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Complexes of cell-penetrating peptides with oligonucleotides: Structure, binding and translocation in lipid membranes
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0001-7522-8964
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The fundamental element of life known to man is the gene. The information contained in genes regulates all cellular functions, in health and disease. The ability to selectively alter genes or their transcript intermediates with designed molecular tools, as synthetic oligonucleotides, represents a paradigm shift in human medicine.

The full potential of oligonucleotide therapeutics is however dependent on the development of efficient delivery vectors, due to their intrinsic characteristics, as size, charge and low bioavailability. Cell-penetrating peptides are short sequences of amino acids that are capable of mediating the transport of most types of oligonucleotide therapeutics to the cell interior. It is the interaction of cell-penetrating peptides with oligonucleotides and the transport of their non-covalently formed complexes across the cellular membrane, that constitutes the main subject of this thesis.

In Paper I we studied the effects of different types of oligonucleotide cargo in the capacity of cationic and amphipathic peptides to interact with lipid membranes. We found that indeed the cargo sequesters some of the peptide’s capacity to interact with membranes. In Paper II we revealed the simultaneous interaction of different molecular and supramolecular peptide and peptide/oligonucleotide species in equilibrium, with the cellular membrane. In Paper III we developed a series of peptides with improved affinity for oligonucleotide cargo as well as enhanced endosomal release and consequently better delivery capacity. In Paper IV we investigated the effect of saturated fatty acid modifications to a cationic cell-penetrating peptide. The varying amphipathicity of the peptide correlated with the complex physicochemical properties and with its delivery efficiency.

This thesis contributes to the field with a set of characterized mechanisms and physicochemical properties for the components of the ternary system – cell-penetrating peptide, oligonucleotide and cell membrane – that should be considered for the future development of gene therapy.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University , 2017. , 79 p.
Keyword [en]
Cell-penetrating peptide, oligonucleotide, transfection, non-covalent complexes, membrane interaction
National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-141881ISBN: 978-91-7649-727-2 (print)ISBN: 978-91-7649-728-9 (electronic)OAI: oai:DiVA.org:su-141881DiVA: diva2:1089643
Public defence
2017-06-16, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.

Available from: 2017-05-22 Created: 2017-04-20 Last updated: 2017-05-18Bibliographically approved
List of papers
1. Effects of cargo molecules on membrane perturbation caused by transportan10 based cell-penetrating peptides
Open this publication in new window or tab >>Effects of cargo molecules on membrane perturbation caused by transportan10 based cell-penetrating peptides
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2014 (English)In: Biochimica et Biophysica Acta, ISSN 0006-3002, Vol. 1838, no 12, 3118-3129 p.Article in journal (Refereed) Published
Abstract [en]

Cell-penetrating peptides with the ability to escape endosomes and reach the target are of great value as delivery vectors for different bioactive cargoes and future treatment of human diseases. We have studied two such peptides, NickFect1 and NickFect51, both originated from stearylated transportan10 (PF3). To obtain more insight into the mechanism(s) of peptide delivery and the biophysical properties of an efficient vector system, we investigated the effect of different bioactive oligonucleotide cargoes on peptide-membrane perturbation and peptide structural induction. We studied the membrane interactions of the peptides with large unilamellar vesicles and compared their effects with parent peptides transportan10 and PF3. In addition, cellular uptake and peptide-mediated oligonucleotide delivery were analyzed. Calcein leakage experiments showed that similar to transportan10, NickFect51 caused a significant degree of membrane leakage, whereas NickFect1, similar to PF3, was less membrane perturbing. The results are in agreement with previously published results indicating that NickFect51 is a more efficient endosomal escaper. However, the presence of a large cargo like plasmid DNA inhibited NickFect's membrane perturbation and cellular uptake efficiency of the peptide was reduced. We conclude that the pathway for cellular uptake of peptide complexes is cargo dependent, whereas the endosomal escape efficacy depends on peptide hydrophobicity and chemical structure. For small interfering RNA delivery, NickFect51 appears to be optimal. The biophysical signature shows that the peptide alone causes membrane perturbation, but the cargo complex does not. These two biophysical characteristics of the peptide and its cargo complex may be the signature of an efficient delivery vector system.

National Category
Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-109101 (URN)10.1016/j.bbamem.2014.08.011 (DOI)000343847200015 ()25135660 (PubMedID)
Available from: 2014-11-12 Created: 2014-11-12 Last updated: 2017-04-20Bibliographically approved
2. Simultaneous membrane interaction of amphipathic peptide monomers, self-aggregates and cargo complexes detected by Fluorescence Correlation Spectroscopy
Open this publication in new window or tab >>Simultaneous membrane interaction of amphipathic peptide monomers, self-aggregates and cargo complexes detected by Fluorescence Correlation Spectroscopy
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2017 (English)Manuscript (preprint) (Other academic)
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 oligonucleotide cargo is mostly driven by electrostatic and hydrophobic interactions, which may result in polydisperse and polymorphic particles. Here we investigate the coexistence of distinct molecular and supramolecular 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. 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’s monomer as well as of peptide aggregates and complexes. Strategies to avoid peptide adsorption to solid surfaces and self-aggregation were developed and allowed successful FCS measurements on peptide/oligonucleotide complexes 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 molecular and supramolecular species, distributed unevenly at the plasma membrane.

Keyword
Cell-penetrating peptide, Fluorescence correlation spectroscopy, PepFect14, siRNA, amphipathic peptide, peptide aggregates, plasma membrane
National Category
Chemical Sciences Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-141095 (URN)
Funder
Swedish Research Council, 521-2011-2461, 2012-2595Swedish Cancer Society, 2014/259Swedish Foundation for Strategic Research , SBE13-0115Knut and Alice Wallenberg Foundation, 2011.0218EU, FP7, Seventh Framework Programme, GLORIA-602919EU, FP7, Seventh Framework Programme, FP7/2007-2013
Available from: 2017-03-30 Created: 2017-03-30 Last updated: 2017-05-16Bibliographically approved
3. pH-responsive PepFect cell-penetrating peptides
Open this publication in new window or tab >>pH-responsive PepFect cell-penetrating peptides
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2016 (English)In: International Journal of Pharmaceutics, ISSN 0378-5173, E-ISSN 1873-3476, Vol. 501, no 1-2, 32-38 p.Article in journal (Refereed) Published
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.

Keyword
Cell-penetrating peptide, Oligonucleotide delivery, Calcein leakage, PepFect, Cellular uptake, pH-responsive
National Category
Chemical Sciences Pharmaceutical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-128503 (URN)10.1016/j.ijpharm.2016.01.055 (DOI)000370845300004 ()26821060 (PubMedID)
Available from: 2016-04-11 Created: 2016-03-30 Last updated: 2017-04-20Bibliographically approved
4. Saturated Fatty Acid Analogues of Cell-Penetrating Peptide PepFect14: Role of Fatty Acid Modification in Complexation and Delivery of Splice-Correcting Oligonucleotides
Open this publication in new window or tab >>Saturated Fatty Acid Analogues of Cell-Penetrating Peptide PepFect14: Role of Fatty Acid Modification in Complexation and Delivery of Splice-Correcting Oligonucleotides
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2017 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 28, no 3, 782-792 p.Article in journal (Refereed) Published
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.

Keyword
peptide, delivery, oligonucleotide
National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-140331 (URN)10.1021/acs.bioconjchem.6b00680 (DOI)000396801500012 ()
Available from: 2017-03-06 Created: 2017-03-06 Last updated: 2017-05-03Bibliographically approved

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