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  • 1. Arukuusk, Piret
    et al.
    Pärnaste, Ly
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. Tartu University, Estonia.
    PepFects and NickFects for the Intracellular Delivery of Nucleic Acids2015In: Cell-Penetrating Peptides: Methods and Protocols / [ed] Ülo Langel, New York: Springer, 2015, Vol. 1324, p. 303-315Chapter in book (Refereed)
    Abstract [en]

    Nucleic acids can be utilized in gene therapy to restore, alter, or silence gene functions. In order to reveal the biological activity nucleic acids have to reach their intracellular targets by passing through the plasma membrane, which is impermeable for these large and negatively charged molecules. Cell-penetrating peptides (CPPs) condense nucleic acids into nanoparticles using non-covalent complexation strategy and mediate their delivery into the cell, whereas the physicochemical parameters of the nanoparticles determine the interactions with the membranes, uptake mechanism, and subsequent intracellular fate. The nanoparticles are mostly internalized by endocytosis that leads to the entrapment of them in endosomal vesicles. Therefore design of new CPPs that are applicable for non-covalent complex formation strategy and harness endosomolytic properties is highly vital. Here we demonstrate that PepFects and NickFects are efficient vectors for the intracellular delivery of various nucleic acids.This chapter describes how to form CPP/pDNA nanoparticles, evaluate stable nanoparticles formation, and assess gene delivery efficacy.

  • 2.
    Dowaidar, Moataz
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Abdelhamid, Hani Nasser
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Freimann, Krista
    Kurrikof, Kaido
    Zou, Xiaodong
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Magnetic Nanoparticle Assisted Self-assembly of Cell Penetrating Peptides-Oligonucleotides Complexes for Gene Delivery2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 9159Article in journal (Refereed)
    Abstract [en]

    Magnetic nanoparticles (MNPs, Fe3O4) incorporated into the complexes of cell penetrating peptides (CPPs)-oligonucleotides (ONs) promoted the cell transfection for plasmid transfection, splice correction, and gene silencing efficiencies. Six types of cell penetrating peptides (CPPs; PeptFect220 (denoted PF220), PF221, PF222, PF223, PF224 and PF14) and three types of gene therapeutic agents (plasmid (pGL3), splicing correcting oligonucleotides (SCO), and small interfering RNA (siRNA) were investigated. Magnetic nanoparticles incorporated into the complexes of CPPs-pGL3, CPPs-SCO, and CPPs-siRNA showed high cell biocompatibility and efficiently transfected the investigated cells with pGL3, SCO, and siRNA, respectively. Gene transfer vectors formed among PF14, SCO, and MNPs (PF14-SCO-MNPs) showed a superior transfection efficiency (up to 4-fold) compared to the noncovalent PF14-SCO complex, which was previously reported with a higher efficiency compared to commercial vector called Lipofectamine™2000. The high transfection efficiency of the new complexes (CPPs-SCO-MNPs) may be attributed to the morphology, low cytotoxicity, and the synergistic effect of MNPs and CPPs. PF14-pDNA-MNPs is an efficient complex for in vivo gene delivery upon systemic administration. The conjugation of CPPs-ONs with inorganic magnetic nanoparticles (Fe3O4) may open new venues for selective and efficient gene therapy.

  • 3.
    Dowaidar, Moataz
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Abdelhamid, Hani Nasser
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK). Assuit University, Egypt.
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Zou, Xiaodong
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Chitosan enhances gene delivery of oligonucleotide complexes with magnetic nanoparticles–cell-penetrating peptide2018In: Journal of biomaterials applications, ISSN 0885-3282, E-ISSN 1530-8022, Vol. 33, no 3, p. 392-401Article in journal (Refereed)
    Abstract [en]

    Gene-based therapies, including the delivery of oligonucleotides, offer promising methods for the treatment of cancer cells. However, they have various limitations including low efficiency. Herein, cell-penetrating peptides (CPPs)-conjugated chitosan-modified iron oxide magnetic nanoparticles (CPPs-CTS@MNPs) with high biocompatibility as well as high efficiency were tested for the delivery of oligonucleotides such as plasmid pGL3, splice correction oligonucleotides, and small-interfering RNA. A biocompatible nanocomposite, in which CTS@MNPs was incorporated in non-covalent complex with CPPs-oligonucleotide, is developed. Modifying the surface of magnetic nanoparticles with cationic chitosan-modified iron oxide improved the performance of magnetic nanoparticles-CPPs for oligonucleotide delivery. CPPs-CTS@MNPs complexes enhance oligonucleotide transfection compared to CPPs@MNPs or CPPs. The hydrophilic character of CTS@MNPs improves complexation with plasmid pGL3, splice correction oligonucleotides, and small-interfering RNA payload, which consequently resulted in not only strengthening the colloidal stability of the constructed complex but also improving their biocompatibility. Transfection using PF14-splice correction oligonucleotides-CTS@MNPs showed sixfold increase of the transfection compared to splice correction oligonucleotides-PF14 that showed higher transfection than the commercially available lipid-based vector Lipofectamine™ 2000. Nanoscaled CPPs-CTS@MNPs comprise a new family of biomaterials that can circumvent some of the limitations of CPPs or magnetic nanoparticles.

  • 4.
    Dowaidar, Moataz
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Abdelhamid, Hani Nasser
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Zou, Xiaodong
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Graphene oxide nanosheets in complex with cell penetrating peptides for oligonucleotides delivery2017In: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1861, no 9, p. 2334-2341Article in journal (Refereed)
    Abstract [en]

    A new strategy for gene transfection using the nanocarrier of cell penetrating peptides (CPPs; PepFect14 (PF14) or PepFect14 (PF14) (PF221)) in complex with graphene oxide (GO) is reported. GO complexed with CPPs and plasmid (pGL3), splice correction oligonucleotides (SCO) or small interfering RNA (siRNA) are performed. Data show adsorption of CPPs and oligonucleotides on the top of the graphenic lamellar without any observed change of the particle size of GO. GO mitigates the cytotoxicity of CPPs and improves the material biocompatibility. Complexes of GO-pGL3-CPPs (CPPs; PF14 or PF221) offer 2.1–2.5 fold increase of the cell transfection compared to pGL3-CPPs (CPPs; PF14 or PF221). GO-SCO-PF14 assemblies effectively transfect the cells with an increase of > 10–25 fold compared to the transfection using PF14. The concentration of GO plays a significant role in the material nanotoxicity and the transfection efficiency. The results open a new horizon in the gene treatment using CPPs and offer a simple strategy for further investigations.

  • 5.
    Dowaidar, Moataz
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gestin, Maxime
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Cerrato, Carmine Pasquale
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Jafferali, Mohammed Hakim
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Margus, Helerin
    Kivistik, Paula Ann
    Ezzat, Kariem
    Hallberg, Einar
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Pooga, Margus
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Role of autophagy in cell-penetrating peptide transfection model2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 12635Article in journal (Refereed)
    Abstract [en]

    Cell-penetrating peptides (CPPs) uptake mechanism is still in need of more clarification to have a better understanding of their action in the mediation of oligonucleotide transfection. In this study, the effect on early events (1 h treatment) in transfection by PepFect14 (PF14), with or without oligonucleotide cargo on gene expression, in HeLa cells, have been investigated. The RNA expression profile was characterized by RNA sequencing and confirmed by qPCR analysis. The gene regulations were then related to the biological processes by the study of signaling pathways that showed the induction of autophagy-related genes in early transfection. A ligand library interfering with the detected intracellular pathways showed concentration-dependent effects on the transfection efficiency of splice correction oligonucleotide complexed with PepFect14, proving that the autophagy process is induced upon the uptake of complexes. Finally, the autophagy induction and colocalization with autophagosomes have been confirmed by confocal microscopy and transmission electron microscopy. We conclude that autophagy, an inherent cellular response process, is triggered by the cellular uptake of CPP-based transfection system. This finding opens novel possibilities to use autophagy modifiers in future gene therapy.

  • 6.
    Dowaidar, Moataz
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gestin, Maxime
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Cerrato, Carmine Pasquale
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Margus, Helerin
    Kivistik, Paula Ann
    Pooga, Margus
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Role of autophagy in PepFect14 transfection2017Manuscript (preprint) (Other academic)
    Abstract [en]

    Cell-penetrating peptides (CPP) uptake mechanism is still to be clarified to have a better understanding of their action in the mediation of oligonucleotide transfection. In this study, the effect on early events (1 h treatment) in transfection by Pepfect 14, with or without oligonucleotide cargo on gene expression, on HeLa cells, have been investigated. The RNA expression profile was characterized by RNA sequencing and confirmed with qPCR analysis. The gene regulations were then related to the biological process by the study of signaling pathways that showed the induction of autophagy-related genes in early transfection. A ligand library interfering with the detected intracellular pathways showed concentration-dependent effects on the transfection of splice correction oligonucleotide complexed with Pepfect 14 confirming the induction of autophagy process by the uptake of complexes. Finally, colocalization of nucleic acid cargo and autophagosomes, as well as the autophagosome production induced by the treatment, have been shown by confocal microscopy and transmission electron microscopy. We conclude that autophagy is an important response process triggered by the cellular uptake of CPP-based transfection system. This conclusion opens a possibility to use autophagy modifiers in future gene therapy.

  • 7.
    Dowaidar, Moataz
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Regberg, Jakob
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Dobchev, Dimitar A.
    Lehto, Tõnis
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Karelson, Mati
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Refinement of a Quantitative Structure–Activity Relationship Model for Prediction of Cell-Penetrating Peptide Based Transfection Systems2017In: International Journal of Peptide Research and Therapeutics, E-ISSN 1573-3904, Vol. 23, no 1, p. 91-100Article in journal (Refereed)
    Abstract [en]

    Cell-penetrating peptide (CPP) based transfection systems (PBTS) are a promising class of drug delivery vectors. CPPs are short mainly cationic peptides capable of delivering cell non-permeant cargo to the interior of the cell. Some CPPs have the ability to form non-covalent complexes with oligonucleotides for gene therapy applications. In this study, we use quantitative structure–activity relationships (QSAR), a statistical method based on regression data analysis. Here, a fragment QSAR (FQSAR) model is developed to predict new peptides based on standard alpha helical conformers and Assisted Model Building with Energy Refinement molecular mechanics simulations of previous peptides. These new peptides were examined for plasmid transfection efficiency and compared with their predicted biological activity. The best predicted peptides were capable of achieving plasmid transfection with significant improvement compared to the previous generation of peptides. Our results demonstrate that FQSAR model refinement is an efficient method for optimizing PBTS for improved biological activity.

  • 8.
    Erlandsson, Mikael
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Metallic zinc reduction of disulfide bonds between cysteine residues in peptides and proteins2005In: International Journal of Peptide Research and Therapeutics, ISSN 1573-3149, Vol. 11, no 4, p. 261-265Article in journal (Refereed)
    Abstract [en]

    The use of powdered metallic zinc in acidic solution for the reduction of disulfide bonds in peptides and proteins has been investigated. The method has several advantages over the traditional mereapto based reducing methods currently used; the reducing agent is readily available and inexpensive; reduction can be performed in weakly acidic solutions of water and/or acetonitrile; work up simply consists of a centrifugation step followed by pipeting the supernatant from the metal pellet, thereby greatly diminishing the risk of reoxidation as a more elaborate work up procedure could result in. As no mercapto compounds are added, there is no risk that the reducing agent will interfere in subsequent modification of the thiol functionality. Disulfides in a model peptide are reduced within 5 min in any mixture of water/acetonitrile containing 1% TFA, all disulfides in insulin is reduced within I h in any mixture of water/acetonitrile containing 5% acetic acid. To stress the convenience of the metallic zinc reduction method, the resulting thiol compound was subjected to two commonly employed reactions in peptide chemistry: Cys(Npys) directed disulfide formation (70% yield) and native chemical ligation between the reduced model peptide and Boc-Ala-p-metylthiobenzyl ester (65% yield of the ligation product plus disulfide formation between Cys and p-thiocresol).

  • 9.
    Ezzat, Kariem
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    EL Andaloussi, Samir
    Zaghloul, Eman M.
    Lehto, Taavi
    Lindberg, Staffan
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Moreno, Pedro M. D.
    Viola, Joana R.
    Magdy, Tarek
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Abdo, Rania
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Guterstam, Peter
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Sillard, Rannar
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hammond, Suzan M.
    Wood, Matthew J. A.
    Arzumanov, Andrey A.
    Gait, Michael J.
    Smith, C. I. Edvard
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    PepFect 14, a novel cell-penetrating peptide for oligonucleotide delivery in solution and as solid formulation2011In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, no 12, p. 5284-5298Article in journal (Refereed)
    Abstract [en]

    Numerous human genetic diseases are caused by mutations that give rise to aberrant alternative splicing. Recently, several of these debilitating disorders have been shown to be amenable for splice-correcting oligonucleotides (SCOs) that modify splicing patterns and restore the phenotype in experimental models. However, translational approaches are required to transform SCOs into usable drug products. In this study, we present a new cell-penetrating peptide, PepFect14 (PF14), which efficiently delivers SCOs to different cell models including HeLa pLuc705 and mdx mouse myotubes; a cell culture model of Duchenne's muscular dystrophy (DMD). Non-covalent PF14-SCO nanocomplexes induce splice-correction at rates higher than the commercially available lipid-based vector Lipofectamine™ 2000 (LF2000) and remain active in the presence of serum. Furthermore, we demonstrate the feasibility of incorporating this delivery system into solid formulations that could be suitable for several therapeutic applications. Solid dispersion technique is utilized and the formed solid formulations are as active as the freshly prepared nanocomplexes in solution even when stored at an elevated temperatures for several weeks. In contrast, LF2000 drastically loses activity after being subjected to same procedure. This shows that using PF14 is a very promising translational approach for the delivery of SCOs in different pharmaceutical forms.

  • 10.
    Holm, Tina
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Netzereab, Semharai
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Hansen, Mats
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Uptake of cell-penetrating peptides in yeasts2005In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 579, no 23, p. 5217-5222Article in journal (Refereed)
    Abstract [en]

    The uptake of different cell-penetrating peptides (CPPs) in two yeast species, Saccharomyces cerevisiae and Candida albicans, was studied using fluorescence HPLC-analyses of cell content. Comparison of the ability of penetratin, pVEC and (KFF)(3)K to traverse the yeast cell envelope shows that the cellular uptake of the peptides varies widely. Moreover, the intracellular degradation of the CPPs studied varies from complete stability to complete degradation. We show that intracellular degradation into membrane impermeable products can significantly contribute to the fluorescence signal. pVEC displayed highest internalizing capacity, and considering its stability in both yeast species, it is an attractive candidate for further studies.

  • 11.
    Hällbrink, Mattias
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Karelson, Mati
    Prediction of Cell-Penetrating Peptides2015In: Cell-Penetrating Peptides: Methods and Protocols / [ed] Ülo Langel, Springer-Verlag New York, 2015, p. 39-58Chapter in book (Refereed)
    Abstract [en]

    The in silico methods for the prediction of the cell-penetrating peptides are reviewed. Those include the multivariate statistical methods, machine-learning methods such as the artificial neural networks and support vector machines, and molecular modeling techniques including molecular docking and molecular dynamics.

    The applicability of the methods is demonstrated on the basis of the exemplary cases from the literature.

  • 12.
    Hällbrink, Mattias
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Prediction of cell-penetrating peptides2007In: Handbook of cell-penetrating peptides / [ed] Ülo Langel, Boca Raton: CRC Press, 2007, 2, p. 77-85Chapter in book (Refereed)
  • 13.
    Järver, Peter
    et al.
    Medical Research Council , Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, United Kingdom.
    Zaghloul, Eman M
    Department of Laboratory Medicine, Karolinska Institute, Karolinska University Hospital, Huddinge, Sweden.
    Arzumanov, Andrey A
    Medical Research Council, Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, United Kingdom.
    Saleh, Amer F
    Medical Research Council, Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, United Kingdom.
    McClorey, Graham
    Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom.
    Hammond, Suzan M
    Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom.
    Hällbrink, Mattias
    Department of Laboratory Medicine, Karolinska Institute, Karolinska University Hospital, Huddinge, Sweden.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Smith, C I Edvard
    Department of Laboratory Medicine, Karolinska Institute, Karolinska University Hospital, Huddinge, Sweden.
    Wood, Matthew J A
    Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom.
    Gait, Michael J
    Medical Research Council, Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, United Kingdom.
    El Andaloussi, Samir
    Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom.
    Peptide nanoparticle delivery of charge-neutral splice-switching morpholino oligonucleotides.2015In: Nucleic Acid Therapeutics, ISSN 2159-3337, E-ISSN 2159-3345, Vol. 25, no 2, p. 65-77Article in journal (Refereed)
    Abstract [en]

    Oligonucleotide analogs have provided novel therapeutics targeting various disorders. However, their poor cellular uptake remains a major obstacle for their clinical development. Negatively charged oligonucleotides, such as 2'-O-Methyl RNA and locked nucleic acids have in recent years been delivered successfully into cells through complex formation with cationic polymers, peptides, liposomes, or similar nanoparticle delivery systems. However, due to the lack of electrostatic interactions, this promising delivery method has been unsuccessful to date using charge-neutral oligonucleotide analogs. We show here that lipid-functionalized cell-penetrating peptides can be efficiently exploited for cellular transfection of the charge-neutral oligonucleotide analog phosphorodiamidate morpholino. The lipopeptides form complexes with splice-switching phosphorodiamidate morpholino oligonucleotide and can be delivered into clinically relevant cell lines that are otherwise difficult to transfect while retaining biological activity. To our knowledge, this is the first study to show delivery through complex formation of biologically active charge-neutral oligonucleotides by cationic peptides.

  • 14.
    Lehto, Tõnis
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Vasconcelos, Luis
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Margus, Helerin
    Figueroa, Ricardo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Pooga, Margus
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Saturated Fatty Acid Analogues of Cell-Penetrating Peptide PepFect14: Role of Fatty Acid Modification in Complexation and Delivery of Splice-Correcting Oligonucleotides2017In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 28, no 3, p. 782-792Article in journal (Refereed)
    Abstract [en]

    Modifying cell-penetrating peptides (CPPs) with fatty acids has long been used to improve peptide-mediated nucleic acid delivery. In this study we have revisited this phenomenon with a systematic approach where we developed a structure activity relationship to describe the role of the acyl chain length in the transfection process. For that we took a well studied CPP, PepFectl4, as the basis and varied its N-terminal acyl chain length from 2 to 22 carbons. To evaluate the delivery efficiency, the peptides were noncovalently complexed with a splice-correcting oligonucleotide (SCO) and tested in HeLa pLuc705 reporter cell line. Our results demonstrate that biological splice-correction activity emerges from acyl chain of 12 carbons and increases linearly with each additional carbon. To assess the underlying factors regarding how the transfection efficacy of these complexes is dependent on hydrophobicity, we used an array of different methods. For the functionally active peptides (C12-22) there was no apparent difference in their physicochemical properties, including complex formation efficiency, hydrodynamic size, and zeta potential. Moreover, membrane activity studies with peptides and their complexes with SCOs confirmed that the toxicity of the complexes at higher molar ratios is mainly caused by the free fraction of the peptide which is not incorporated into the peptide/oligonucleotide complexes. Finally, we show that the increase in splice-correcting activity correlates with the ability of the complexes to associate with the cells. Collectively these studies lay the ground work for how to design highly efficient CPPs and how to optimize their oligonucleotide complexes for lowest toxicity without losing efficiency.

  • 15.
    Lindgren, Maria
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Rosenthal-Aizman, Katri
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Saar, Külliki
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Eiríksdóttir, Emelía
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Jiang, Yang
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Sassian, Meeri
    Östlund, Pernilla
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Overcoming methotrexate resistance in breast cancer tumour cells by the use of a new cell-penetrating peptide2006In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 71, no 4, p. 416-425Article in journal (Refereed)
    Abstract [en]

    Resistance to chemotherapy limits the effectiveness of anti-cancer drug treatment. Here, we present a new approach to overcome the setback of drug resistance by designing a conjugate of a cell-penetrating peptide and the cytostatic agent methotrexate (MTX). Two different peptides, YTA2 and YTA4, were designed and their intracellular delivery efficiency was characterized by fluorescence microscopy and quantified by fluorometry. MTX was conjugated to the transport peptides and the ability of the peptide–MTX conjugates to inhibit dihydrofolate reductase, the target enzyme of MTX, was found to be 15 and 20 times less potent than MTX. In addition, in vitro studies were performed in a drug resistant cell model using the 100-fold MTX resistant breast cancer cells MDA-MB-231. At a concentration of 1 mM, the peptide–MTX conjugates were shown to overcome MTX resistance and kill the cells more efficiently than MTX alone. Estimated EC50’s were determined for MTX, MTXYTA2 and YTA2 to be 18.5, 3.8 and 20 µM, respectively. In summary, cell-penetrating peptide conjugation of MTX is a new way of increasing delivery, and thereby, the potency of already well-characterized therapeutic molecules into drug resistant tumour cells.

  • 16. Lorents, Annely
    et al.
    Kodavali, Praveen Kumar
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Oskolkov, Nikita
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Pooga, Margus
    Cell-penetrating Peptides Split into Two Groups Based on Modulation of Intracellular Calcium Concentration2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 20, p. 16880-16889Article in journal (Refereed)
    Abstract [en]

    Cell-penetrating peptides (CPPs) promote the uptake of different cargo molecules, e.g. therapeutic compounds, making the harnessing of CPPs a promising strategy for drug design and delivery. However, the internalization mechanisms of CPPs are still under discussion, and it is not clear how cells compensate the disturbances induced by peptides in the plasma membrane. In this study, we demonstrate that the uptake of various CPPs enhances the intracellular Ca2+ levels in Jurkat and HeLa cells. The elevated Ca2+ concentration in turn triggers plasma membrane blebbing, lysosomal exocytosis, and membrane repair response. Our results indicate that CPPs split into two major classes: (i) amphipathic CPPs that modulate the plasma membrane integrity inducing influx of Ca2+ and activating downstream responses starting from low concentrations; (ii) non-amphipathic CPPs that do not evoke changes at relevant concentrations. Triggering of the membrane repair response may help cells to replace distorted plasma membrane regions and cells can recover from the influx of Ca2+ if its level is not drastically elevated.

  • 17.
    Lundberg, Pontus
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Magzoub, Mazin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lindberg, Mattias
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Jarvet, Jüri
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Eriksson, L. E. Göran
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Cell membrane translocation of the N-terminal (1-28) part of the prion protein2002In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 299, no 1, p. 85-90Article in journal (Refereed)
    Abstract [en]

    The N-terminal (1-28) part of the mouse prion protein (PrP) is a cell penetrating peptide, capable of transporting large hydrophilic cargoes through a cell membrane. Confocal fluorescence microscopy shows that it transports the protein avidin (67 kDa) into several cell lines. The (1-28) peptide has a strong tendency for aggregation and P-structure formation, particularly in interaction with negatively charged phospholipid membranes. The findings have implications for how prion proteins with uncleaved signal peptides in the N-termini may enter into cells, which is important for infection. The secondary structure conversion into beta-structure may be relevant as a seed for the conversion into the scrapie (PrPSc) form of the protein and its arnyloidic transformation.

  • 18. Nordin, Joel Z.
    et al.
    Lee, Yi
    Vader, Pieter
    Mäger, Imre
    Johansson, Henrik J.
    Heusermann, Wolf
    Wiklander, Oscar P. B.
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Seow, Yiqi
    Bultema, Jarred J.
    Gilthorpe, Jonathan
    Davies, Tim
    Fairchild, Paul J.
    Gabrielsson, Susanne
    Meisner-Kober, Nicole C.
    Lehtio, Janne
    Smith, C. I. Edvard
    Wood, Matthew J. A.
    Andaloussi, Samir E. L.
    Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties2015In: Nanomedicine: Nanotechnology, Biology and Medicine, ISSN 1549-9634, E-ISSN 1549-9642, Vol. 11, no 4, p. 879-883Article in journal (Refereed)
    Abstract [en]

    Extracellular vesicles (EVs) are natural nanoparticles that mediate intercellular transfer of RNA and proteins and are of great medical interest; serving as novel biomarkers and potential therapeutic agents. However, there is little consensus on the most appropriate method to isolate high-yield and high-purity EVs from various biological fluids. Here, we describe a systematic comparison between two protocols for EV purification: ultrafiltration with subsequent liquid chromatography (UF-LC) and differential ultracentrifugation (UC). A significantly higher EV yield resulted from UF-LC as compared to UC, without affecting vesicle protein composition. Importantly, we provide novel evidence that, in contrast to UC-purified EVs, the biophysical properties of UF-LC-purified EVs are preserved, leading toadifferent in vivo biodistribution, with less accumulation in lungs. Finally, we show that UF-LC is scalable and adaptable for EV isolation from complex media types such as stem cell media, which is of huge significance for future clinical applications involving EVs.

  • 19. Padari, Kart
    et al.
    Koppel, Kaida
    Lorents, Annely
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Mano, Miguel
    Pedroso de Lima, Maria C.
    Pooga, Margus
    S4(13)-PV Cell-Penetrating Peptide Forms Nanoparticle-Like Structures to Gain Entry Into Cells2010In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 21, no 4, p. 774-783Article in journal (Refereed)
    Abstract [en]

    Despite increasing interest in cell-penetrating peptides (CPPs) as carriers for drugs and in gene therapy, the current understanding of their exact internalization mechanism is still far from complete. The cellular translocation of CPPs and their payloads has been mostly described by fluorescence- and activity-based methods, leaving the more detailed characterization at the ultrastructural level almost out of attention. Herein, we used transmission electron microscopy to characterize the membrane interaction and internalization of a cell-penetrating peptide S4(13)-PV. We demonstrate that S4(13)-PV peptide forms spherical nanoparticle-like regular structures upon association with cell surface glycosaminoglycans on the plasma membrane. Insertion of S4(13)-PV particles into plasma membrane induces disturbances and leads to the vesicular uptake of peptides by cells. We propose that for efficient cellular translocation S4(13)-PV peptides have to assemble into particles of specific size and shape. The spherical peptide particles are not dissociated in intracellular vesicles but often retain their organization and remain associated with the membrane of vesicles, destabilizing them and promoting the escape of peptides into cytosol. Lowering the temperature and inhibition of dynamins' activity reduce the internalization of S4(13)-PV peptides, but do not block it completely. Our results provide an ultrastructural insight into the interaction mode of CPPs with the plasma membrane and the distribution in cells, which might help to better understand how CPPs cross the biological membranes and gain access into cells.

  • 20.
    Palm, Caroline
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Jayamanne, Mala
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Kjellander, Marcus
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Peptide degradation is a critical determinant for cell-penetrating peptide uptake2007In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1768, no 7, p. 1769-1776Article in journal (Refereed)
    Abstract [en]

    Cell-penetrating peptide mediated uptake of labels appears to follow an equilibrium-like process. However, this assumption is only valid if the peptides are stabile. Hence, in this study we investigate intracellular and extracellular peptide degradation kinetics of two fluorescein labeled cell-penetrating peptides, namely MAP and penetratin, in Chinese hamster ovarian cells. The degradation and uptake kinetics were assessed by RP-HPLC equipped with a fluorescence detector. We show that MAP and penetratin are rapidly degraded both extracellularly and intracellularly giving rise to several degradation products. Kinetics indicates that intracellularly, the peptides exist in (at least) two distinct pools: one that is immediately degraded and one that is stabile. Moreover, the degradation could be decreased by treating the peptides with BSA and phenanthroline and the uptake was significantly reduced by cytochalasin B, chloroquine and energy depletion. The results indicate that the extracellular degradation determines the intracellular peptide concentration in this system and therefore the stability of cell-penetrating peptides needs to be evaluated.

  • 21.
    Palm, Caroline
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Netzereab, Semharai
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Quantitatively determined uptake of cell-penetrating peptides in non-mammalian cells with an evaluation of degradation and antimicrobial effects2006In: Peptides, ISSN 0196-9781, E-ISSN 1873-5169, Vol. 27, no 7, p. 1710-1716Article in journal (Refereed)
    Abstract [en]

    Cell-penetrating peptides (CPPs) are carriers developed to improve mammalian cell uptake of important research tools such as antisense oligonucleotides and short interfering RNAs. However, the data on CPP uptake into non-mammalian cells are limited. We have studied the uptake and antimicrobial effects of the three representative peptides penetratin (derived from a non-mammalian protein), MAP (artificial peptide) and pVEC (derived from a mammalian protein) using fluorescence HPLC in four common model systems: insect cells (Sfg), gram-positive bacteria (Bacillus megaterium), gram-negative bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae). We demonstrate that non-mammalian cells internalize CPPs and a comparison of the uptake of the peptides show that the intracellular concentration and degradation of the peptides varies widely among organisms. In addition, these CPPs showed antimicrobial activity.

  • 22.
    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.

  • 23.
    Palm-Apergi, Caroline
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lorents, Annely
    Padari, Kärt
    Pooga, Margus
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    The membrane repair response masks membrane disturbances caused by cell-penetrating peptide uptake2009In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 23, no 1, p. 214-223Article in journal (Refereed)
    Abstract [en]

    Although cell-penetrating peptides are able to deliver cargo into cells, their uptake mechanism is still not fully understood and needs to be elucidated to improve their delivery efficiency. Herein, we present evidence of a new mechanism involved in uptake, the membrane repair response. Recent studies have suggested that there might be a direct penetration of peptides in parallel with different forms of endocytosis. The direct penetration of hydrophilic peptides through the hydrophobic plasma membrane, however, is highly controversial. Three proteins involved in target cell apoptosis—perforin, granulysin, and granzymes—share many features common in uptake of cell-penetrating peptides (e.g., they bind proteoglycans). During perforin uptake, the protein activates the membrane repair response, a resealing mechanism triggered in cells with injured plasma membrane, because of extracellular calcium influx. On activation of the membrane repair response, internal vesicles are mobilized to the site of the disrupted plasma membrane, resealing it within seconds. In this study, we have used flow cytometry, fluorescence, and electron microscopy, together with high-performance liquid chromatography and mass spectrometry, to present evidence that the membrane repair response is able to mask damages caused during cell-penetrating peptide uptake, thus preventing leakage of endogenous molecules out of the cell.—Palm-Apergi, C., Lorents, A., Padari, K., Pooga, M., and Hällbrink, M. The membrane repair response masks membrane disturbances caused by cell-penetrating peptide uptake.

  • 24.
    Pooga, Margus
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology. Estonian Biocentre, Estonia.
    Kut, Cecilia
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Kihlmark, Madeleine
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Fernaeus, Sandra
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Raid, Raivo
    Land, Tiit
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Hallberg, Einar
    Bartfai, Tamas
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology. Scripps Research Institute, California.
    Cellular translocation of proteins by transportan2001In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 15, no 6, p. 1451-1453Article in journal (Refereed)
    Abstract [en]

    Proteins with molecular masses ranging from 30 kDa. (green fluorescent protein, GFP) to 150 kDa (monoclonal and polyclonal antibodies) were coupled to the cellular translocating peptide transportan. We studied the ability of the resulting protein-peptide constructs to penetrate into Bowes melanoma, BRL, and COS-7 cells. After 0.5-3 h incubation with recombinant GFP coupled to transportan, most of the GFP fluorescence was found in intracellular membranes of BRL and COS-7 cells, which suggests that transportan could internalize covalently linked proteins of about 30 kDa in a folded state. Transportan could internalize covalently coupled molecules of even larger size; that is, avidin and antibodies, (up to 150 kDa). The covalent bond between the transport peptide and its cargo is not obligatory because streptavidin was translocated into the cells within 15 min as a noncovalent complex with biotinylated transportan. Inside the cells, the delivered streptavidin was first located mainly in close proximity to the plasma membrane and was later distributed to the perinuclear region. Most of the internalized streptavidin was confined to vesicular structures, but a significant fraction of the protein was distributed in the cytoplasm. Our data suggest that transportan can deliver proteins and other hydrophilic macromolecules into intact mammalian cells, and this finding demonstrates good potential as powerful cellular delivery vector for scientific and therapeutic purposes.

  • 25.
    Regberg, Jakob
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Luis Daniel, Ferreira Vasconcelos
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Madani, Fatemeh
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    pH-responsive PepFect cell-penetrating peptides2016In: International Journal of Pharmaceutics, ISSN 0378-5173, E-ISSN 1873-3476, Vol. 501, no 1-2, p. 32-38Article in journal (Refereed)
    Abstract [en]

    A series of cell-penetrating PepFect peptide analogues was developed by substitutions of the galanin-derived N-terminal sequence. Histidine modifications were incorporated in order to make the peptides pH-responsive. The peptides were all able to form non-covalent complexes with an oligonucleotide cargo by co-incubation in buffer. The complexes were characterized by dynamic light scattering and circular dichroism, and an assay to evaluate the peptide-cargo affinity was developed. Cellular bioactivity was studied in HeLa cells using a luciferase-based splice correction assay. In addition, the membrane interactions of the peptides in large unilammelar vesicles was studied using a calcein leakage assay. The effects of substitutions were found to be dependent of the non-modified, C-terminal sequence of the peptides; for analogues of PepFect 3 we observed an increase in membrane activity and bioactivity for histidine-containing analogues, whereas the same modifications introduced to PepFect 14 lead to a decreased bioactivity. Peptides modified with a leucine/histidine sequence were found to be pH responsive, complexes formed from these peptides were small at pH 7 and grew under acidic conditions. The most promising of the novel PepFect 3 analogues, PepFect 132 has a significantly higher bioactivity and membrane activity than the parent peptide PepFect 3.

  • 26. Sork, Helena
    et al.
    Nordin, Joel Z.
    Turunen, Janne J.
    Wiklander, Oscar P. B.
    Bestas, Burcu
    Zaghloul, Eman M.
    Margus, Helerin
    Padari, Kärt
    Duru, Adil D.
    Corso, Giulia
    Bost, Jeremy
    Vader, Pieter
    Pooga, Margus
    Smith, C. I. Edvard
    Wood, Matthew J. A.
    Schiffelers, Raymond M.
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Andaloussi, Samir E. L.
    Lipid-based Transfection Reagents Exhibit Cryo-induced Increase in Transfection Efficiency2016In: Molecular Therapy - Nucleic Acids, ISSN 2162-2531, E-ISSN 2162-2531, Vol. 5, article id e290Article in journal (Refereed)
    Abstract [en]

    The advantages of lipid-based transfection reagents have permitted their widespread use in molecular biology and gene therapy. This study outlines the effect of cryo-manipulation of a cationic lipid-based formulation, Lipofectamine 2000, which, after being frozen and thawed, showed orders of magnitude higher plasmid delivery efficiency throughout eight different cell lines, without compromising cell viability. Increased transfection efficiency with the freeze-thawed reagent was also seen with 2'-O-methyl phosphorothioate oligonucleotide delivery and in a splice-correction assay. Most importantly, a log-scale improvement in gene delivery using the freeze-thawed reagent was seen in vivo. Using three different methods, we detected considerable differences in the polydispersity of the different nucleic acid complexes as well as observed a clear difference in their surface spreading and sedimentation, with the freeze-thawed ones displaying substantially higher rate of dispersion and deposition on the glass surface. This hitherto overlooked elevated potency of the freeze-thawed reagent facilitates the targeting of hard-to-transfect cells, accomplishes higher transfection rates, and decreases the overall amount of reagent needed for delivery. Additionally, as we also saw a slight increase in plasmid delivery using other freeze-thawed transfection reagents, we postulate that freeze-thawing might prove to be useful for an even wider variety of transfection reagents.

  • 27.
    Srimanee, Artita
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Arvanitidou, Maria
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Kim, Kumjee
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Cell-penetrating peptides for siRNA delivery to glioblastomas2018In: Peptides, ISSN 0196-9781, E-ISSN 1873-5169, Vol. 104, p. 62-69Article in journal (Refereed)
    Abstract [en]

    Delivery of small interfering RNA (siRNA) to suppress glioblastoma growth is a hurdle due to the critical obstacles of the blood-brain barrier and the siRNA properties of such as high negative charges and instability in serum. Therefore, the passage of siRNA to targeted cells is limited. Several siRNA carriers have been constructed using cell-penetrating peptides (CPPs) since the CPPs have shown a high potential for oligonucleotide delivery into the cells. In this study, two CPPs, PepFect 14 (PF14) and the amphipathic peptide PepFect 28 (PF28), were modified with targeting peptides by covalent conjugation and non-covalent complex formation to improve glioma-targeted specificity and gene-silencing efficiency. In conclusion, we have established an efficient non-covalently complexed carrier (PF14:TG1) for siRNA delivery to human glioblastoma cells (U87), showing a significant two-fold increase in gene-silencing efficiency compared to the parent peptide PF14 and also improved specificity to U87 cells compared to non-glioma targeted cells.

  • 28.
    Srimanee, Artita
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Arvanitidou, Maria
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Kim, Kumjee
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    New strategy for siRNA delivery to glioblastoma cells by cell-penetrating peptideManuscript (preprint) (Other academic)
  • 29.
    Srimanee, Artita
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry. Mahidol University, Thailand.
    Regberg, Jakob
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Kurrikoff, Kaido
    Veiman, Kadi-Liis
    Vajragupta, Opa
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Peptide-Based Delivery of Oligonucleotides Across Blood-Brain Barrier Model2014In: International Journal of Peptide Research and Therapeutics, ISSN 1573-3904, Vol. 20, no 2, p. 169-178Article in journal (Refereed)
    Abstract [en]

    Delivery of pharmaceutical agents across a blood–brain barrier (BBB) is a challenge for brain cancer therapy. In this study, an in vitro BBB model was utilized to study the delivery of oligonucleotides across brain endothelial cells targeting to glioma cells in a Transwell™ setup. A series of novel peptides were synthesized by covalent conjugation of cell-penetrating peptides with targeting peptides for delivery of gene-based therapeutics. These peptides were screened for passage across the Transwell™ and we found the most efficient peptide PepFect32 from originating PepFect 14 coupled with the targeting peptide angiopep-2. PepFect32/pDNA nanocomplexes exhibited high transcytosis across the BBB in vitro model and the highest transfection efficiency to glioma cells. In conclusion, PepFect32 revealed the most efficient peptide-based vector for pDNA delivery across in vitro BBB model.

  • 30.
    Srimanee, Artita
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry. Mahidol University, Thailand.
    Regberg, Jakob
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Vajragupta, Opa
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Role of scavenger receptors in peptide-based delivery of plasmid DNA across a blood-brain barrier model2016In: International Journal of Pharmaceutics, ISSN 0378-5173, E-ISSN 1873-3476, Vol. 500, no 1-2, p. 128-135Article in journal (Refereed)
    Abstract [en]

    Receptor-mediated transcytosis remains a major route for drug delivery across the blood-brain barrier (BBB). PepFect 32 (PF32), a peptide-based vector modified with targeting ligand (Angiopep-2) binding to low-density lipoprotein receptor-related protein-1 (LRP-1), was previously found to be a promising vector for plasmid delivery across an in vitro model of the BBB. Cellular uptake of PF32/plasmid DNA (pDNA) complexes was speculated the internalization via LRP-1 receptor. In this study, we prove that PF32/pDNA nanocomplexes are not only transported into brain endothelial cells via LRP-1 receptor-mediated endocytosis, but also via scavenger receptor class A and B (SCARA3, SCARA5, and SR-BI)-mediated endocytosis. SCARA3, SCARA5, and SR-BI are found to be expressed in the brain endothelial cells. Inhibition of these receptors leads to a reduction of the transfection. In conclusion, this study shows that scavenger receptors also play an essential role in the cellular uptake of the PF32/pDNA nanocomplexes.

  • 31. van Asbeck, Alexander H.
    et al.
    Beyerle, Andrea
    McNeill, Hesta
    Bovee-Geurts, Petra H. M.
    Lindberg, Staffan
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Verdurmen, Wouter P. R.
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Heidenreich, Olaf
    Brock, Roland
    Molecular Parameters of siRNA-Cell Penetrating Peptide Nanocomplexes for Efficient Cellular Delivery2013In: ACS Nano, ISSN 1936-0851, E-ISSN 1936-086X, Vol. 7, no 5, p. 3797-3807Article in journal (Refereed)
    Abstract [en]

    Cell-penetrating peptides (CPPs) are versatile tools for the intracellular delivery of various biomolecules, including siRNA. Recently, CPPs were introduced that showed greatly enhanced delivery efficiency. However, the molecular basis of this increased activity is poorly understood. Here, we performed a detailed analysis of the molecular and physicochemical properties of seven different siRNA-CPP nanoparticles. In addition, we determined which complexes are internalized most efficiently into the leukemia cell-line SKNO-1, and subsequently inhibited the expression of a luctferase reporter gene. We demonstrated effective complexation of siRNA for all tested CPPs, and optimal encapsulation of the siRNA was achieved at very similar molar ratios independent of peptide charge. However, CPPs with an extreme high or low overall charge proved to be exceptions, suggesting an optimal range of charge for CPP-siRNA nanoparticle formation based on opposite charge. The most active CPP (PepFect6) displayed high serum resistance but also high sensitivity to decomplexation by polyanionic macromolecules, indicating the necessity for partial decomplexation for efficient uptake. Surprisingly, CPP-siRNA complexes acquired a negative zeta-potential in the presence of serum. These novel insights shed light on the observation that cell association is necessary but not sufficient for activity and motivate new research into the nature of the nanoparticle-cell interaction. Overall, our results provide a comprehensive molecular basis for the further development of peptide-based olipnudeotide transfection agents.

  • 32.
    Vasconcelos, Luis
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lehto, Tõnis
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Madani, Fatemeh
    Radoi, Vlad
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Vukojević, Vladana
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Simultaneous membrane interaction of amphipathic peptide monomers, self-aggregates and cargo complexes detected by fluorescence correlation spectroscopy2018In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1860, no 2, p. 491-504Article in journal (Refereed)
    Abstract [en]

    Peptides able to translocate cell membranes while carrying macromolecular cargo, as cell-penetrating peptides (CPPs), can contribute to the field of drug delivery by enabling the transport of otherwise membrane impermeable molecules. Formation of non-covalent complexes between amphipathic peptides and oligonucleotides is driven by electrostatic and hydrophobic interactions. Here we investigate and quantify the coexistence of distinct molecular species in multiple equilibria, namely peptide monomer, peptide self-aggregates and peptide/oligonucleotide complexes. As a model for the complexes, we used a stearylated peptide from the PepFect family, PF14 and siRNA. PF14 has a cationic part and a lipid part, resembling some characteristics of cationic lipids. Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) were used to detect distinct molecular entities in solution and at the plasma membrane of live cells. For that, we labeled the peptide with carboxyrhodamine 6G and the siRNA with Cyanine 5. We were able to detect fluorescent entities with diffusional properties characteristic of the peptide monomer as well as of peptide aggregates and peptide/oligonucleotide complexes. Strategies to avoid peptide adsorption to solid surfaces and self-aggregation were developed and allowed successful FCS measurements in solution and at the plasma membrane. The ratio between the detected molecular species was found to vary with pH, peptide concentration and the proximity to the plasma membrane. The present results suggest that the diverse cellular uptake mechanisms, often reported for amphipathic CPPs, might result from the synergistic effect of peptide monomers, self-aggregates and cargo complexes, distributed unevenly at the plasma membrane.

  • 33. Verdurmen, Wouter P. R.
    et al.
    Bovee-Geurts, Petra H.
    Wadhwani, Parvesh
    Ulrich, Anne S.
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    van Kuppevelt, Toin H.
    Brock, Roland
    Preferential Uptake of L- versus D-Amino Acid Cell-Penetrating Peptides in a Cell Type-Dependent Manner2011In: Chemistry and Biology, ISSN 1074-5521, E-ISSN 1879-1301, Vol. 18, no 8, p. 1000-1010Article in journal (Refereed)
    Abstract [en]

    The use of protease-resistant D-peptides is a prominent strategy for overcoming proteolytic sensitivity in the use of cell-penetrating peptides (CPPs) as delivery vectors. So far, no major differences have been reported for the uptake of L- and D-peptides. Here we report that cationic L-CPPs are taken up more efficiently than their D-counterparts in MC57 fibrosarcoma and He La cells but not in Jurkat T leukemia cells. Reduced uptake of D-peptides co-occurred with persistent binding to heparan sulfates (HS) at the plasma membrane. In vitro binding studies of Land D-peptides with HS indicated similar binding affinities. Our results identify two key events in the uptake of CPPs: binding to HS chains and the initiation of internalization. Only the second event depends on the chirality of the CPP. This knowledge may be exploited for a stereochemistry-dependent preferential targeting of cells.

  • 34. Zaghloul, Eman M.
    et al.
    Gissberg, Olof
    Moreno, Pedro M. D.
    Siggens, Lee
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Jørgensen, Anna S.
    Ekwall, Karl
    Zain, Rula
    Wengel, Jesper
    Lundin, Karin E.
    Smith, C. I. Edvard
    CTG repeat-targeting oligonucleotides for down-regulating Huntingtin expression2017In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, no 9, p. 5153-5169Article in journal (Refereed)
    Abstract [en]

    Huntington's disease (HD) is a fatal, neurodegenerative disorder in which patients suffer from mobility, psychological and cognitive impairments. Existing therapeutics are only symptomatic and do not significantly alter the disease progression or increase life expectancy. HD is caused by expansion of the CAG trinucleotide repeat region in exon 1 of the Huntingtin gene (HTT), leading to the formation of mutant HTT transcripts (muHTT). The toxic gain-of-function of muHTT protein is a major cause of the disease. In addition, it has been suggested that the muHTT transcript contributes to the toxicity. Thus, reduction of both muHTT mRNA and protein levels would ideally be the most useful therapeutic option. We herein present a novel strategy for HD treatment using oligonucleotides (ONs) directly targeting the HTT trinucleotide repeat DNA. A partial, but significant and potentially long-term, HTT knock-down of both mRNA and protein was successfully achieved. Diminished phosphorylation of HTT gene-associated RNA-polymerase II is demonstrated, suggestive of reduced transcription downstream the ON-targeted repeat. Different backbone chemistries were found to have a strong impact on the ON efficiency. We also successfully use different delivery vehicles as well as naked uptake of the ONs, demonstrating versatility and possibly providing insights for in vivo applications.

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