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Design and evaluation of drug delivery vehicles
Stockholm University, Faculty of Science, Department of Neurochemistry.
Responsible organisation
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A crucial aspect of drug delivery is efficient transport to the site of action. Thus, there is a need to design and evaluate new delivery vehicles. In this thesis two delivery vehicles, cell-penetrating peptides and bacterial ghosts, were evaluated. The understanding of the internalization and degradation kinetics of cell-penetrating peptides is important for the practical aspects of cargo delivery since peptides have a notorious reputation of being rapidly degraded. If the cell-penetrating peptide remains intact inside the cellular environment, there is a possibility that the peptide-cargo conjugate leaks back to the extracellular environment. However, if it is degraded outside the cell, the cargo will never be delivered. In order to improve uptake efficiency and to be able to foresee side effects, the translocation mechanism needs to be fully elucidated. Data gathered from the first two papers led to the proposal of a new me-chanism involved in cell-penetrating peptide uptake: the membrane repair response, a resealing mechanism rapidly patching up broken membranes. This mechanism could explain the divergence in perception concerning the uptake pathways. Furthermore a new assay to produce the second delivery vehicle, bacterial ghosts, was developed based on data from the cell-penetrating peptide investigations. Bacterial ghosts are dead bacteria devoid of cytoplasmic contents but still retaining their structural and morphological characteristics, after protein E lysis of the bacterial cell membrane. By using a cell-penetrating peptide with antimicrobial effects, a new rapid peptide-based strategy to produce ghosts was developed and the capability to deliver plasmid DNA into the cell for expression was evaluated.

Place, publisher, year, edition, pages
Stockholm: Institutionen för neurokemi , 2008. , 77 p.
National Category
Neurosciences
Research subject
Neurochemistry and Neurotoxicology
Identifiers
URN: urn:nbn:se:su:diva-8256ISBN: 978-91-7155-754-4 (print)OAI: oai:DiVA.org:su-8256DiVA: diva2:199915
Public defence
2008-11-14, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 13:00
Opponent
Supervisors
Available from: 2008-10-23 Created: 2008-10-07Bibliographically approved
List of papers
1. Quantitatively determined uptake of cell-penetrating peptides in non-mammalian cells with an evaluation of degradation and antimicrobial effects
Open this publication in new window or tab >>Quantitatively determined uptake of cell-penetrating peptides in non-mammalian cells with an evaluation of degradation and antimicrobial effects
2006 (English)In: Peptides, ISSN 0196-9781, E-ISSN 1873-5169, Vol. 27, no 7, 1710-1716 p.Article in journal (Refereed) Published
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.

Keyword
cell-penetrating peptides, non-mammalian cells, uptake, degradation
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-25530 (URN)10.1016/j.peptides.2006.01.006 (DOI)000238780300016 ()
Available from: 2008-10-23 Created: 2008-10-07 Last updated: 2017-12-13Bibliographically approved
2. Peptide degradation is a critical determinant for cell-penetrating peptide uptake
Open this publication in new window or tab >>Peptide degradation is a critical determinant for cell-penetrating peptide uptake
2007 (English)In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1768, no 7, 1769-1776 p.Article in journal (Refereed) Published
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.

Keyword
cell-penetrating peptide, protein transduction domains, uptake, degradation, endocytosis inhibitor
National Category
Biochemistry and Molecular Biology Biophysics
Identifiers
urn:nbn:se:su:diva-25531 (URN)10.1016/j.bbamem.2007.03.029 (DOI)000247716200009 ()
Available from: 2008-10-23 Created: 2008-10-07 Last updated: 2017-12-13Bibliographically approved
3. The membrane repair response masks membrane disturbances caused by cell-penetrating peptide uptake
Open this publication in new window or tab >>The membrane repair response masks membrane disturbances caused by cell-penetrating peptide uptake
Show others...
2009 (English)In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 23, no 1, 214-223 p.Article in journal (Refereed) Published
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.

Keyword
protein transduction domains, perforin, granzyme B, granulysin, cellular wound healing
National Category
Neurosciences
Identifiers
urn:nbn:se:su:diva-25532 (URN)10.1096/fj.08-110254 (DOI)000262095500024 ()
Available from: 2008-10-23 Created: 2008-10-07 Last updated: 2017-12-13Bibliographically approved
4. A new rapid cell-penetrating peptide based strategy to produce bacterial ghosts for plasmid delivery
Open this publication in new window or tab >>A new rapid cell-penetrating peptide based strategy to produce bacterial ghosts for plasmid delivery
2008 (English)In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 132, no 1, 49-54 p.Article in journal (Refereed) Published
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.

Keyword
Cell-penetrating peptides, Protein transduction domains, Bacterial ghosts, Delivery vehicle, Gene delivery
National Category
Chemical Sciences
Identifiers
urn:nbn:se:su:diva-25533 (URN)10.1016/j.jconrel.2008.08.011 (DOI)000261354400008 ()
Available from: 2008-10-23 Created: 2008-10-07 Last updated: 2017-12-13Bibliographically approved

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