Sara Trabulo

Cell-Penetrating Peptides - Mechanisms of Cellular Uptake and Generation of Delivery Systems

The successful clinical application of nucleic acid-based therapeutic strategies has been limited by the poor delivery efficiency achieved by existing vectors. The development of alternative delivery systems for improved biological activity is, therefore, mandatory. Since the seminal observations two decades ago that the Tat protein, and derived peptides, can translocate across biological membranes, cell-penetrating peptides (CPPs) have been considered one of the most promising tools to improve non-invasive cellular delivery of therapeutic molecules. Despite extensive research on the use of CPPs for this purpose, the exact mechanisms underlying their cellular uptake and that of peptide conjugates remain controversial. Over the last years, our research group has been focused on the S413-PV cell-penetrating peptide, a prototype of this class of peptides that results from the combination of 13-amino-acid cell penetrating sequence derived from the Dermaseptin S4 peptide with the SV40 large T antigen nuclear localization signal. By performing an extensive biophysical and biochemical characterization of this peptide and its analogs, we have gained important insights into the mechanisms governing the interaction of CPPs with cells and their translocation across biological membranes. More recently, we have started to explore this peptide for the intracellular delivery of nucleic acids (plasmid DNA, siRNA and oligonucleotides). In this review we discuss the current knowledge of the mechanisms responsible for the cellular uptake of cell-penetrating peptides, including the S413-PV peptide, and the potential of peptide-based formulations to mediate nucleic acid delivery.

Survivin Silencing as a Promising Strategy To Enhance the Sensitivity of Cancer Cells to Chemotherapeutic Agents

Since clinical application of conventional cancer therapies is usually limited by drug resistance and toxic side effects, combination of chemotherapeutic agents with gene therapy appears as an attractive therapeutic strategy to overcome these issues. Being selectively expressed in tumor tissues, survivin is a promising target for the development of anticancer strategies aimed at eliminating tumor cells while sparing normal tissues. In this work, we achieved substantial protein knockdown in a number of human cell lines, namely, A549, HeLa and MCF-7 cells which overexpress survivin, after treatment with anti-survivin siRNAs, which was associated with a significant reduction of cell viability, when compared to treatment with control siRNAs. Interestingly, when the survivin-silencing approach was combined with a chemotherapeutic agent, an enhancement of the therapeutic effect was achieved. Treatment with anti-survivin siRNAs resulted in high levels of caspase 3/7 activation, and an enhancement of this effect was observed when survivin silencing was combined with vinblastine. In addition, we showed that for A549 and HeLa cells survivin silencing contributes to the reversion of cell resistance to doxorubicin. Overall, we demonstrate that the combination of a survivin-directed silencing strategy with chemotherapeutic agents constitutes a valuable approach for cancer treatment.

S4 13 - PV cell penetrating peptide and cationic liposomes act synergistically to mediate intracellular delivery of plasmid DNA

Cell penetrating peptides have been successfully used to mediate the intracellular delivery of a wide variety of molecules of pharmacological interest. The main aim of the present work was to evaluate the potential of the S413‐PV cell penetrating peptide to mediate the intracellular delivery of plasmid DNA, aiming at its use in gene therapy applications. The S413‐PV cell penetrating peptide is a chimeric peptide that results from the combination of a cell penetrating sequence derived from the Dermaseptin S4 peptide with the nuclear localization signal present in the Simian Virus 40 (SV40) large T antigen.

A non-covalent strategy combining cationic lipids and CPPs to enhance the delivery of splice correcting oligonucleotides

Modulation of pre-mRNA splicing by steric-block oligonucleotides constitutes a promising strategy for the treatment of many diseases, but requires efficient delivery to cell nuclei. In the present study, we evaluated the efficacy of a non-covalent strategy that combines a cell penetrating peptide with a lipoplex-based formulation to mediate the delivery of splice-switching oligonucleotides. The splice correcting ability of these new formulations was assessed using splice-switching oligonucleotides targeted towards the mutated splicing site of human beta-globin pre-mRNA in the HeLa pLuc/705 splice correction model. Importantly, the optimal splice correcting activity was exhibited by the formulation containing both lipid and peptide components, the order of component addition in these formulations being crucial for their efficacy. Our results demonstrate that the inclusion of cationic liposomes in the formulation provides the ability to improve release from endocytic vesicles, a barrier that severely limits the efficiency of oligonucleotide delivery by cell penetrating peptides. On the other hand, cell penetrating peptides potentiate the cellular uptake and delivery of the oligonucleotides by the lipoplexes. Moreover, when combining cell penetrating peptides with the lipoplex formulations, a significant reduction in the amount of required cationic lipid could be achieved, while maintaining or even enhancing biological activity.

S4(13)-PV cell-penetrating peptide induces physical and morphological changes in membrane-mimetic lipid systems and cell membranes: implications for cell internalization.

The present work aims to gain insights into the role of peptide-lipid interactions in the mechanisms of cellular internalization and endosomal escape of the S4(13)-PV cell-penetrating peptide, which has been successfully used in our laboratory as a nucleic acid delivery system. A S4(13)-PV analogue, S4(13)-PVscr, displaying a scrambled amino acid sequence, deficient cell internalization and drug delivery inability, was used in this study for comparative purposes. Differential scanning calorimetry, fluorescence polarization and X-ray diffraction at small and wide angles techniques showed that both peptides interacted with anionic membranes composed of phosphatidylglycerol or a mixture of this lipid with phosphatidylethanolamine, increasing the lipid order, shifting the phase transition to higher temperatures and raising the correlation length between the bilayers. However, S4(13)-PVscr, in contrast to the wild-type peptide, did not promote lipid domain segregation and induced the formation of an inverted hexagonal lipid phase instead of a cubic phase in the lipid systems assayed. Electron microscopy showed that, as opposed to S4(13)-PVscr, the wild-type peptide induced the formation of a non-lamellar organization in membranes of HeLa cells. We concluded that lateral phase separation and destabilization of membrane lamellar structure without compromising membrane integrity are on the basis of the lipid-driven and receptor-independent mechanism of cell entry of S4(13)-PV peptide. Overall, our results can contribute to a better understanding of the role of peptide-lipid interactions in the mechanisms of cell-penetrating peptide membrane translocation, helping in the future design of more efficient cell-penetrating peptide-based drug delivery systems.

Cell-penetrating peptides as nucleic acid delivery systems: from biophysics to biological applications.

The increasing knowledge on the genetic basis of disease has allowed the development of promising gene-targeted therapies that can be applied to numerous diseases. Such genetic-based approaches involve the use of nucleic acids as therapeutic agents, either for the insertion or repair and regulation of specific genes. However, the clinical application of these large and charged molecules remains highly dependent on the development of delivery systems capable of mediating efficient cellular uptake. Since the first observations, two decades ago, that some protein-derived domains can translocate across biological membranes, a wide group of peptides called cell-penetrating peptides (CPPs) have been considered one of the most promising tools to improve non-invasive cellular delivery of therapeutic molecules. The mechanistic basis of CPP and CPP conjugate cellular uptake remains controversial. However, biophysical studies on the interactions of CPPs with membrane models have contributed to unravel the mechanisms underlying CPP membrane translocation as well as to propose relationships between those mechanisms and CPP efficiency in mediating cargo delivery. In this review, representative examples of CPPs were gathered from the most recent literature in order to emphasize the contributions of chemists, biophysicists and cell biologists towards the rational design of increasingly more efficient delivery systems. In this context, the present review aims at giving an overview of some of the most significant CPP families and their biological applications as nucleic acid delivery systems.

Targeted lipoplexes for siRNA delivery.

RNA interference provides a powerful technology for silencing any single protein within a target cell. Therapeutic applications of small interfering RNAs (siRNAs), however, require vehicles for stable and efficient delivery of these nucleic acid molecules, both in vitro and in vivo. Targeted lipoplexes have been used as a promising system to mediate siRNA delivery and to achieve gene silencing. Electrostatic association of transferrin (Tf) to cationic liposomes enhances the transfection of siRNA. We describe the methods used for the preparation of Tf-lipoplexes and evaluation of their biocompatibility. Approaches to assess the complexation of siRNAs, the ability of Tf-lipoplexes to mediate siRNA protection, and intracellular delivery, as well as to achieve both mRNA and protein knockdown, are also described. We illustrate the efficiency of Tf-lipoplexes in mediating the knockdown of both green fluorescent protein (GFP) and luciferase in cell lines stably expressing these reporter genes.

Comparison of the efficiency of complexes based on S4(13)-PV cell-penetrating peptides in plasmid DNA and siRNA delivery.

The successful application of gene therapy approaches is highly dependent on the efficient delivery of nucleic acids into target cells. In the present study, new peptide-based nonviral systems were developed to enhance plasmid DNA and siRNA delivery, aiming at generating appropriate gene delivery and gene silencing tools for preclinical and clinical application. For this purpose, a new cell-penetrating peptide derived from the wild-type S4(13)-PV peptide was synthesized through the addition of a five-histidine tail to its N-terminus (H5-S4(13)-PV), and its ability to mediate gene expression and gene silencing was evaluated and compared to that of the wild-type peptide. The histidine-enriched peptide, H5-S4(13)-PV, proved to be generally more efficient and less toxic than the wild-type peptide in the delivery of plasmid DNA. In addition, complexes of H5-S4(13)-PV with siRNAs, but not of S4(13)-PV, were efficiently internalized by cells and presented high knockdown activity (63%). Interestingly, systems containing the S4(13)-PV or the H5-S4(13)-PV peptide exhibited superior biological activity when compared to those containing the reverse NLS or scrambled peptides, suggesting that both the cell-penetrating sequence and the NLS of the S4(13)-PV peptide influence the competence of binary and ternary complexes to accomplish nucleic acid delivery. In order to unravel the cancer therapeutic potential of formulations with the histidine-enriched peptide, their efficiency to mediate silencing of the oncogenic protein survivin was evaluated. As opposed to complexes with the wild-type peptide, H5-S4(13)-PV complexes showed the ability to promote a high survivin knockdown at the level of both protein (44%) and mRNA (73%), in HT1080 cells.

Cell-Penetrating Peptide-Based Systems for Nucleic Acid Delivery: A Biological and Biophysical Approach

The increasing knowledge on the genetic basis of disease provides a platform for the development of promising gene-targeted therapies that can be applied to numerous pathological conditions, including cancer. Such genetic-based approaches involve the use of nucleic acids as therapeutic agents, either for the insertion or for the repair and regulation of specific genes. However, despite the huge pharmacological potential of these molecules, their application remains highly dependent on the development of delivery systems capable of mediating efficient cellular uptake. The discovery of a class of small peptides, the so-called cell-penetrating peptides (CPPs), which are able to very efficiently cross cell membranes through a mechanism that is independent of membrane receptors or transporters and avoids lysosomal enzymatic degradation, has been enthusiastically considered of key interest to improve noninvasive cellular delivery of therapeutic molecules. A large number of CPPs have been applied successfully to mediate the intracellular delivery of nucleic acids, including the S4(13)PV peptide for which interactions with membranes and resulting biological effects are illustrated in this chapter. Here, we provide a description of the experimental procedures for the preparation of CPP-based nucleic acid complexes and assessment of their formation, the selection of those protocols leading to the most efficient complexes, the biophysical characterization of CPP membrane interactions, and the evaluation of the biological and cytotoxic activity of the complexes.

Cationic Liposome-Based Systems for Nucleic Acid Delivery: From the Formulation Development to Therapeutic Applications

Significant progress has been made in the development of different types of nucleic acids, including plasmid DNA, siRNA and oligonucleotides with the potential to form the basis of new treatment options for genetic and acquired diseases. However, the lack of suitable vectors for efficient delivery of nucleic acids into target cells represents a major hurdle for the successful application of gene therapy.