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BBA - Biomembranes (v.1758, #3)
Special issue: Mechanisms of carrier-mediated intracellular delivery of therapeutics
by G. Divita; F. Heitz (pp. 259-259).
Cell-penetrating peptides—A brief introduction by Peter Järver; Ülo Langel (pp. 260-263).
Cargo delivery; Cell-penetrating peptides; Endocytosis; Membrane transduction
Mechanistic aspects of CPP-mediated intracellular drug delivery: Relevance of CPP self-assembly by SÃlvia Pujals; Jimena Fernández-Carneado; Carmen López-Iglesias; Marcelo J. Kogan; Ernest Giralt (pp. 264-279).
In recent years, cell-penetrating peptides have proven to be an efficient intracellular delivery system. The mechanism for CPP internalisation, which first involves interaction with the extracellular matrix, is followed in most cases by endocytosis and finally, depending on the type of endocytosis, an intracellular fate is reached. Delivery of cargo attached to a CPP requires endosomal release, for which different methods have recently been proposed. Positively charged amino acids, hydrophobicity and/or amphipathicity are common to CPPs. Moreover, some CPPs can self-assemble. Herein is discussed the role of self assembly in the cellular uptake of CPPs. Sweet Arrow Peptide (SAP) CPP has been shown to aggregate by CD and TEM (freeze-fixation/freeze-drying), although the internalised species have yet to be identified as either the monomer or an aggregate.
Keywords: Amphipathic peptide; Cell-penetrating peptide; Drug delivery; Internalisation mechanism; Self-assembly
The effect of the structure of branched polypeptide carrier on intracellular delivery of daunomycin by Judit Reményi; Gabriella CsÃk; Péter Kovács; Francesca Reig; Ferenc Hudecz (pp. 280-289).
The conjugate of acid labile cis-aconityl-daunomycin (cAD) with branched chain polypeptide, poly[Lys(Glui-DL-Alam)] (EAK) was very effective against L1210 leukemia in mice. However, Dau attached to a polycationic polypeptide, poly[Lys(Seri-DL-Alam)] (SAK) exhibited no in vivo antitumor effect. In order to understand this difference we have performed comparative in vitro studies to dissect properties related to interaction with the whole body (e.g., biodistribution) from those present at cellular or even molecular level. We report here (a) the kinetics of acid-induced Dau liberation, (b) interaction with DPPC phospholipid bilayer, (c) in vitro cytotoxic effect on different tumor cells, and (d) intracellular distribution in HL-60 cells of polycationic (cAD-SAK) and amphoteic (cAD-EAK) conjugates. Fluorescence properties of the two conjugates are also reported. Our findings demonstrate that the kinetics of the drug release, intracellular distribution and in vitro cytotoxic effect are rather similar, while the effect on DPPC phospholipid bilayer and fluorescence properties of the two conjugates are not the same. We also found that the in vitro cytotoxicity is cell line dependent. These observations suggest that the structure of the polypeptide carrier could have marked influence on drug uptake related events.
Keywords: Polypeptide carrier; Daunomycin; pH-dependent drug liberation; Altered conjugate cytotoxicity; Uptake of daunomycin conjugate
Targeting the HIV-1 RNA leader sequence with synthetic oligonucleotides and siRNA: Chemistry and cell delivery by John J. Turner; Martin Fabani; Andrey A. Arzumanov; Gabriela Ivanova; Michael J. Gait (pp. 290-300).
New candidates for development as potential drugs or virucides against HIV-1 infection and AIDS continue to be needed. The HIV-1 RNA leader sequence has many essential functional sites for virus replication and regulation that includes several highly conserved sequences. The review describes the historical context of targeting the HIV-1 RNA leader sequence with antisense phosphorothioate oligonucleotides, such as GEM 91, and goes on to describe modern approaches to targeting this region with steric blocking oligonucleotide analogues having newer and more advantageous chemistries, as well as recent studies on siRNA, towards the attainment of antiviral activity. Recent attempts to obtain improved cell delivery are highlighted, including exciting new developments in the use of peptide conjugates of peptide nucleic acid (PNA) as potential virucides.
Keywords: Oligonucleotide; siRNA; HIV-1; AIDS; Virucide; Antiviral agent
Cationic amphipathic histidine-rich peptides for gene delivery by Antoine Kichler; A. James Mason; Burkhard Bechinger (pp. 301-307).
Besides being a useful tool in research, gene transfer has a high potential as treatment for a variety of genetic and acquired diseases. However, in order to enable a gene to become a pharmaceutical, efficient and safe methods of delivery have to be developed. We recently found that cationic amphipathic histidine-rich peptide antibiotics can efficiently deliver DNA into mammalian cells. Our lead compound, LAH4 (KKALLALALHHLAHLALHLALALKKA), demonstrated in vitro transfection efficiencies comparable to those of commercially available reagents. Synthesis and evaluation of LAH mutants provided evidence that the transfection efficiency depends on the number and positioning of histidine residues in the peptide as well as on the pH at which the in-plane to transmembrane transition takes place. Moreover, recent results suggest that binding of the DNA complexes to the plasma membrane is mediated by heparan sulfate proteoglycans and that anionic phospholipids may be involved in the endosomal destabilization process. Finally, we also describe in this review the rationale that led to the development of LAH4 as a DNA carrier as well as the biophysical methods that have allowed us to propose a model which could explain the way this peptide destabilizes the endosomal bilayer.
Keywords: Amphipathic peptide; Gene transfer; Membrane destabilization; Antibiotic; Solid-state NMR
Cell penetration properties of maurocalcine, a natural venom peptide active on the intracellular ryanodine receptor by Sylvie Boisseau; Kamel Mabrouk; Narendra Ram; Nicolas Garmy; Véronique Collin; Abir Tadmouri; Mohamad Mikati; Jean-Marc Sabatier; Michel Ronjat; Jacques Fantini; Michel De Waard (pp. 308-319).
Maurocalcine (MCa) is a 33-amino acid residue peptide toxin initially isolated from the scorpion Scorpio maurus maurus. Its structural and functional features make it resembling many Cell Penetrating Peptides. In particular, MCa exhibits a characteristic positively charged face that may interact with membrane lipids. External application of MCa is known to produce Ca2+-release from intracellular stores within seconds. MCa binds directly to the skeletal muscle isoform of the ryanodine receptor, an intracellular channel target of the endoplasmic reticulum, and induces long-lasting channel openings in a mode of smaller conductance. The binding sites for MCa have been mapped within the cytoplasmic domain of the ryanodine receptor. In this manuscript, we further investigated how MCa proceeds to cross biological membranes in order to reach its target. A biotinylated derivative of MCa (MCab) was chemically synthesized, coupled to a fluorescent streptavidin indicator (Cy3 or Cy5) and the cell penetration of the entire complex followed by confocal microscopy and FACS analysis. The data provide evidence that MCa allows the penetration of the macro proteic complex and therefore may be used as a vector for the delivery of proteins in the cytoplasm as well as in the nucleus. Using both FACS and confocal analysis, we show that the cell penetration of the fluorescent complex is observed at concentrations as low as 10Â nM, is sensitive to membrane potential and is partly inhibited by heparin. We also show that MCa interacts with the disialoganglioside GD3, the most abundant charged lipid in natural membranes. Despite its action on ryanodine receptor, MCa showed no sign of cell toxicity on HEK293 cells suggesting that it may have a wider application range. These data indicate that MCa may cross the plasma membrane directly by cell translocation and has a promising future as a carrier of various drugs and agents of therapeutic, diagnostic and technological value.
Keywords: Abbreviations; MCa; maurocalcine; MCa; b; biotinylated maurocalcine; Strep; streptavidine; Cy3; cyanine 3; Cy5; cyanine5; GD3; disialoganglioside NeuAcα2-8NeuAcα2-3Galβ1-4Glcβ1-Cer; DPPC; dipalmitoylphosphatidylcholine; DHPR; dihydropyridine receptor; RyR; ryanodine receptor; SR; sarcoplasmic reticulum; PBS; phosphate-buffered saline; CPP; cell penetrating peptideCell-penetrating peptide; Cellular uptake; Maurocalcine; Cargo delivery
Rational design of complementary peptides to the βAmyloid 29–42 fusion peptide: An application of PepDesign by Marc Decaffmeyer; Laurence Lins; Benoit Charloteaux; Marie Hélène VanEyck; Annick Thomas; Robert Brasseur (pp. 320-327).
Peptides in solution currently exist under several conformations; an equilibrium which varies with solvent polarity. Despite or because of this structure versatility, peptides can be selective biological tools: they can adapt to a target, vary conformation with solvents and so on. These capacities are crucial for cargo carriers. One promising way of using peptides in biotechnologies is to decipher their medium–sequence–structure–function relationships and one approach is molecular modelling. Only few “in silico� methods of peptide design are described in the literature. Most are used in support of experimental screening of peptide libraries. However, the way they are made does not teach us much for future researches. In this paper, we describe an “in silico� method (PepDesign) which starts by analysing the native interaction of a peptide with a target molecule in order to define which points are important. From there, a modelling protocol for the design of ‘better’ peptides is set. The PepDesign procedure calculates new peptides fulfilling the hypothesis, tests the conformational space of these peptides in interaction with the target by angular dynamics and goes up to the selection of the best peptide based on the analysis of complex structure properties. Experimental biological assays are finally used to test the selected peptides, hence to validate the approach. Applications of PepDesign are wide because the procedure will remain similar irrespective of the target which can be a protein, a drug or a nucleic acid. In this paper, we describe the design of peptides which binds to the fusogenic helical form of the C-terminal domain of the Aβ peptide (Aβ29–42).
Keywords: Alzheimer; Amyloid; Angular dynamic; ApoE; Complementary peptide; Computer-aided design; In silico design; Peptide interaction; Tilted peptide
Interactions of amphipathic CPPs with model membranes by Sébastien Deshayes; May C. Morris; Gilles Divita; Frédéric Heitz (pp. 328-335).
We have investigated the interactions between two carrier peptides and model membrane systems as well as the conformational consequences of these interactions. Studies performed with lipid monolayers at the air–water interface have enabled identification of the nature of the lipid–peptide interactions and characterization of the influence of phospholipids on the ability of these peptides to penetrate into lipidic media. Penetration experiments reveal that both peptides interact strongly with phospholipids. Conformational investigations indicate that the lipid–peptide interaction govern the conformational state of the peptides. Based on the ability of both peptides to promote ion permeabilization of both natural and artificial membranes, we propose a model illustrating the translocation process. For MPG, it is based on the formation of a β-barrel pore-like structure, while for Pep-1, it is based on association of helices.
Keywords: Cell-penatrating peptide; Amphipatic peptide; Interactions with membrane; Conformation; Spectroscopic analysis
Cellular uptake of S413-PV peptide occurs upon conformational changes induced by peptide–membrane interactions by Miguel Mano; Ana Henriques; Artur Paiva; Manuel Prieto; Francisco Gavilanes; Sérgio Simões; Maria C. Pedroso de Lima (pp. 336-346).
In face of accumulated reports demonstrating that uptake of some cell-penetrating peptides occurs through previously described endocytic pathways, or is a consequence of cell fixation artifacts, we conducted a systematic analysis on the mechanism responsible for the cellular uptake of the S413-PV karyophilic cell-penetrating peptide. The results reviewed here show that the S413-PV peptide is able to very efficiently accumulate inside live cells in a rapid, non-toxic and dose-dependent manner, through a mechanism distinct from endocytosis. Comparative analysis of peptide uptake by mutant cells lacking heparan sulfate proteoglycans demonstrates that, although not mandatory, their presence at cell surface facilitates the cellular uptake of the S413-PV peptide. Furthermore, we demonstrate that upon interaction with lipid vesicles, the S413-PV peptide undergoes significant conformational changes that are consistent with the formation of helical structures. Such conformational changes occur concomitantly with a penetration of the peptide into the lipid bilayer, strongly suggesting that the resulting helical structures are crucial for the non-endocytic cellular uptake of the S413-PV peptide. Overall, our data support that, rather than endocytosis, the cellular uptake of the S413-PV cell-penetrating peptide is a consequence of its direct translocation through cell membranes following conformational changes induced by peptide–membrane interactions.
Keywords: Abbreviations; CPP; cell-penetrating peptide; GAG; glycosaminoglycan; HSPG; heparan sulfate proteoglycan; LUV; large unilamellar vesicle; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-[phospho-; rac; -(1-glycerol)]; CD; circular dichroismCell-penetrating peptide; Protein transduction domain; Peptide–membrane interaction; Tryptophan fluorescence; Circular dichroism; Amphipathic alpha-helix
Developing novel hCT derived cell-penetrating peptides with improved metabolic stability by Robert Rennert; Christian Wespe; Annette G. Beck-Sickinger; Ines Neundorf (pp. 347-354).
Many promising therapeutics are currently awaiting their clinical application. Due to their low capability of cell membrane crossing, these compounds do not reach their site of action. One way to overcome this problem might be the fusion of these agents to cell-penetrating peptides (CPP), which are able to shuttle various cargoes across cellular membranes. One disadvantage in using CPP in drug delivery is their low metabolic stability. The aim of our work was to increase the proteolytic resistance of the CPP hCT(9–32), a truncated C-terminal fragment of human calcitonin. Thus, we synthesised six modified N-terminally carboxyfluorescein labelled hCT(9–32) derivatives by replacing positions 12 and/or 16 of hCT(9–32) with either N-methylphenylalanine ord-phenylalanine, respectively. By using confocal laser scanning microscopy we showed that the modifications did neither affect the peptide internalisation efficiency in HeLa nor HEK 293T cells. The metabolic stability of the peptides was investigated in human blood plasma and HEK 293T cell culture supernatant. To analyse the degradation patterns, we used RP-HPLC and MALDI-TOF mass spectrometry. However, we found for all of the new derivatives high metabolic stabilities. In blood plasma, the half-lives for five of the six peptides increased compared to unmodified hCT(9–32). The degradation patterns showed a distinct stabilisation in the N-terminal part of the modified peptides, in the C-terminal part, we found some cleavage to a minor extent. Furthermore, we studied the conformation of the peptides by CD spectroscopy and demonstrated that they possess no cell toxicity. Since our metabolically more stable compounds are still able to pass the cell membrane they provide powerful tools as drug delivery vectors.
Keywords: Abbreviations; CD; circular dichroism; CF; carboxyfluorescein; CLSM; confocal laser scanning microscopy; CPP; cell-penetrating peptide; DIC; diisopropylcarbodiimide; HATU; O; -(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; hCT; human calcitonin; HOBt; 1-hydroxybenzotriazole; MALDI-TOF; matrix assisted laser desorption ionisation-time of flight; MS; mass spectrometry; RP-HPLC; reversed phase - high performance liquid chromatography; SPPS; solid phase peptide synthesis; TBTU; O; -(benzotriazol-1-yl)1,1,3,3-tetramethyluronium tetrafluoroborate; TFA; trifluoroacetic acid; TFE; trifluoroethanole; XTT; 2,3-bis(methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilideDrug delivery; Cell-penetrating peptide; Human calcitonin; Metabolic degradation; Fragmentation pattern
The importance of valency in enhancing the import and cell routing potential of protein transduction domain-containing molecules by Michael Sung; Gregory M.K. Poon; Jean Gariépy (pp. 355-363).
Protein transduction domains (PTDs) are peptides that afford the internalization of cargo macromolecules (including plasmid DNA, proteins, liposomes, and nanoparticles). In the case of polycationic peptides, the efficiency of PTDs to promote cellular uptake is directly related to their molecular mass or their polyvalent presentation. Similarly, the efficiency of routing to the nucleus increases with the number of nuclear localization signals (NLS) associated with a cargo. The quantitative enhancement, however, depends on the identity of the PTD sequence as well as the targeted cell type. Thus the choice and multivalent presentation of PTD and NLS sequences are important criteria guiding the design of macromolecules intended for specific intracellular localization. This review outlines synthetic and recombinant strategies whereby PTDs and signal sequences can be assembled into multivalent peptide dendrimers and promote the uptake and routing of their cargoes. In particular, the tetramerization domain of the tumour suppressor p53 (p53tet) is emerging as a useful scaffold to present multiple routing and targeting moieties. Short cationic peptides fused to the 31-residue long p53tet sequence resulted in tetramers displaying a significant enhancement (up to 1000 fold) in terms of their ability to be imported into cells and delivered to the cell nucleus in relation to their monomeric analogues. The design of future polycationic peptide dendrimers as effective delivering vehicles will need to incorporate selective cell targeting functions and provide solutions to the issue of endosomal entrapment.
Keywords: Abbreviations; Antp; Drosophila; antennapedia homeodomain; NLS; nuclear localization sequence; Np; nucleoplasmin; p53; mono; monomeric form of human p53 tetramerization domain (residues 325–355) harbouring a L334P mutation; p53; tet; human p53 tetramerization domain (residues 325–355); PTD; protein transduction domain; scF; v; single-chain antibody; Tat; HIV-1 transactivator of transcription
Comparison of basic peptides- and lipid-based strategies for the delivery of splice correcting oligonucleotides by Alain R. Thierry; Said Abes; Sarah Resina; Adrian Travo; Jean Philippe Richard; Paul Prevot; Bernard Lebleu (pp. 364-374).
Expression of alternatively spliced mRNA variants at specific stages of development or in specific cells and tissues contributes to the functional diversity of the human genome. Aberrations in alternative splicing were found as a cause or a contributing factor to the development, progression, or maintenance of numerous diseases. The use of antisense oligonucleotides (ON) to modify aberrant expression patterns of alternatively spliced mRNAs is a novel means of potentially controlling such diseases. Oligonucleotides can be designed to repair genetic mutations, to modify genomic sequences in order to compensate for gene deletions, or to modify RNA processing in order to improve the effects of the underlying gene mutation. Steric block ON approach have proven to be effective in experimental model for various diseases. Here, we describe our experience in investigating two strategies for ON delivery: ON conjugation with basic peptides and lipid-based particulate system (lipoplex). Basic peptides or Cell Penetrating Peptides (CPP) such as the TAT-derived peptide appear to circumvent many problems associated with ON and drug delivery. This strategy may represent the next paradigm in our ability to modulate cell function and offers a unique avenue for the treatment of disease. Lipoplexes result from the intimate interaction of ON with cationic lipids leading to ON carrying particles able to be taken up by cells and to release ON in the cytoplasm. We have used as an experimental model the correction of a splicing alteration of the mutated β-globin intron causing thalassemia. Data on cell penetration and efficacy of correction of specific steric block ON delivered either by basic peptides or lipoplex are described. A comparison of the properties of both delivery systems is made respective to the use of this new class of therapeutic molecules.
Keywords: Antisense oligonucleotide; PNA; Delivery; Cell penetrating peptide; Liposome; Splicing correction
Quantification of the efficiency of cargo delivery by peptidic and pseudo-peptidic Trojan carriers using MALDI-TOF mass spectrometry by Baptiste Aussedat; Sandrine Sagan; Gérard Chassaing; Gérard Bolbach; Fabienne Burlina (pp. 375-383).
We have measured the efficiencies of two novel pseudo-peptidic carriers and various cell-penetrating peptides (Penetratin, (Arg)9 and the third helix of the homeodomain of Knotted-1) to deliver the same cargo inside cells. The cargo that was studied corresponds to the pseudo-substrate of protein kinase C. Cargo delivery was quantified using a recent method based on isotope labeling and MALDI-TOF MS. Results of cargo delivery were compared to the amounts of free CPP internalized inside cells. The third helix of Knotted gave the best results concerning free CPP cellular uptake. It was also found to be the most efficient carrier. This peptide thus emerges as a new CPP with very promising properties.
Keywords: Abbreviations; Acm; acetamidomethyl; Boc; tert; -butoxycarbonyl; BSA; bovine serumalbumin; CHCA; α-cyano-4-hydroxycinnamic acid; CHO; Chinese hamster ovary; CPP; cell-penetrating peptide; H-CPP; non-deuterated cell-penetrating peptide; D-CPP; deuterated cell-penetrating peptide; 2Cl-Cbz; 2-chlorobenzyloxycarbonyl; DCC; dicyclohexylcarbodiimide; DMEM; Dubelcco's modified eagle medium; DIEA; diisopropylethylamine; DMF,; N; N; -dimethylformamide; DTT; dithiotreitol; EDTA; ethylenediamine tetraacetic acid; HATU; N; -[(dimethylamino)-1; H; -1,2,3-triazolo[4,5-; b; ]pyridin-1-ylmethylene]-; N; -methylmethanaminium hexafluorophosphate; N; -oxide; HBTU; N; -[(1; H; -benzotriazol-1-yl)(dimethylamino)methylene]-; N; -methylmethanaminium hexafluorophosphate; N; -oxide; HOAt; 1-hydroxy-7-aza-benzotriazole; HOBt; 1-hydroxybenzotriazole; HPLC; high performance liquid chromatography; MALDI-TOF MS; matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MBHA-PS; 4-methylbenzhydrylamine polystyrene; 4-MeOBzl; p-methoxybenzyl; Npys; 3-nitro-2-pyridinesulphenyl; PyAOP; 7-Azabenzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate; PKC; protein kinase C; PKCi; proteins kinase C inhibitor; PyBOP; benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexa-fluorophosphate; SEM; standard error of the mean; TFA; trifluoroacetamide; TFFH; tetramethylfluoroformamidinium hexafluorophosphate; THF; tetrahydrofuranCell-penetrating peptide; Cargo delivery; Quantification; MALDI-TOF MS
A non-covalent peptide-based strategy for protein and peptide nucleic acid transduction by Edwige Gros; Sebastien Deshayes; May C. Morris; Gudrun Aldrian-Herrada; Julien Depollier; Frederic Heitz; Gilles Divita (pp. 384-393).
The development of therapeutic peptides and proteins is limited by the poor permeability and the selectivity of the cell membrane. The discovery of protein transduction domains has given a new hope for administration of large proteins and peptides in vivo. We have developed a non-covalent strategy for protein transduction based on an amphipathic peptide, Pep-1, that consists of a hydrophobic domain and a hydrophilic lysine-rich domain. Pep-1 efficiently delivers a variety of fully biologically active peptides and proteins into cells, without the need for prior chemical cross-linking or chemical modifications. The mechanism through which Pep-1 delivers active macromolecules does not involve the endosomal pathway and the dissociation of the Pep-1/macromolecule particle occurs immediately after it crosses the cell membrane. Pep-1 has been successfully applied to the screening of therapeutic peptides in vivo and presents several advantages: stability in physiological buffer, lack of toxicity and of sensitivity to serum. In conclusion, Pep-1 technology could contribute significantly to the development of fundamental and therapeutic applications and be an alternative to covalent protein transduction domain-based technologies.
Keywords: Cell-penetrating peptide; Therapeutic protein; Peptide delivery; Peptide nucleic acid; Amphipathic peptide; Peptide-based nanoparticle
Light controllable siRNAs regulate gene suppression and phenotypes in cells by Quan N. Nguyen; Rajesh V. Chavli; Joao T. Marques; Peter G. Conrad II; Die Wang; Weihai He; Barbara E. Belisle; Aiguo Zhang; Larry M. Pastor; Frank R. Witney; May Morris; Frederic Heitz; Gilles Divita; Bryan R.G. Williams; Gary K. McMaster (pp. 394-403).
Small interfering RNA (siRNA) is widely recognized as a powerful tool for targeted gene silencing. However, siRNA gene silencing occurs during transfection, limiting its use is in kinetic studies, deciphering toxic and off-target effects and phenotypic assays requiring temporal, and/or spatial regulation. We developed a novel controllable siRNA (csiRNA) that is activated by light. A single photo removable group is coupled during oligonucleotide synthesis to the 5′ end of the antisense strand of the siRNA, which blocks the siRNA's activity. A low dose of light activates the siRNA, independent of transfection resulting in knock down of specific target mRNAs and proteins (GAPDH, p53, survivin, hNuf2) without stimulating non-specific effects such as regulated protein kinase PKR and induction of the interferon response. We demonstrate survivin and hNuf2 csiRNAs temporally knockdown their mRNAs causing multinucleation and cell death by mitotic arrest, respectively. Furthermore, we demonstrate a dose-dependent light regulation of hNuf2 csiRNA activity and resulting phenotype. The light controllable siRNAs are introduced into cells using commercially available reagents including the MPG peptide based delivery system. The csiRNAs are comparable to standard siRNAs in their transfection efficiency and potency of gene silencing. This technology should be of interest for phenotypic assays such as cell survival, cell cycle regulation, and cell development.
Keywords: Light controllable siRNAs to regulate gene expression; MPG delivery for difficult to transfect cell types; Branch DNA quantitation of mRNA expression; Non-specific effects of the double-stranded RNA (dsRNA) regulated protein kinase PKR kinase and induction of the IFN response; Immunofluorescence detection of protein expression; Western blot detection of protein expression; Apoptosis and multinucleation phenotypic assays
Functionalized carbon nanotubes as emerging nanovectors for the delivery of therapeutics by Cédric Klumpp; Kostas Kostarelos; Maurizio Prato; Alberto Bianco (pp. 404-412).
Functionalized carbon nanotubes ( f-CNT) are emerging as a new family of nanovectors for the delivery of different types of therapeutic molecules. The application of CNT in the field of carrier-mediated delivery has become possible after the recent discovery of their capacity to penetrate into the cells. CNT can be loaded with active molecules by forming stable covalent bonds or supramolecular assemblies based on noncovalent interactions. Once the cargos are carried into various cells, tissues and organs they are able to express their biological function. In this review, we will describe the potential of f-CNT to deliver different types of therapeutic molecules.
Keywords: Carbon nanotube; Vector; Vaccine; Gene; Drug delivery; Peptide
Enhanced cellular binding of concatemeric oligonucleotide complexes by Olesya N. Simonova; Albina V. Vladimirova; Marina A. Zenkova; Valentin V. Vlassov (pp. 413-418).
Interaction of oligonucleotides condensed into long concatemeric complexes with cancer cells was investigated. Pairs of 24- and 25-mer oligodeoxyribonucleotides were designed so that they could hybridize and form concatemeric structures. Pre-assembling of the oligonucleotides into concatemers considerably enhanced their ability to bind to human embryo kidney 293 cells and neuroblastoma IMR-32 cells as compared to free oligonucleotides. Efficiency of concatemers binding to the cells is improved with increase of the length and concentration of concatemeric complexes. The obtained results suggest incorporation of pharmacologically active oligonucleotides into concatemeric complexes as an approach to improvement of their cellular interaction.
Keywords: Abbreviations; DMEM; Dulbecco's modified Eagle's medium; PAAG; polyacrylamide gel; PAGE; polyacrylamide gel electrophoresisOligonucleotide complex; Oligonucleotide delivery; Cellular uptake; Antisense oligonucleotide
Polyplex-embedding in polyelectrolyte multilayers for gene delivery by Florent Meyer; Vincent Ball; Pierre Schaaf; Jean Claude Voegel; Joelle Ogier (pp. 419-422).
In this work, incorporation of plasmid DNA, pre-complexed with PEI, into polyelectrolyte multilayers has been studied to further develop platforms for local gene delivery. Polyplex embedding in synthetic and naturally degradable architectures was efficient for transfection of human hepato-cellular carcinoma cells.
Keywords: Polyelectrolyte multilayer; Gene delivery; Polyplex
Electropermeabilization, a physical method for the delivery of therapeutic molecules into cells by Marie-Pierre Rols (pp. 423-428).
Electropermeabilization designates the use of short high-voltage pulses to overcome the barrier of the cell membrane. A position-dependent reversible local membrane permeabilization is induced leading to an exchange of hydrophilic molecules across the membrane. This permeabilized state can be used to load cells with therapeutic molecules. In the case of small molecules, such as anticancer drugs, transfer occurs through simple diffusion. In the case of DNA, transfer occurs through a multi-step mechanism, a process that involves the electrophoretically driven association of the DNA molecule with the destabilised membrane and then its passage.
Keywords: Electric field; Electroporation; Electrotransfection; Electrochemotherapy; Electrogenotherapy