Stearoyl CoA Desaturase-1 Silencing in Glioblastoma Cells: Phospholipid Remodeling and Cytotoxicity Enhanced upon Autophagy Inhibition.

Modulation of lipid metabolism is a well-established cancer hallmark, and SCD1 has been recognized as a key enzyme in promoting cancer cell growth, including in glioblastoma (GBM), the deadliest brain tumor and a paradigm of cancer resistance. The central goal of this work was to identify, by MS, the phospholipidome alterations resulting from the silencing of SCD1 in human GBM cells, in order to implement an innovative therapy to fight GBM cell resistance. With this purpose, RNAi technology was employed, and low serum-containing medium was used to mimic nutrient deficiency conditions, at which SCD1 is overexpressed. Besides the expected increase in the saturated to unsaturated fatty acid ratio in SCD1 silenced-GBM cells, a striking increase in polyunsaturated chains, particularly in phosphatidylethanolamine and cardiolipin species, was noticed and tentatively correlated with an increase in autophagy (evidenced by the increase in LC3BII/I ratio). The contribution of autophagy to mitigate the impact of SCD1 silencing on GBM cell viability and growth, whose modest inhibition could be correlated with the maintenance of energetically associated mitochondria, was evidenced by using autophagy inhibitors. In conclusion, SCD1 silencing could constitute an important tool to halt GBM resistance to the available treatments, especially when coupled with a mitochondria disrupter chemotherapeutic.

Gemini surfactant dimethylene-1,2-bis(tetradecyldimethylammonium bromide)-based gene vectors: a biophysical approach to transfection efficiency.

Cationic liposomes have been proposed as biocompatible gene delivery vectors, able to overcome the barriers imposed by cell membranes. Besides lipids, other surfactant molecules have been successfully used in the composition of gene carriers. In the present work, we used a Gemini surfactant, represented by the general structure [C(14)H(29)(CH(3))(2)N(+)(CH(2))(2)N(+)(CH(3))(2)C(14)H(29)]2Br(-) and herein designated 14-2-14, to prepare cationic gene carriers, both as the sole component and in combination with neutral helper lipids, cholesterol and DOPE. The effectiveness of three Gemini-based formulations, namely neat 14-2-14, 14-2-14:Chol (1:1 molar ratio) and 14-2-14:Chol:DOPE (2:1:1 molar ratio), to mediate gene delivery was evaluated in DNA mixtures of +/- charge ratios ranging from 1/1 to 12/1. After ruling out cytotoxicity as responsible for the differences observed in the transfection competence, structural and physical properties of the vector were investigated, using several techniques. The size and surface charge density (zeta potential) of surfactant-based structures were determined by conventional techniques and the thermotropic behaviour of aqueous dispersions of surfactant/lipid/DNA formulations was monitored by fluorescence polarization of DPH and DPH-PA probes. The capacity of lipoplexes to interact with membrane-mimicking lipid bilayers was evaluated, using the PicoGreen assay and a FRET technique. Our data indicate inefficiency of the neat 14-2-14 formulation for gene delivery, which could result from the large dimensions of the particles and/or from its relative incompetence to release DNA upon interaction with anionic lipids. The addition of cholesterol or cholesterol and DOPE conferred to Gemini-based gene carrier transfection activity at specific ranges of +/- charge ratios. Fluorescence polarization data suggest that an order parameter within a specific range was apparently needed for complexes to display maximal transfection efficiency. The transfection-competent formulations showed to be efficiently destabilized by interaction with different anionic and zwitterionic bilayers, including those containing PS and cardiolipin. These data are discussed in terms of the potential of these formulations to address different intracellular targets.

Targeted and intracellular triggered delivery of therapeutics to cancer cells and the tumor microenvironment: impact on the treatment of breast cancer.

Limiting tumor invasion to the surrounding healthy tissues has proven to be clinically relevant for anticancer treatment options. We have demonstrated that, within a solid tumor, it is possible to achieve such a goal with the same nanoparticle by intracellular and triggered targeted drug delivery to more than one cell population. We have identified the nucleolin receptor in endothelial and cancer cells in tissue samples from breast cancer patients, which enabled the design of a F3-peptide-targeted sterically stabilized pH-sensitive liposome. The clinical potential of such strategy was demonstrated by the successful specific cellular association by breast cancer cells harvested from tumors of patients submitted to mastectomy. In vitro, the nanoparticle targeted the nucleolin receptor on a cell and ligand-specific manner and improved cytotoxicity of doxorubicin (used as a model drug) towards breast cancer and endothelial cells by 177- and 162-fold, respectively, relative to the commercially available non-targeted non-pH-sensitive liposomes. Moreover, active accumulation of F3-targeted pH-sensitive liposomes into human orthotopic tumors, implanted in the mammary fat pad of nude mice, was registered for a time point as short as 4 h, reaching 48% of the injected dose/g of tissue. Twenty-four hours post-injection the accumulation of the dual-targeted pH-sensitive nanoparticle in the tumor tissue was 33-fold higher than the non-targeted non-pH-sensitive counterpart. In mice treated with the developed targeted nanoparticle significant decrease of the tumor viable rim area and microvascular density, as well as limited invasion to surrounding healthy tissues were observed (as opposed to other tested controls), which may increase the probability of tumors falling in the category of "negative margins" with reduced risk of relapse.

Design of peptide-targeted liposomes containing nucleic acids.

Anticancer systemic gene silencing therapy has been so far limited by the inexistence of adequate carrier systems that ultimately provide an efficient intracellular delivery into target tumor cells. In this respect, one promising strategy involves the covalent attachment of internalizing-targeting ligands at the extremity of PEG chains grafted onto liposomes. Therefore, the present work aims at designing targeted liposomes containing nucleic acids, with small size, high encapsulation efficiency and able to be actively internalized by SCLC cells, using a hexapeptide (antagonist G) as a targeting ligand. For this purpose, the effect of the liposomal preparation method, loading material (ODN versus siRNA) and peptide-coupling procedure (direct coupling versus post-insertion) on each of the above-mentioned parameters was assessed. Post-insertion of DSPE-PEG-antagonist G conjugates into preformed liposomes herein named as stabilized lipid particles, resulted in targeted vesicles with a mean size of about 130 nm, encapsulation efficiency close to 100%, and a loading capacity of approximately 5 nmol siRNA/mumol of total lipid. In addition, the developed targeted vesicles showed increased internalization in SCLC cells, as well as in other tumor cells and HMEC-1 microvascular endothelial cells. The improved cellular association, however, did not correlate with enhanced downregulation of the target protein (Bcl-2) in SCLC cells. These results indicate that additional improvements need to be performed in the future, namely by ameliorating the access of the nucleic acids to the cytoplasm of the tumor cells following receptor-mediated endocytosis.

Striatal and nigral pathology in a lentiviral rat model of Machado-Joseph disease.

Machado-Joseph disease (MJD) is a fatal, dominant neurodegenerative disorder. MJD results from polyglutamine repeat expansion in the MJD-1 gene, conferring a toxic gain of function to the ataxin-3 protein. In this study, we aimed at overexpressing ataxin-3 in the rat brain using lentiviral vectors (LV), to generate an in vivo MJD genetic model and, to study the disorder in defined brain regions: substantia nigra, an area affected in MJD, cortex and striatum, regions not previously reported to be affected in MJD. LV encoding mutant or wild-type human ataxin-3 was injected in the brain of adult rats and the animals were tested for behavioral deficits and neuropathological abnormalities. Striatal pathology was confirmed in transgenic mice and human tissue. In substantia nigra, unilateral overexpression of mutant ataxin-3 led to: apomorphine-induced turning behavior; formation of ubiquitinated ataxin-3 aggregates; alpha-synuclein immunoreactivity; and loss of dopaminergic markers (TH and VMAT2). No neuropathological changes were observed upon wild-type ataxin-3 overexpression. Mutant ataxin-3 expression in striatum and cortex, resulted in accumulation of misfolded ataxin-3, and within striatum, loss of neuronal markers. Striatal pathology was confirmed by observation in MJD transgenic mice of ataxin-3 aggregates and substantial reduction of DARPP-32 immunoreactivity and, in human striata, by ataxin-3 inclusions, immunoreactive for ubiquitin and alpha-synuclein. This study demonstrates the use of LV encoding mutant ataxin-3 to produce a model of MJD and brings evidence of striatal pathology, suggesting that this region may contribute to dystonia and chorea observed in some MJD patients and may represent a target for therapies.

Co-encapsulation of anti-BCR-ABL siRNA and imatinib mesylate in transferrin receptor-targeted sterically stabilized liposomes for chronic myeloid leukemia treatment.

Chronic myeloid leukemia (CML) is triggered by the BCR-ABL oncogene. Imatinib is the first-line treatment of CML; however imatinib resistance and intolerance have been detected in many patients. Therefore, new therapeutic approaches are required. The present work aimed at the development and application of transferrin receptor (TrfR) targeted liposomes co-encapsulating anti-BCR-ABL siRNA and imatinib at different molar ratios. The encapsulation yields and drug loading of each molecule was evaluated. Anti-leukemia activity of the developed formulations co-encapsulating siRNA and imatinib and of the combination of Trf-liposomes carrying siRNA and free imatinib under two different treatment schedules of pre-sensitization was assessed. The results obtained demonstrate that the presence of imatinib significantly decreases the encapsulation yields of siRNA, whereas imatinib encapsulation yields are increased by the presence of siRNA. Cytotoxicity assays demonstrate that the formulations co-encapsulating siRNA and imatinib promote a 3.84-fold reduction on the imatinib IC(50) (from 3.49 to 0.91 µM), whereas a 8.71-fold reduction was observed for the pre-sensitization protocols (from 42.7 to 4.9 nM). It was also observed that the formulations with higher siRNA to imatinib molar ratios promote higher cell toxicity. Thus, the present work describes a novel triple targeting strategy with one single system: cellular targeting (through the targeting ligand, transferrin) and molecular targeting at the BCR-ABL mRNA and Bcr-Abl protein level.

Polyethylenimine of various molecular weights as adjuvant for transfection mediated by cationic liposomes.

Over the last years significant progress has been made in non-viral gene delivery mediated by cationic liposomes. However, the results obtained are still far from being satisfactory regarding transfection efficiency, particularly when compared to that achieved using viral vectors. We have previously demonstrated that association of transferrin with cationic liposomes significantly improves transfection in a large variety of cells, both in vitro and in vivo. In this work, several strategies have been explored in order to further improve transfection mediated by transferrin-associated lipoplexes. To this regard, the effect on transfection of pre-condensation of DNA with polyethylenimine of low MWs (2.7, 2.0 and 0.8 KDa) at various N/P ratios, lipid composition, cationic lipid/DNA (+/-) charge ratio and the presence of a surfactant in the lipoplexes was investigated. Two different modes for preparing the liposomes were tested and the extent of cell association of their complexes with DNA as well as their capacity to protect the carried DNA were evaluated. Our results show that complexes generated from cationic liposomes prepared by the ethanol injection method in which the carried DNA was pre-condensed with low MW polyethylenimine are highly efficient in mediating transfection. The differential modulating effect observed upon association of transferrin to various liposome formulations on transfection mediated by the polyethylenimine-complexes suggests that these complexes enter into the cells through different pathways (involving clathrin versus caveolin), most likely by taking advantage of their intrinsic biophysical properties to escape from the endosome to the cytosol.

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

BACKGROUND: 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 S4(13)-PV cell penetrating peptide to mediate the intracellular delivery of plasmid DNA, aiming at its use in gene therapy applications. The S4(13)-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. METHODS: S4(13)-PV cell penetrating peptide and cationic liposomes composed of 1,2-dioleoyl-3-trimethylammonium-propane:1,2-dioleoyl-sn-glycero-3-phosphoethanolamine were complexed with pDNA at different charge ratios. Complexation of pDNA was assessed by gel electrophoresis. Luciferase assay, fluorescence microscopy and fluorescence-activated cell sorting analysis were used to evaluate reporter gene delivery to TSA and HeLa cells. Cytotoxicity of the pDNA complexes was assessed by Alamar blue assay. RESULTS: Complexes obtained through electrostatic association of the S4(13)-PV cell penetrating peptide with plasmid DNA are able to very efficiently mediate transfection, particularly at high peptide/DNA charge ratios. Additionally, our results clearly demonstrate that, both in HeLa and TSA cells, ternary complexes, resulting from association of cationic liposomes to peptide/DNA complexes, are significantly more efficient in mediating transfection than the corresponding peptide/DNA or cationic liposome/DNA complexes. CONCLUSIONS: Overall, our data highlight the potential of cell penetrating peptides for the development of improved nonviral gene delivery systems.

Resveratrol stimulates the metabolic reprogramming of human CD4+ T cells to enhance effector function.

The polyphenol resveratrol activates the deacetylase Sirt1, resulting in various antioxidant, chemoprotectant, neuroprotective, cardioprotective, and anti-inflammatory properties. We found that at high concentrations of resveratrol, human CD4+ T cells showed defective antigen receptor signaling and arrest at the G1 stage of the cell cycle, whereas at low concentrations, cells were readily activated and exhibited enhanced Sirt1 deacetylase activity. Nevertheless, low-dose resveratrol rapidly stimulated genotoxic stress in the T cells, which resulted in engagement of a DNA damage response pathway that depended on the kinase ATR [ataxia telangiectasia-mutated (ATM) and Rad3-related], but not ATM, and subsequently in premitotic cell cycle arrest. The concomitant activation of p53 was coupled to the expression of gene products that regulate cell metabolism, leading to a metabolic reprogramming that was characterized by decreased glycolysis, increased glutamine consumption, and a shift to oxidative phosphorylation. These alterations in the bioenergetic homeostasis of CD4+ T cells resulted in enhanced effector function, with both naïve and memory CD4+ T cells secreting increased amounts of the inflammatory cytokine interferon-γ. Thus, our data highlight the wide range of metabolic adaptations that CD4+ T lymphocytes undergo in response to genomic stress.

Role of microRNAs in the regulation of innate immune cells under neuroinflammatory conditions.

MiRNAs are short, evolutionary conserved noncoding RNA molecules with the ability to control the magnitude of inflammation. The immunosuppressive nature of the brain is sustained by miRNA-dependent regulation of microglial cells, which become activated under neuroinflammatory conditions, such as brain injury and neurodegeneration. The pro-inflammatory and suppressive role of the most studied neuroimmune miRNAs, miR-155 and miR-146a, has been recently challenged. Although the molecular targets of these miRNAs remain unchanged across brain diseases, different kinetics of miRNA expression and degradation can produce different immune outcomes and change microglia phenotypes. Here, we discuss current knowledge regarding the implications of disruption of miRNA networks in neuroinflammation and in the pathophysiology of acute and chronic CNS diseases.

MicroRNA deregulation and chemotaxis and phagocytosis impairment in Alzheimer's disease.

INTRODUCTION: Mononuclear phagocytes play a critical role during Alzheimer's disease (AD) pathogenesis due to their contribution to innate immune responses and amyloid beta (Aß) clearance mechanisms. METHODS: Blood-derived monocytes (BDMs) and monocyte-derived macrophages (MDMs) were isolated from blood of AD, mild cognitive impairment (MCI) patients, and age-matched healthy controls for molecular and phenotypic comparisons. RESULTS: The chemokine/chemokine receptor CCL2/CCR2 axis was impaired in BDMs from AD and MCI patients, causing a deficit in cell migration. Changes were also observed in MDM-mediated phagocytosis of Aß fibrils, correlating with alterations in the expression and processing of the triggering receptor expressed on myeloid cells 2 (TREM2). Finally, immune-related microRNAs (miRNAs), including miR-155, -154, -200b, -27b, and -128, were found to be differentially expressed in these cells. DISCUSSION: This work provides evidence that chemotaxis and phagocytosis, two crucial innate immune functions, are impaired in AD and MCI patients. Correlations with miRNA levels suggest an epigenetic contribution to systemic immune dysfunction in AD.

Liposomal and viral vectors for gene therapy of the central nervous system.

Due to the presence of the blood-brain barrier, the central nervous system (CNS) is not easily accessible to systemically delivered macromolecules with therapeutic activity such as growth factors, cytokines or enzymes. Therefore, the expression of exogenously administered genes in the brain has been proposed for a wide variety of inherited and acquired diseases of the CNS, for which classical pharmacotherapy is unavailable or not easily applicable. Gene therapy to the CNS has been the target of a great number of studies aiming at finding a viable therapeutic strategy for the treatment of neurological disorders. This approach has already been used as a promising tool for brain protection and repair from neuronal insults and degeneration in several animal models, and is currently being applied in clinical trials. The choice of an appropriate vector system for transferring the desired gene into the affected brain area is an important issue for developing a safe and efficient gene therapy approach for the CNS. In this review, we focus on the various types of vectors that have been used for gene delivery into the CNS. Particular emphasis is given to their mode of preparation, biological activity, safety and in vivo behavior. Examples illustrating the potential of both viral and non-viral vectors in therapeutic applications to brain disorders are provided. In addition, the use of lentiviral vectors for in vivo modeling of genetic disorders of the CNS is discussed.

MicroRNAs in glioblastoma: role in pathogenesis and opportunities for targeted therapies.

Glioblastoma (GBM) is among the most lethal human cancers, being generally characterized by rapid diffuse and infiltrative growth and high level of cellular heterogeneity associated with therapeutic resistance. Despite remarkable advances in cancer theranostics, which resulted in significant improvement of clinical outcomes, patient survival remains under one year. In recent years, considerable progress has been made in understanding the role of small non-coding RNAs, designated microRNAs, in the pathogenesis of GBM. Indeed, microRNAs were found to play a critical role in multiple steps of the tumorigenic process, including cellular proliferation, apoptosis evasion, invasion, angiogenesis, and stemness. Moreover, the modulation of microRNA expression, using either antisense oligonucleotides or precursor/mimic sequences, revealed a tremendous potential for application in GBM-targeted therapeutic approaches, either per se or in combination with chemo- and/or radiotherapy. In this manuscript, we review the regulatory role of microRNAs in key cellular processes underlying GBM tumorigenesis, including migration and invasion, apoptosis evasion, angiogenesis and GBM stem-like cell proliferation/differentiation, and discuss the current knowledge on their potential as diagnostic, prognostic and predictive biomarkers in this disease. We also address the latest advances in microRNA-based therapeutic approaches for GBM, by summarizing the major achievements in in vitro and pre-clinical studies. The trends identified by these studies are highlighted in order to provide new prospects for future developments towards the successful treatment of GBM.

New serine-derived gemini surfactants as gene delivery systems.

Gemini surfactants have been extensively used for in vitro gene delivery. Amino acid-derived gemini surfactants combine the special aggregation properties characteristic of the gemini surfactants with high biocompatibility and biodegradability. In this work, novel serine-derived gemini surfactants, differing in alkyl chain lengths and in the linker group bridging the spacer to the headgroups (amine, amide and ester), were evaluated for their ability to mediate gene delivery either per se or in combination with helper lipids. Gemini surfactant-based DNA complexes were characterized in terms of hydrodynamic diameter, surface charge, stability in aqueous buffer and ability to protect DNA. Efficient formulations, able to transfect up to 50% of the cells without causing toxicity, were found at very low surfactant/DNA charge ratios (1/1-2/1). The most efficient complexes presented sizes suitable for intravenous administration and negative surface charge, a feature known to preclude potentially adverse interactions with serum components. This work brings forward a new family of gemini surfactants with great potential as gene delivery systems.

On the formulation of pH-sensitive liposomes with long circulation times.

Strategies used to enhance liposome-mediated drug delivery in vivo include the enhancement of stability and circulation time in the bloodstream, targeting to specific tissues or cells, and facilitation of intracytoplasmic delivery. pH-sensitive liposomes have been developed to mediate the introduction of highly hydrophilic molecules or macromolecules into the cytoplasm. These liposomes destabilize under acidic conditions found in the endocytotic pathway, and usually contain phosphatidylethanolamine (PE) and titratable stabilizing amphiphiles. Formulations without PE have also been developed. Encapsulated compounds are thought to be transported into the cytoplasm through destabilization of or fusion with the endosome membrane. Incorporation of a low mole percentage of poly(ethylene glycol) (PEG)-conjugated lipids into pH-sensitive liposomes confers prolonged circulation times to these liposomes, which are otherwise cleared rapidly. While the incorporation of PEG-lipids reduces the pH-dependent release of encapsulated fluorescent markers in vitro, it does not hinder the cytoplasmic delivery of the markers per cell-associated liposome. This suggests that intracellular delivery is not dictated simply by the destabilization of the liposomes. Antibodies or ligands to cell surface receptors can be coupled to pH-sensitive or sterically stabilized pH-sensitive liposomes for targeting. pH-sensitive liposomes have been used to deliver anticancer drugs, antibiotics, antisense oligonucleotides, ribozymes, plasmids, proteins and peptides to cells in culture or in vivo.

Sustained release of naltrexone from poly(n-isopropylacrylamide) microgels.

The release of the opioid antagonist naltrexone from neutral poly(N-isopropylacrylamide) (PNIPAAM) microgels and negatively charged PNIPAAM microgels containing acrylic acid groups (PNIPAAM-co-PAA) has been studied at various microgel and drug concentrations. The release curves were found to be well represented by the Weibull equation. The release rates were observed to be dependent on the microgel concentration. At most conditions, the release from the charged microgels was slower than for the neutral microgels. In addition, the charged microgels exhibited a release lag time, which was dependent on the microgel concentration. No significant lag time could be observed for the neutral microgels. Increasing the naltrexone concentration did not significantly affect the release rates from the neutral microgels, but the release from the charged microgels became faster. The microgels did not exhibit any significant cytotoxic effect on HeLa cells at the tested concentrations.

Bis-quaternary gemini surfactants as components of nonviral gene delivery systems: a comprehensive study from physicochemical properties to membrane interactions.

Gemini surfactants have been successfully used as components of gene delivery systems. In the present work, a family of gemini surfactants, represented by the general structure [CmH2m+1(CH3)2N(+)(CH2)sN(+)(CH3)2CmH2m+1]2Br(-), or simply m-s-m, was used to prepare cationic gene carriers, aiming at their application in transfection studies. An extensive characterization of the gemini surfactant-based complexes, produced with and without the helper lipids cholesterol and DOPE, was carried out in order to correlate their physico-chemical properties with transfection efficiency. The most efficient complexes were those containing helper lipids, which, combining amphiphiles with propensity to form structures with different intrinsic curvatures, displayed a morphologically labile architecture, putatively implicated in the efficient DNA release upon complex interaction with membranes. While complexes lacking helper lipids were translocated directly across the lipid bilayer, complexes containing helper lipids were taken up by cells also by macropinocytosis. This study contributes to shed light on the relationship between important physico-chemical properties of surfactant-based DNA vectors and their efficiency to promote gene transfer, which may represent a step forward to the rational design of gene delivery systems.

In vitro cytotoxicity of a thermoresponsive gel system combining ethyl(hydroxyethyl) cellulose and lysine-based surfactants.

The cytotoxicity of three lysine-derived surfactants with a gemini-like structure was evaluated on HeLa cells. The half maximal effective concentration (EC(50)) was estimated from the dose-response curves and the values indicated an increase in toxicity with the increase in alkyl chain length. The shorter chain length surfactant (C(6)) was shown to be less cytotoxic than sodium dodecyl sulfate (SDS), and all the lysine-derived surfactants were less toxic than the cationic cetyl trimethylammonium bromide (CTAB). The presence of ethyl (hydroxyethyl) cellulose (EHEC), shown previously to form thermoresponsive gels in combination with these surfactants, was found to contribute to a lower toxicity on HeLa cells. The conjecture is that the polymer-surfactant interactions in forming mixed micelles are the key contributors to the enhanced biocompatibility of the hydrogels. The most promising results were obtained in the presence of either the most hydrophilic surfactant or in the presence of the longest chain-length surfactant. For the latter, very low concentrations are needed to induce a sol-gel transition of EHEC semi-dilute solutions.

Suicide gene therapy in cancer: where do we stand now?

Suicide gene therapy is based on the introduction into tumor cells of a viral or a bacterial gene, which allows the conversion of a non-toxic compound into a lethal drug. Although suicide gene therapy has been successfully used in a large number of in vitro and in vivo studies, its application to cancer patients has not reached the desirable clinical significance. However, recent reports on pre-clinical cancer models demonstrate the huge potential of this strategy when used in combination with new therapeutic approaches. In this review, we summarize the different suicide gene systems and gene delivery vectors addressed to cancer, with particular emphasis on recently developed systems and associated bystander effects. In addition, we review the different strategies that have been used in combination with suicide gene therapy and provide some insights into the future directions of this approach, particularly towards cancer stem cell eradication.

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.