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Cell-penetrating peptides; chemical modification, mechanism of uptake and formulation development
Stockholm University, Faculty of Science, Department of Neurochemistry.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Gene therapy holds the promise of revolutionizing the way we treat diseases. By using recombinant DNA and oligonucleotides (ONs), gene functions can be restored, altered or silenced according to the therapeutic need. However, gene therapy approaches require the delivery of large and charged nucleic acid-based molecules to their intracellular targets across the plasma membrane, which is inherently impermeable to such molecules. In this thesis, two chemically modified cell-penetrating peptides (CPPs) that have superior delivery properties for several nucleic acid-based therapeutics are developed. These CPPs can spontaneously form nanoparticles upon non-covalent complexation with the nucleic acid cargo, and the formed nanoparticles mediate efficient cellular transfection. In paper I, we show that an N-terminally stearic acid-modified version of transportan-10 (PF3) can efficiently transfect different cell types with plasmid DNA and mediates efficient gene delivery in-vivo when administrated intra muscularly (i.m.) or intradermaly (i.d.). In paper II, a new peptide with ornithine modification, PF14, is shown to efficiently deliver splice-switching oligonucleotides (SSOs) in different cell models including mdx mouse myotubes; a cell culture model of Duchenne’s muscular dystrophy (DMD). Additionally, we describe a method for incorporating the PF14-SSO nanoparticles into a solid formulation that is active and stable even when stored at elevated temperatures for several weeks. In paper III, we demonstrate the involvement of class-A scavenger receptor subtypes (SCARA3 & SCARA5) in the uptake of PF14-SSO nanoparticles, which possess negative surface charge, and suggest for the first time that some CPP-based systems function through scavenger receptors. In paper IV, the ability of PF14 to deliver siRNA to different cell lines is shown and their stability in simulated gastric acidic conditions is highlighted.

Taken together, these results demonstrate that certain chemical modifications can drastically enhance the activity and stability of CPPs for delivering nucleic acids after spontaneous nanoparticle formation upon non-covalent complexation. Moreover, we show that CPP-based nanoparticles can be formulated into convenient and stable solid formulations that can be suitable for several therapeutic applications. Importantly, the involvement of scavenger receptors in the uptake of such nanoparticles is presented, which could yield novel possibilities to understand and improve the transfection by CPPs and other gene therapy nanoparticles.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University Stockholm University , 2012. , 86 p.
Keyword [en]
cell penetrating peptides, gene therapy, scavenger receptors, pharmaceutical formulation, solid dispersion
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-75537ISBN: 978-91-7447-464-0 (print)OAI: oai:DiVA.org:su-75537DiVA: diva2:517044
Public defence
2012-06-11, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
At the time of doctoral defence the following paper was unpublished and had a status as follows: Paper nr 4: SubmittedAvailable from: 2012-05-10 Created: 2012-04-20 Last updated: 2012-04-24Bibliographically approved
List of papers
1. A Peptide-based Vector for Efficient Gene Transfer In Vitro and In Vivo
Open this publication in new window or tab >>A Peptide-based Vector for Efficient Gene Transfer In Vitro and In Vivo
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2011 (English)In: Molecular Therapy, ISSN 1525-0016, E-ISSN 1525-0024, Vol. 19, no 8, 1457-1467 p.Article in journal (Refereed) Published
Abstract [en]

Finding suitable nonviral delivery vehicles for nucleic acid-based therapeutics is a landmark goal in gene therapy. Cell-penetrating peptides (CPPs) are one class of delivery vectors that has been exploited for this purpose. However, since CPPs use endocytosis to enter cells, a large fraction of peptides remain trapped in endosomes. We have previously reported that stearylation of amphipathic CPPs, such as transportan 10 (TP10), dramatically increases transfection of oligonucleotides in vitro partially by promoting endosomal escape. Therefore, we aimed to evaluate whether stearyl-TP10 could be used for the delivery of plasmids as well. Our results demonstrate that stearyl-TP10 forms stable nanoparticles with plasmids that efficiently enter different cell-types in a ubiquitous manner, including primary cells, resulting in significantly higher gene expression levels than when using stearyl-Arg9 or unmodified CPPs. In fact, the transfection efficacy of stearyl-TP10 almost reached the levels of Lipofectamine 2000 (LF2000), however, without any of the observed lipofection-associated toxicities. Most importantly, stearyl-TP10/plasmid nanoparticles are nonimmunogenic, mediate efficient gene delivery in vivo, when administrated intramuscularly (i.m.) or intradermally (i.d.) without any associated toxicity in mice.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:su:diva-56148 (URN)10.1038/mt.2011.10 (DOI)000293378500010 ()21343913 (PubMedID)
Available from: 2011-04-11 Created: 2011-04-11 Last updated: 2017-12-11Bibliographically approved
2. PepFect 14, a novel cell-penetrating peptide for oligonucleotide delivery in solution and as solid formulation
Open this publication in new window or tab >>PepFect 14, a novel cell-penetrating peptide for oligonucleotide delivery in solution and as solid formulation
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2011 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, no 12, 5284-5298 p.Article in journal (Refereed) Published
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.

National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-56147 (URN)10.1093/nar/gkr072 (DOI)000292564900040 ()21345932 (PubMedID)
Available from: 2011-04-11 Created: 2011-04-11 Last updated: 2017-12-11Bibliographically approved
3. Scavenger receptor-mediated uptake of cell-penetrating peptide nanoparticles with oligonucleotides
Open this publication in new window or tab >>Scavenger receptor-mediated uptake of cell-penetrating peptide nanoparticles with oligonucleotides
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2012 (English)In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 26, no 3, 1172-1180 p.Article in journal (Refereed) Published
Abstract [en]

Cell-penetrating peptides (CPPs) are shortcationic peptides that penetrate cells by interacting withthe negatively charged plasma membrane; however, thedetailed uptake mechanism is not clear. In contrary to theconventional mode of action of CPPs, we show here thata CPP, PepFect14 (PF14), forms negatively charged nanocomplexeswith oligonucleotides and their uptake is mediatedby class-A scavenger receptors (SCARAs). Specificinhibitory ligands of SCARAs, such as fucoidin, polyinosinicacid, and dextran sulfate, totally inhibit the activityof PF14-oligonucleotide nanocomplexes in the HeLapLuc705 splice-correction cell model, while nonspecific,chemically related molecules do not. Furthermore, RNAinterference (RNAi) knockdown of SCARA subtypes(SCARA3 and SCARA5) that are expressed in this cell lineled to a significant reduction of the activity to <50%. Inline with this, immunostaining shows prevalent colocalizationof the nanocomplexes with the receptors, and electronmicroscopy images show no binding or internalizationof the nanocomplexes in the presence of theinhibitory ligands. Interestingly, naked oligonucleotidesalso colocalize with SCARAs when used at high concentrations.These results demonstrate the involvement ofSCARA3 and SCARA5 in the uptake of PF14-oligonucleotidenanocomplexes and suggest for the first time thatsome CPP-based systems function through scavenger receptors,which could yield novel possibilities to understandand improve the transfection by CPPs.

Keyword
drug delivery, splice correction, cellular uptake
National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-75534 (URN)10.1096/fj.11-191536 (DOI)000300949300020 ()22138034 (PubMedID)
Available from: 2012-04-20 Created: 2012-04-20 Last updated: 2017-12-07Bibliographically approved
4. Solid formulation of cell-penetrating peptide nanoparticles with siRNA and their stability in simulated gastric conditions
Open this publication in new window or tab >>Solid formulation of cell-penetrating peptide nanoparticles with siRNA and their stability in simulated gastric conditions
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2012 (English)In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 162, no 1, 1-8 p.Article in journal (Refereed) Published
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.

Keyword
Cell penetrating peptide, siRNA, Solid formulation, Acid stability, Gastric fluid
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:su:diva-75482 (URN)10.1016/j.jconrel.2012.06.006 (DOI)000307769200001 ()
Available from: 2012-04-19 Created: 2012-04-19 Last updated: 2017-12-07Bibliographically approved

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