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PEG-stabilized lipid disks as carriers for amphiphilic antimicrobial peptides
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Physical Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Physical Chemistry.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
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2011 (English)In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 156, no 3, 323-328 p.Article in journal (Refereed) Published
Abstract [en]

Antimicrobial peptides hold potential as a possible alternative, or complement, to conventional antibiotics but new, safe and efficient means are needed for formulation and administration of the peptides. In this study we have investigated the utility of a novel type of lipid particles, the polyethylene glycol-stabilized lipid-disks, as carriers for the model peptide melittin. The structural integrity of the carrier particle when loaded with the peptide was investigated using cryo-transmission electron microscopy. Liposome leakage upon addition of the peptide-lipid-disks was monitored as a means to verify the membrane lytic effect of the formulation. The susceptibility of melittin to tryptic digestion was studied and compared in the absence and presence of lipid-disks. Finally, the antibacterial effect of the peptide-lipid-disk formulation was compared to that of free melittin after both single and repeated exposure to Escherichia coli. The results show that melittin can redistribute from the disk into a new host membrane and that formulation in the disks does not compromise melittin's membrane permeabilizing ability. Further, the peptide was found to be fully protected against degradation when bound to the disks. Time-kill experiments revealed that all the antibacterial effect of melittin administered in free form was gone after a single exposure to E. coli. In contrast, the disk formulation showed significant cell-killing effect also upon a second exposure to bacteria, indicating an extended release of peptide from the lipid-disks. These results suggest that the lipid-disks constitute a new class of promising carriers for peptide antibiotics.

Place, publisher, year, edition, pages
2011. Vol. 156, no 3, 323-328 p.
Keyword [en]
PEG-stabilized lipid disk, Antimicrobial peptide, Melittin, E. coli, Peptide delivery
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-158960DOI: 10.1016/j.jconrel.2011.08.029ISI: 000298555000007PubMedID: 21903146OAI: oai:DiVA.org:uu-158960DiVA: diva2:441882
Available from: 2011-09-19 Created: 2011-09-19 Last updated: 2017-12-08Bibliographically approved
In thesis
1. Development and Evaluation of Lipodisks Intended for Use as Biomimetic Membranes and Drug Carriers
Open this publication in new window or tab >>Development and Evaluation of Lipodisks Intended for Use as Biomimetic Membranes and Drug Carriers
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Polyethylene glycol-stabilized lipodisks have emerged as a novel type of lipid-based nanoparticles with high potential as both drug carriers and biomimetic membranes. In this thesis we assess both of these applications, and show how the properties of the lipodisks can be further developed and optimized.

Initially, we show that the antimicrobial peptides melittin, alamethicin and magainin 2, in spite of their very different physico-chemical properties and suggested modes of action on membranes, all have high affinity to lipodisks. Using melittin as a model peptide, we confirm a maintained antimicrobial effect of disk-formulated peptides. We also show that melittin dissociates slowly from the disks, resulting in extended drug release and prolonged antibacterial effect. Additionally, we present evidence that the peptide is protected against enzymatic degradation when formulated in the disks.

Further, we develop a stable HPLC-MS system with immobilized lipodisks as model membranes. The stability of the system is confirmed by drug partitioning analysis using 15 different drug compounds. We also show how the lipodisk column can be supplemented with cyclooxygenase by in situ incorporation of the protein in the lipodisks. The specific binding of the protein to the disks is confirmed using QCM-D.

Finally, by changing the polymer length and applying a new preparation protocol, we have optimized the lipodisks for use as drug carriers and biomimetic membranes. Previous lipodisk studies have been conducted on systems containing PEG-lipids with polymer molecular weights of 2000 or 5000 Da. Also, conventional protocols for the preparation of lipodisks typically require a PEG-lipid concentration of 15 mol% or more. Here we show that stable lipodisks can also be produced using PEG-lipids with a 1000 Da molecular weight polymer and that the use of shorter PEG-lipids dramatically improve the amount of lipodisks that can be immobilized on silica surfaces. Moreover, through the development of a method in which lipid mixtures are sonicated at low temperatures, we produce lipodisks containing as little as 2 mol% PEG-lipid. We present data verifying that these disks are superior to disks with higher PEG-lipid content in terms of their ability to incorporate externally added PEG-lipids functionalized with targeting agents.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. 58 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1340
Keyword
model membranes, drug delivery, drug partitioning, antimicrobial peptides, nanocarriers, cryo-TEM, polymer-stabilized bilayer disks
National Category
Physical Chemistry
Research subject
Chemistry with specialization in Physical Chemistry
Identifiers
urn:nbn:se:uu:diva-268667 (URN)978-91-554-9466-7 (ISBN)
Public defence
2016-03-18, B22, BMC, Husargatan 3, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2016-02-26 Created: 2015-12-09 Last updated: 2016-03-09
2. Development of lipodisks as carriers for cationic amphiphilic peptides
Open this publication in new window or tab >>Development of lipodisks as carriers for cationic amphiphilic peptides
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Antibiotics have made a tremendous contribution to mankind. They are one of the most successful medicines in human history. However, more and more bacterial strains develop resistance and the risk to public health can hardly be overstated. New types of antibiotics are urgently needed. Antimicrobial peptides (AMPs) have emerged as potential antibiotics because of their broad-spectrum activities and non-conventional mechanism of action. More recently, they have also received attention as promising anticancer agents. The clinical and commercial development of AMPs as a new generation of antibiotics and anticancer drugs is hampered, however, by issues concerning the toxicity, specificity and stability of the peptides.

The aim of this thesis has been to explore if formulation in a novel type of nanocarriers, referred to as lipodisks, can be used to increase the therapeutic potential of AMPs as antimicrobial and anticancer agents. Focus has been on AMPs classified as cationic amphiphilic peptides.

Encouragingly, the data presented suggests that the therapeutic potential of the AMP melittin as an antimicrobial and anticancer agent can be substantially increased by formulation in lipodisks. When formulated in the lipodisk, melittin is protected against enzymatic degradation. The lipodisk also offer a slow-release effect that sustains the bacterial cell-killing effect. We also show that specific delivery of melittin to tumour cells can be obtained by formulating the peptide in small EGF-targeting lipodisks.

Melittin contains a tryptophan residue and its interaction with lipodisks can be characterized by means of fluorimetric binding assays. In order to investigate the binding behavior also for peptides that lack intrinsic fluorescence, we developed a method based on measurements using the QCM-D technique. Studies using this, and other techniques, confirmed that it is a general behavior for cationic amphiphilic peptides to preferentially bind to the highly curved rim of lipodisks. Results of our binding studies show that the peptide to lipid ratio in the lipodisks can be tuned and optimized by varying the size and charge of the disks.

Taken together, the findings in this thesis point towards PEG-stabilized lipodisks as promising nanocarriers for antibacterial and anticancer peptides.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. 48 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1442
National Category
Natural Sciences Physical Chemistry
Identifiers
urn:nbn:se:uu:diva-305443 (URN)978-91-554-9729-3 (ISBN)
Public defence
2016-12-02, B41, BMC, Husargatan 3, Uppsala, 10:15 (Swedish)
Opponent
Supervisors
Available from: 2016-11-11 Created: 2016-10-18 Last updated: 2016-11-16

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Zetterberg, Malin M.Reijmar, KarinPränting, MariaEngström, ÅkeAndersson, Dan I.Edwards, Katarina
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