystyrene and polyacrylamide gels of varying stiffness for cell culture, and flow cytometry quantification and enzyme-linked immunoassays (ELISA) for uptake assays, we showed that the amount of uptake of the peptide is increased on soft substrates. Further, peptide uptake per cell increased at lower cell density. The improved uptake seen on soft substrates in vitro better correlates with in vivo functional studies where 10-100 µM concentrations of the MK2 inhibitor cell penetrating peptide demonstrated functional activity in several disease models. Additional characterization showed actin polymerization did not affect uptake, while microtubule polymerization had a profound effect on uptake. This work demonstrates that cell culture substrate stiffness can play a role in endocytic uptake, and may be an important consideration to improve correlations between in vitro and in vivo drug efficacy

Conjugation of an oligonucleotide to Tat, a cell penetrating peptide, via click chemistry

Uptake of diagnostic and therapeutic oligonucleotides that specifically target disease can be enhanced by attachment of a cell penetrating peptide. Here we describe the covalent attachment of an oligonucleotide to Tat, a biologically important cell-penetrating peptide, via click chemistry

Cell-penetrating peptide exploited syndecans

AbstractCell-penetrating peptides (CPPs) are short peptides capable of translocating across the plasma membrane of live cells and transporting conjugated compounds intracellularly. Fifteen years after discovering the first model cationic CPPs, penetratin and TAT, CPP internalization is still challenging many questions. Particularly it has been unknown whether CPPs enter the cells with or without mediation of a specific surface receptor. Here we report that syndecan-4, the universally expressed isoform of the syndecan family of transmembrane proteoglycans, binds and mediates transport of the three most frequently utilized cationic CPPs (penetratin, octaarginine and TAT) into the cells. Quantitative uptake studies and mutational analyses demonstrate that attachment of the cationic CPPs is mediated by specific interactions between the heparan sulfate chains of syndecan-4 and the CPPs. Protein kinase C alpha is also heavily involved in the uptake mechanism. The collected data give the first direct evidence on the receptor-mediated uptake of cationic CPPs and may replace the long-thought, but already contradicted membrane penetration hypothesis. Thus our study might give an answer for a decade long debate and foster the development of rationalized, syndecan-4 targeted novel delivery technologies

Intracellular delivery of a catalytic organometallic complex

A homogeneous carbene-based palladium catalyst was conjugated to a cell-penetrating peptide, allowing intracellular delivery of catalytically active Pd complexes that demonstrated bioorthogonal activation of a profluorophore within prostate cancer cells

Enhanced intracellular translocation and cytokine profiling of gold nanoparticles conjugated with human papilloma virus based (HPV-R9) cell-penetrating peptide in cancer cells

515-521Functionalized gold nanoparticles are emerging as a promising nanocarrier for targeted delivery of therapeutic molecules. The present study describes synthesis, characterization and intracellular delivery of gold nanoparticles (AuNPs) functionalized with L1 protein of human papilloma virus peptide (HPV) derived cell-penetrating peptide (CPP) conjugated with R9. HPV-R9 cell penetrating peptide conjugated with AuNPs (fAuNPs) was characterized using UV-visible and Fourier Transform Infrared (FTIR) spectroscopy, transmission electron microscopy, dynamic light scattering and zeta potential. The cellular uptake of fAuNPs was studied in Vero cells using fluorescence microscopy and flow cytometry. The fAuNPs displayed reversible low intracellular aggregation, ~20% cytotoxicity but high efficiency and stability in Vero cells. The cytokines study by real-time PCR was performed in 4T1 cells of the IL-4, IL-12 and IFN-ᵞ, found to be upregulated in comparison to control. The functionalized gold nanoparticles cell penetrating peptide show good intracellular transport to thecellular destinations.Results of this study demonstrate that cell penetrating peptide can be used along with gold nanoparticles for therapeutics application, particularly cancer

Triple-targeting Gram-negative selective antimicrobial peptides capable of disrupting the cell membrane and lipid A biosynthesis

Early lipid A biosynthesis is a potential antimicrobial target as this is an essential component of lipid A producing Gram-negative bacteria. Herein, we introduce a potent and highly synergistic Gram-negative selective triple-targeting antimicrobial peptide. The peptide contains a dual lipid A biosynthesis inhibiting sequence and an antimicrobial sequence

Potential applications of the cell-penetrating peptide crotamine

A doença renal crônica é um importante problema de saúde pública, que afeta em torno de 10% da população mundial e que determina aproximadamente 1 milhão de mortes por ano, devido principalmente à falta de acesso ao tratamento adequado especialmente em países de baixa renda. A perda da função renal está frequentemente associada à fibrose túbulo-intersticial, e as células epiteliais do túbulo proximal (CETPs) desempenham um papel crucial para a sua progressão. Embora as abordagens existentes sejam eficazes para o retardo desta doença renal terminal, ainda não há cura para a fibrose renal, possivelmente porque os tratamentos atuais atuem principalmente a nível vascular e glomerular, mas não a nível tubular. Neste projeto, propomos estudar estratégias de intervenção específicas para as CETPs, visando modular a expressão de genes específicos e/ou de proteínas terapêuticas que possam retardar a perda da função renal. Mais especificamente, propomos avaliar a viabilidade de uso da crotamina como vetor de transporte direcionado para a terapia de disfunções renais. A crotamina é um polipeptídeo célula penetrante originalmente isolado da peçonha da cascavel Crotalus durissus terrificus, que é internalizada por diversos tipos celulares e com capacidade comprovada de transportar genes para o interior das células in vitro e in vivo. Neste projeto demonstramos que a injeção intraperitoneal (IP) em camundongos saudáveis leva a um acúmulo preferencial da crotamina nos rins, mais especificamente nas CETPs. Demonstramos ainda a ausência de efeitos tóxicos evidentes após o tratamento prolongado por via IP com baixas doses da crotamina (1 μg/animal/dia), por até 21 dias, visto que não foram observadas alterações significativas na massa corpórea, nos marcadores bioquímicos de função renal e nas análises histopatológicas dos tecidos de animais tratados comparados aos controles que receberam veículo. Foi demonstrado pela primeira vez aqui o potencial de uso da crotamina como um carreador não-viral de moléculas terapêuticas para o tratamento de disfunções renais, já que o complexo formado pela crotamina-siRNA (do inglês, small interfering RNA) mostrou-se resistente à degradação por nucleases e com especificidade e internalização seletiva pelas CETPs tanto in vitro quanto in vivo, como demonstrado para a crotamina livre não-complexada também. Adicionalmente o complexo crotamina-siRNA para sindecan-1 suprimiu eficientemente a expressão do proteoglicano sindecan-1, que é super-expresso nos túbulos proximais em condições de lesão renal, contribuindo então para a ativação da via alternativa do sistema complemento e levando à fibrose tubulointersticial. A supressão direcionada de sindecan-1 em CETPs pode potencialmente reduzir a ativação do processo inflamatório e a progressão da injúria renal. Portanto, os resultados aqui apresentados nos permitem sugerir que a viabilidade do emprego in vivo do complexo crotamina-siRNA pode abrir novos caminhos para a terapia gênica direcionada para doenças renais, mais precisamente, em tubulopatias.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Flexible synthesis of cationic peptide-porphyrin derivatives for light-triggered drug delivery and photodynamic therapy

Efficient syntheses of cell-penetrating peptide-porphyrin conjugates are described using a variety of bioconjugation chemistries. This provides a flexible means to convert essentially hydrophobic tetrapyrolle photosensitisers into amphiphilic derivatives which are well-suited for use in light-triggered drug delivery by photochemical internalisation (PCI) and targeted photodynamic therapy (PDT)

Carrier PNA for shRNA delivery into cells

A peptide nucleic acid (PNA)-cell-penetrating peptide (CPP) conjugate (carrier PNA) was used as 'bridgebuilder' to connect a CPP with an shRNA. The carrier PNA successfully formed a hybrid with an shRNA bearing complementary dangling bases and the shRNA was introduced into cells by the carrier PNA, and RNAi was induced by the shRNA