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Development and Evaluation of Lipodisks Intended for Use as Biomimetic Membranes and Drug Carriers
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
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 [en]
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: urn:nbn:se:uu:diva-268667ISBN: 978-91-554-9466-7 (print)OAI: oai:DiVA.org:uu-268667DiVA: diva2:898122
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
List of papers
1. Effect of α-helical peptides on liposome structure: A comparative study of melittin and alamethicin
Open this publication in new window or tab >>Effect of α-helical peptides on liposome structure: A comparative study of melittin and alamethicin
2010 (English)In: Journal of Colloid and Interface Science, ISSN 0021-9797, E-ISSN 1095-7103, Vol. 346, no 1, 127-135 p.Article in journal (Refereed) Published
Abstract [en]

Cryo-transmission electron microscopy was used in combination with turbidity and leakage measurements to explore and compare the membrane perturbing effects of melittin and alamethicin on POPC-based liposomes of varying composition. The results show that the two peptides, despite their differences in physico-chemical properties and proposed mode of action, induce similar structural effects on the liposomes. Importantly, whereas low peptide concentrations leave pure POPC liposomes intact and seemingly unperturbed, POPC liposomes supplemented with 40 mol.% cholesterol change their shape, rupture and fuse in response to the addition of both melittin and alamethicin. In the case of alamethicin, but not melittin, fusion is effectively prevented by inclusion of 10 mol.% POPG in the liposome membranes. By means of a competitive binding assay we furthermore show that alamethicin, in line with earlier findings for melittin, possess high affinity for positively curved lipid surfaces. Moreover, results from the present study show that magainin 2 has a similar preference for curved surfaces.

Place, publisher, year, edition, pages
Elsevier, 2010
Keyword
Melittin, Alamethicin, Magainin, Liposomes, Affinity, Cryo-transmission electron microscopy
National Category
Physical Chemistry
Research subject
Chemistry with specialization in Physical Chemistry
Identifiers
urn:nbn:se:uu:diva-124618 (URN)10.1016/j.jcis.2010.02.032 (DOI)000277132700020 ()20226468 (PubMedID)
Available from: 2010-05-05 Created: 2010-05-05 Last updated: 2017-12-12Bibliographically approved
2. PEG-stabilized lipid disks as carriers for amphiphilic antimicrobial peptides
Open this publication in new window or tab >>PEG-stabilized lipid disks as carriers for amphiphilic antimicrobial peptides
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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.

Keyword
PEG-stabilized lipid disk, Antimicrobial peptide, Melittin, E. coli, Peptide delivery
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-158960 (URN)10.1016/j.jconrel.2011.08.029 (DOI)000298555000007 ()21903146 (PubMedID)
Available from: 2011-09-19 Created: 2011-09-19 Last updated: 2017-12-08Bibliographically approved
3. Immobilized lipodisks as model membranes in high-throughput HPLC-MS analysis
Open this publication in new window or tab >>Immobilized lipodisks as model membranes in high-throughput HPLC-MS analysis
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2013 (English)In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 405, no 14, 4859-4869 p.Article in journal (Refereed) Published
Abstract [en]

Lipodisks, also referred to as polyethylene glycol (PEG)-stabilized bilayer disks, have previously been demonstrated to hold great potential as model membranes in drug partition studies. In this study, an HPLC-MS system with stably immobilized lipodisks is presented. Functionalized lipodisks were immobilized on two different HPLC support materials either covalently by reductive amination or by streptavidin-biotin binding. An analytical HPLC column with immobilized lipodisks was evaluated by analysis of mixtures containing 15 different drug compounds. The efficiency, reproducibility, and stability of the system were found to be excellent. In situ incorporation of cyclooxygenase-1 (COX-1) in immobilized lipodisks on a column was also achieved. Specific binding of COX-1 to the immobilized lipodisks was validated by interaction studies with QCM-D. These results, taken together, open up the possibility of studying ligand interactions with membrane proteins by weak affinity chromatography.

Keyword
Lipodisks, COX-1, HPLC-MS, Model membrane, Drug partition studies, Membrane protein, WAC, Weak affinity chromatography
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-197337 (URN)10.1007/s00216-013-6892-3 (DOI)000318312400018 ()
Funder
Swedish Research Council
Note

De två (2) första författarna delar förstaförfattarskapet.

Available from: 2013-03-22 Created: 2013-03-22 Last updated: 2017-12-06Bibliographically approved
4. Optimization of Lipodisk Properties by Modification of the Extent and Density of the PEG Corona
Open this publication in new window or tab >>Optimization of Lipodisk Properties by Modification of the Extent and Density of the PEG Corona
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2016 (English)In: Journal of Colloid and Interface Science, ISSN 0021-9797, E-ISSN 1095-7103, Vol. 484, 86-96 p.Article in journal (Refereed) Published
Abstract [en]

Lipodisks are nanosized flat, circular, phospholipid bilayers that are edge-stabilized by polyethylene glycol-conjugated lipids (PEG-lipids). Over the last decade, lipodisks stabilized with PEG of molecular weight 2000 or 5000 have been shown to hold high potential as both biomimetic membranes and drug carriers. In this study we investigate the possibilities to optimize the properties of the lipodisks, and widen their applicability, by reducing the PEG molecular weight and/or the density of the PEG corona. Results obtained by cryo-transmission electron microscopy and dynamic light scattering show that stable, well-defined lipodisks can be produced from mixtures of distearoylphosphatidylcholine (DSPC) and distearoylphosphatidylethanolamine conjugated to PEG of molecular weight 1000 (DSPE-PEG(1000)). Preparations based on the use of DSPE-PEG(750) tend, in contrast, to be polydisperse in size and structure. By comparing immobilization of lipodisks stabilized with DSPE-PEG(1000), DSPE-PEG(2000), and DSPE-PEG(5000) to porous and smooth silica surfaces, we show that the amount of surface bound disks can be considerably improved by the use of PEG-lipids with reduced molecular weight. Further, a modified preparation protocol that enables production of lipodisks with very low PEG-lipid content is described. The reduced PEG density, which facilitates the incorporation of externally added ligand-linked PEG-lipids, is shown to be beneficial for the production of targeting lipodisks.

Keyword
Lipid self-assembly; Bilayer disks; Biomimetic membranes; Surface immobilization; Drug delivery; Nanocarriers; Specific targeting
National Category
Physical Chemistry
Identifiers
urn:nbn:se:uu:diva-274972 (URN)10.1016/j.jcis.2016.08.067 (DOI)000385690200011 ()27592189 (PubMedID)
Funder
Swedish Research Council, 621-2011-3524Swedish Cancer Society, CAN 2014/617
Available from: 2016-01-27 Created: 2016-01-27 Last updated: 2017-11-30Bibliographically approved

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