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  • 1.
    El-Andaloussi, S.
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Johansson, H.
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Magnusdottir, A.
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Järver, P.
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Lundberg, P.
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    TP10, a delivery vector for decoy oligonucleotides targeting the Myc protein2005In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 110, no 1, p. 189-201Article in journal (Refereed)
    Abstract [en]

    One approach to investigate gene function, by silencing the activity of certain proteins, is the usage of double stranded decoy oligodeoxynucleotides (ds decoy ODNs). Decoy, in this sense, is ds ODNs bearing the consensus binding sequence for a DNA-binding protein. This can be used in clinical settings to attenuate the effect of overexpressed transcription factors in tumor cells. We here choose to target the oncogenic protein Myc. Since oligonucleotides are poorly internalized to cells, a cell-penetrating peptide, TP10, was coupled to the Myc decoy, using two different strategies. Either TP10 was simply mixed with ds decoy ODNs forming complexes through non-covalent electrostatic interactions, or by having a nona-nucleotide overhang in one of the decoy strands, and adding a complementary PNA sequence coupled to an NLS sequence and TP10, which could hybridize to the Myc decoy.

    By using these strategies, uptake was significantly enhanced, especially with the co-incubation approach. Interestingly, various endocytosis inhibitors had no effect on the uptake pattern, suggesting that uptake of these complexes is not mediated via endocytosis. Finally, a decreased proliferative capacity was observed when treating the neuroblastoma cell line N2a with TP10–PNA conjugate hybridized to Myc decoy compared to naked Myc decoy and untreated cells. A dose-dependent decrease in proliferation was also observed in MCF-7 cells, when using both strategies. These results suggest an alternative way to efficiently deliver ds ODNs into cells using the cell-penetrating peptide TP10 and prevent tumor growth by targeting the oncogenic protein Myc.

  • 2.
    Ezzat, Kariem
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Zaghloul, Eman M.
    EL Andaloussi, Samir
    Lehto, Taavi
    Hilal, Ramy
    Magdy, Tarek
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Smith, Edvard C. I.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Solid formulation of cell-penetrating peptide nanoparticles with siRNA and their stability in simulated gastric conditions2012In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 162, no 1, p. 1-8Article in journal (Refereed)
    Abstract [en]

    Cell-penetrating peptides (CPPs) are short cationic peptides that have been extensively studied as drug delivery vehicles for proteins, nucleic acids and nanoparticles. However, the formulation of CPP-based therapeutics into different pharmaceutical formulations and their stability in relevant biological environments have not been given the same attention. Here, we show that a newly developed CPP, PepFect 14 (PF14), forms non-covalent nanocomplexes with short interfering RNA (siRNA), which are able to elicit efficient RNA-interference (RNAi) response in different cell-lines. RNAi effect was obtained at low siRNA doses with a unique kinetic profile. Furthermore, we utilized the solid dispersion technique to formulate PF14/siRNA nanocomplexes into solid formulations that were as active as the freshly prepared nanocomplexes in solution. Importantly, the freshly prepared nanocomplexes and solid formulations were stable after incubation with simulated gastric fluid having a pH of 1.2 and containing proteolytic enzymes. These results demonstrate the activity of PF14 in delivering and protecting siRNA in different pharmaceutical forms and biological environments.

  • 3. Freimann, Krista
    et al.
    Arukuusk, Piret
    Kurrikoff, Kaido
    Vasconcelos, Luís Daniel Ferreira
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Veiman, Kadi-Liis
    Uusna, Julia
    Margus, Helerin
    Garcia-Sosa, Alfonso T.
    Pooga, Margus
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Optimization of in vivo DNA delivery with NickFect peptide vectors2016In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 241, p. 135-143Article in journal (Refereed)
    Abstract [en]

    As the field of gene therapy progresses, an increasingly urgent need has arisen for efficient and non-toxic vectors for the in vivo delivery of nucleic acids. Cell-penetrating peptides (CPP) are very efficient transfection reagents in vitro, however, their application in vivo needs improvement. To enhance in vivo transfection we designed various CPPs based on previous knowledge of internalization studies and physiochemical properties of NickFect (NF) nanoparticles. We show that increment of the helicity of these Transportan10 analogues improves the transfection efficiency. We rationally design by modifying the net charge and the helicity of the CPP a novel amphipathic α-helical peptide NF55 for in vivo application. NF55 condenses DNA into stable nanoparticles that are resistant to protease degradation, promotes endosomal escape, and transfects the majority of cells in a large cell population. We demonstrate that NF55 mediates DNA delivery in vivo with gene induction efficiency that is comparable to commercial transfection reagents. In addition to gene induction in healthy mice, NF55/DNA nanoparticles showed promising tumor transfection in various mouse tumor models, including an intracranial glioblastoma model. The efficiency of NF55 to convey DNA specifically into tumor tissue increased even further after coupling a PEG2000 to the peptide via a disulphide-bond. Furthermore, a solid formulation of NF55/DNA displayed an excellent stability profile without additives or special storage conditions. Together, its high transfection efficacy and stability profile make NF55 an excellent vector for the delivery of DNA in vivo.

  • 4. Hassane, Fatouma Said
    et al.
    Abes, Rachida
    EL Andaloussi, Samir
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lehto, Taavi
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Sillard, Rannar
    Langel, Ulo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lebleu, Bernard
    Insights into the cellular trafficking of splice redirecting oligonucleotides complexed with chemically modified cell-penetrating peptides2011In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 153, no 2, p. 163-172Article in journal (Refereed)
    Abstract [en]

    Conjugates of cell-penetrating peptides (CPP) and splice redirecting oligonucleotides (ON) display clinical potential as attested by in vivo experimentation in murine models of Duchenne muscular dystrophy. However, micromolar concentrations of these conjugates are required to obtain biologically relevant responses as a consequence of extensive endosomal sequestration following endocytosis. Recent work from our group has demonstrated that appending stearic acid to CPPs increases their efficiency and that the inclusion of pH titrable entities leads to further improvement. Moreover, these modified CPPs form non covalent complexes with charged ON analogs or siRNAs, which allows decreasing the concentrations of ONs by nearly one log. These modified CPPs and the parent peptides have been compared here in the same in vitro model in terms of cell uptake, trafficking and splicing redirection activity. The increased splicing redirection activity of our modified CPPs cannot be explained by differences in cell uptake but rather by their enhanced ability to escape from endocytic vesicles. Accordingly, a clear correlation between membrane destabilizing activity and splicing redirection was observed using a liposome leakage assay. Studies of cellular trafficking for the most active PF6:ON complexes indicate uptake by clathrin-mediated endocytosis using either FACS cell uptake or a splicing redirection functional assay. Acidification of intracellular vesicles and membrane potential were found important for splicing redirection but not for cell uptake. These results do confirm that the increased potency of PF6:ON complexes is not due to the use of a non endocytic route of cell internalization as proposed for some CPPs.

  • 5.
    Kilk, Kalle
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    EL Andaloussi, Samir
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Järver, Peter
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Meikas, Anne
    Valkna, Andres
    Bartfai, Tamas
    Kogerman, Priit
    Metsis, Madis
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Evaluation of transportan 10 in PEI mediated plasmid delivery assay2005In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 103, no 2, p. 511-23Article in journal (Refereed)
    Abstract [en]

    Cell-penetrating peptides (CPPs) are novel high-capacity delivery vectors for different bioactive cargoes. We have evaluated the CPP transportan 10 (TP10) as a delivery vector in different in vitro plasmid delivery assays. Tested methods include: TP10 crosslinked to a plasmid via a peptide nucleic acid (PNA) oligomer, TP10 conjugation with polyethyleneimine (PEI), and addition of unconjugated TP10 to standard PEI transfection assay. We found that without additional DNA condensing agents, TP10 has poor transfection abilities. However, the presence of TP10 increases the transfection efficiency several folds compared to PEI alone. At as low concentrations as 0.6 nM, TP10–PNA constructs were found to enhance plasmid delivery up to 3.7-fold in Neuro-2a cells. Interestingly, the transfection efficiency was most significant at low PEI concentrations, allowing reduced PEI concentration without loss of gene delivery. No increase in cytotoxicity due to TP10 was observed and the uptake mechanism was determined to be endocytosis, as previously reported for PEI mediated transfection. In conclusion, TP10 can enhance PEI mediated transfection at relatively low concentrations and may help to develop future gene delivery systems with reduced toxicity.

  • 6.
    Lehto, Taavi
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Abes, Rachida
    Oskolkov, Nikita
    Suhorutšenko, Julia
    Copolovici, Dana-Maria
    Mäger, Imre
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Viola, Joana R.
    Simonson, Oscar E.
    Ezzat, Kariem
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Guterstam, Peter
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Eriste, Elo
    Smith, Edvard
    Lebleu, Bernard
    EL Andaloussi, Samir
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Delivery of nucleic acids with a stearylated (RxR)4 peptide using a non-covalent co-incubation strategy2010In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 141, no 1, p. 42-51Article in journal (Refereed)
    Abstract [en]

    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.

  • 7.
    Lindberg, Staffan
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Regberg, Jakob
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Eriksson, Jonas
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Helmfors, Henrik
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Muñoz-Alarcón, Andrés
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Srimanee, Artita
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Figueroa, Ricardo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hallberg, Einar
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Ezzat, Kariem
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    A convergent uptake route for peptide- and polymer-based nucleotide delivery systems2015In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 206, p. 58-66Article in journal (Refereed)
    Abstract [en]

    Cell-penetrating peptides (CPPs) have been used as vehicles to deliver various cargos into cells and are promising as tools to deliver therapeutic biomolecules such as oligonucleotides both in vitro and in vivo. CPPs are positively charged and it is believed that CPPs deliver their cargo in a receptor-independent manner by interactingwith the negatively charged plasmamembrane and thereby inducing endocytosis. In this study we examine the mechanism of uptake of several different, well known, CPPs that form complexes with oligonucleotides.We show that these CPP:oligonucleotide complexes are negatively charged in transfection-media and their uptake is mediated by class A scavenger receptors (SCARA). These receptors are known to promiscuously bind to, and mediate uptake of poly-anionic macromolecules. Uptake of CPP:oligonucleotide complexes was abolished using pharmacological SCARA inhibitors as well as siRNA-mediated knockdown of SCARA. Additionally, uptake of CPP:oligonucleotide was significantly increased by transiently overexpressing SCARA. Furthermore, SCARA inhibitors also blocked internalization of cationic polymer:oligonucleotide complexes.Our results demonstrate that the previous held belief that CPPs act receptor independently does not hold true for CPP:oligonucleotide complexes, as scavenger receptor class A (SCARA) mediates the uptake of all the examined CPP:oligonucleotide complexes in this study.

  • 8. Lundborg, Magnus
    et al.
    Wennberg, Christian L.
    Narangifard, Ali
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Norlen, Lars
    Predicting drug permeability through skin using molecular dynamics simulation2018In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 283, p. 269-279Article in journal (Refereed)
    Abstract [en]

    Understanding and predicting permeability of compounds through skin is of interest for transdermal delivery of drugs and for toxicity predictions of chemicals. We show, using a new atomistic molecular dynamics model of the skin's barrier structure, itself validated against near-native cryo-electron microscopy data from human skin, that skin permeability to the reference compounds benzene, DMSO (dimethyl sulfoxide), ethanol, codeine, naproxen, nicotine, testosterone and water can be predicted. The permeability results were validated against skin permeability data in the literature. We have investigated the relation between skin barrier molecular organization and permeability using atomistic molecular dynamics simulation. Furthermore, it is shown that the calculated mechanism of action differs between the five skin penetration enhancers Azone, DMSO, oleic acid, stearic acid and water. The permeability enhancing effect of a given penetration enhancer depends on the permeating compound and on the concentration of penetration enhancer inside the skin's barrier structure. The presented method may open the door for computer based screening of the permeation of drugs and toxic compounds through skin.

  • 9.
    Mäe, Maarja
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    EL Andaloussi, Samir
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lundin, Per
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Oskolkov, Nikita
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Johansson, Henrik J.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Guterstam, Peter
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    A stearylated CPP for delivery of splice correcting oligonucleotides using a non-covalent co-incubation strategy2009In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 134, no 3, p. 221-227Article in journal (Refereed)
    Abstract [en]

    Aberrations in splicing patterns play a significant role in several diseases, and splice correction, together with other forms of gene regulation, is consequently an emerging therapeutic target. In order to achieve successful oligonucleotide transfection, efficient delivery vectors are generally necessary. In this study we present one such vector, the chemically modified cell-penetrating peptide (CPP) TP10, for efficient delivery of a splice-correcting 2'-OMe RNA oligonucleotide. Utilizing a functional splice correction assay, we assessed the transfection efficiency of non-covalent complexes of oligonucleotides and stearylated or cysteamidated CPPs. Stearylation of the CPPs Arg9 and penetratin, as well as cysteamidation of MPG and TP10, did not improve transfection, whereas the presence of an N-terminal stearyl group on TP10 improved delivery efficiency remarkably compared to the unmodified peptide. The splice correction levels observed with stearyl-TP10 are in fact in parity with the effects seen with the commercially available transfection agent Lipofectamine (TM) 2000. However, the inherent toxicity associated with cationic lipid-based transfections can be completely eliminated when using the stearylated TP10, making this vector highly promising for non-covalent delivery of negatively charged oligonucleotides.

  • 10. Pae, Janely
    et al.
    Saeaelik, Pille
    Liivamaegi, Laura
    Lubenets, Dmitri
    Arukuusk, Piret
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Pooga, Margus
    Translocation of cell-penetrating peptides across the plasma membrane is controlled by cholesterol and microenvironment created by membranous proteins2014In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 192, p. 103-113Article in journal (Refereed)
    Abstract [en]

    Despite the extensive research in the field of CPPs' cell entry the exact mechanisms underlying their cellular uptake and the role of involved cell surface molecules in the internalization process have remained controversial. The present study focused on the interactions between CPPs and plasma membrane compounds using giant plasma membrane vesicles (GPMVs). GPMVs have shown to be a suitable model to study the translocation of CPPs across the plasma membrane in conditions lacking endocytosis. Our results show that higher cholesterol content and tighter packing of membrane predominantly reduce the accumulation of transportan, TP10 and model amphipathic peptide (MAP) in vesicles, indicating that the internalization of CPPs takes place preferentially via the more dynamic membrane regions. The partial digestion of membrane proteins from GPMVs' surface, on the other hand, drastically reduced the accumulation of nona-arginine and Tat peptide into vesicles, suggesting that proteins play a crucial role in the uptake of arginine-rich CPPs.

  • 11.
    Palm-Apergi, Caroline
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    A new rapid cell-penetrating peptide based strategy to produce bacterial ghosts for plasmid delivery2008In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 132, no 1, p. 49-54Article in journal (Refereed)
    Abstract [en]

    The production of bacterial ghosts involves the lysis gene E plasmid in order to lyse and empty the bacteria of their cytoplasmic contents. After lysis the ghosts can either be loaded with new desired DNA and used for delivery to mammalian cells or used in vaccination. Cell-penetrating peptides have been used as delivery vehicles of drugs and oligonucleotides. Although many of them show low toxicity they have been compared to antimicrobial peptides involved in innate immunity. Recently we showed that cell-penetrating peptides also could be antimicrobial. In this study we take advantage of the antimicrobial effect of one cell-penetrating peptide, namely MAP, which is a model amphipathic peptide and treat bacteria with the peptide to produce bacterial ghosts. This new peptide based strategy is not dependent on the lysis gene E plasmid thus; several tiresome steps are removed in the production of ghosts. In addition the ghosts can be preloaded with a desired plasmid or DNA further removing time consuming reprocessing steps. To our knowledge this is the first study that uses a cell-penetrating peptide based strategy to produce bacterial ghosts to be used in plasmid delivery.

  • 12. Räägel, Helin
    et al.
    Säälik, Pille
    Hansen, Mats
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Pooga, Margus
    CPP-protein constructs induce a population of non-acidic vesicles during trafficking through the endo-lysosomal pathway2009In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 139, no 2, p. 108-117Article in journal (Refereed)
    Abstract [en]

    The major limitation in the application of bioactive molecules is their low permeation across plasma membrane. Effective transporters — cell-penetrating peptides (CPPs) — are utilized to enhance uptake of various cargo upon attachment to its sequences. Still, information about relevance of different endocytic routes during CPP–cargo internalization is ambiguous and underlying mechanism(s) of intracellular trafficking is even less understood. We first defined involvement of recycling pathway in trafficking of 3 different CPPs — transportan, oligoarginine and Tat — complexed to avidin-TexasRed in Cos-7 cells in relation to trans-Golgi network spatially constraining recycling endosomes. By confocal microscopy, only a negligible fraction of complexes-containing vesicles were found inside trans-Golgi ring suggesting its marginal role in CPP-mediated delivery. Secondly, we characterized engagement of endo-lysosomal pathway to assess acidity of complexes-containing vesicles. CPPs induced 3 different populations of complexes-containing vesicles which size and proportion depended on CPP, time and concentration. In time, more complexes were targeted to low-pH structures. However, a population of complexes-containing vesicles was observed to retain rather neutral pH. Induction of vesicles with non-acidic pH generated i.e. by caveolin-dependent endocytosis or by CPPs themselves during intracellular trafficking could be the key step in inducement of escape of complexes from endosomal structures, a limiting step in effective cargo delivery by CPPs.

  • 13. Saalik, Pille
    et al.
    Niinep, Aira
    Pae, Janely
    Hansen, Mats
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lubenets, Dmitri
    Langel, Ulo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Pooga, Margus
    Penetration without cells: Membrane translocation of cell-penetrating peptides in the model giant plasma membrane vesicles2011In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 153, no 2, p. 117-125Article in journal (Refereed)
    Abstract [en]

    The cellular internalization of cell-penetrating peptides (CPPs) is proposed to take place by both endocytic processes and by a direct translocation across the plasma membrane. So far only scarce data is available about what determines the choice between the two uptake routes, or the proportion of used pathways when both are active simultaneously. Furthermore, the mechanism(s) of membrane penetration by peptides is itself still a matter of debate. We have introduced the giant plasma membrane vesicles (GPNIVs) to study the interaction of six well-described CPPs (fluorescently labeled nona-arginine, Tat peptide, Penetratin, MAP, Transportan and TP10) in a model system of native plasma membrane without the interference of endocytic processes. The membranes of GPMVs are shown to segregate into liquid-ordered and liquid-disordered phases at low temperatures and we demonstrate here by confocal microscopy that amphipathic CPPs preferentially associate with liquid-disordered membrane areas. Moreover, all tested CPPs accumulate into the lumen of GPMVs both at ambient and low temperature. The uncharged control peptide and dextran, in contrary, do not translocate from the medium into the lumen of vesicles. The absence of energy-dependent cellular processes and the impermeability to hydrophilic macromolecules makes the GPMVs a useful model to study the translocation of CPPs across the plasma membrane in conditions lacking endocyrosis.

  • 14. Urgard, Egon
    et al.
    Lorents, Annely
    Klaas, Mariliis
    Padari, Kart
    Viil, Janeli
    Runnel, Toomas
    Langel, Kent
    Kingo, Kulli
    Tkaczyk, Eric
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Maimets, Toivo
    Jaks, Viljar
    Pooga, Margus
    Rebane, Ana
    Pre-administration of PepFect6-microRNA-146a nanocomplexes inhibits inflammatory responses in keratinocytes and in a mouse model of irritant contact dermatitis2016In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 235, p. 195-204Article in journal (Refereed)
    Abstract [en]

    The skin is a difficult to access tissue for efficient delivery of large and/or chargedmacromolecules, including therapeutic DNA and RNA oligonucleotides. Cell-penetrating peptide PepFect6 (PF6) has been shown to be suitable transport vehicle for siRNAs in cell culture and systemically in vivo in mice. MiR-146a is known as anti-inflammatory miRNA that inhibits multiple factors fromthe nuclear factor (NF)-kappa B pathway in various cell types, including keratinocytes. In this study, PF6 was shown to form unimodal nanocomplexes with miR-146a mimic that entered into human primary keratinocytes, where miR-146a inhibited the expression of its direct targets fromthe NF-kappa B pathway and the genes known to be activated by NF-kappa B, C-C motif ligand (CCL)5 and interleukin (IL)-8. The transfection of miR-146a mimic with PF6 was more efficient in sub-confluent keratinocyte cultures, affected keratinocyte proliferation less and had similar effect on cell viability when compared with a lipid based agent. Subcutaneous pre-administration of PF6-miR-146a nanocomplexes attenuated ear-swelling and reduced the expression of pro-inflammatory cytokines and chemokines IL-6, CCL11, CCL24 and C-X-C motif ligand 1 (CXCL1) in a mouse model of irritant contact dermatitis. Our data demonstrates that PF6-miR-146a nanoparticles might have potential in the development of therapeutics to target inflammatory skin diseases.

  • 15. Veiman, Kadi-Liis
    et al.
    Kuennapuu, Kadri
    Lehto, Tönis
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Kiisholts, Kristina
    Paern, Kalle
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Kurrikoff, Kaido
    PEG shielded MMP sensitive CPPs for efficient and tumor specific gene delivery in vivo2015In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 209, p. 238-247Article in journal (Refereed)
    Abstract [en]

    Gene therapy has great potential to treat a range of different diseases, such as cancer. For that therapeutic gene can be inserted into a plasmid vector and delivered specifically to tumor cells. The most frequently used applications utilize lipoplex and polyplex approaches where DNA is non-covalently condensed into nanoparticles. However, lack of in vivo efficacy is the major concern that hinders translation of such gene therapeutic applications into clinics. In this work we introduce a novel method for in vivo delivery of plasmid DNA (pDNA) and efficient tumor-specific gene induction using intravenous (i.v) administration route. To achieve this, we utilize a cell penetrating peptide (CPP), PepFect14 (PF14), double functionalized with polyethylene glycol (PEG) and a matrix metalloprotease (MMP) substrate. We show that this delivery vector effectively forms nanoparticles, where the condensed CPP and pDNA are shielded by the PEG, in an MMP-reversible manner. Administration of the complexes results in efficient induction of gene expression specifically in tumors, avoiding normal tissues. This strategy is a potent gene delivery platform that can be used for tumor-specific induction of a therapeutic gene.

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