uu.seUppsala University Publications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Microgel Interactions with Peptides and Proteins: Consequence of Peptide and Microgel Properties
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Microgels are lightly cross-linked hydrogel particles in the sub-micrometer to micrometer size range with a capacity to drastically change their volume in response to changes in the external environment. Microgels have an ability to bind and store substances such as biomacromolecular drugs, notably proteins and peptides, and release them upon stimuli, making them potential candidates as drug delivery vehicles and functional biomaterials. This thesis aims at clarifying important factors affecting peptide-microgel interactions. These interactions were studied by micromanipulator-assisted light and fluorescence microscopy focusing on microgel deswelling in response to peptide binding, as well as re-swelling in response to peptide release or enzymatic degradation. To evaluate peptide uptake in microgels, solution depletion measurements were used whereas the peptide secondary structure was investigated by circular dichroism. In addition, the peptide and enzyme distribution within microgels was analyzed with confocal microscopy.

Results presented in this thesis demonstrate that peptide incorporation into microgels, as well as peptide-induced microgel deswelling, increases with peptide length and charge density. In addition, results demonstrate that the peptide charge (length) rather than peptide charge density determines microgels deswelling. End-to-end cyclization is shown to not noticeably influence peptide-microgel interactions, suggesting that peptide cyclization can be used in combination with oppositely charged microgel carriers to improve the proteolytic and chemical stability of the peptide compared to the corresponding linear variant. Peptide secondary structure is found to drastically affect peptide incorporation into, and release from, oppositely charged microgels. Furthermore, it is shown that microgel charge density, peptide molecular weight, and enzyme concentration all greatly influence microgel bound peptide degradation. Of importance for applications, protective effects of microgels against proteolytic peptide degradation are observed only at sufficiently high microgel charge densities. Enzyme-mediated microgel degradation is shown to increase with increasing enzyme concentration, while an increased peptide loading in microgels causes a concentration-dependent decrease in microgel degradation.

Taken together, results obtained in this work have provided some insight into factors of importance for rational use of microgels as delivery systems for protein or peptide drugs, but also in a host of other biomedical applications using weakly cross-linked polymer systems.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. , 65 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 196
Keyword [en]
Binding, Degradation, Enzyme, Gel, Hydrogel, Microgel, Peptide, Protein, Release
National Category
Pharmaceutical Sciences Physical Chemistry Materials Engineering
Research subject
Pharmaceutical Physical Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-242893ISBN: 978-91-554-9157-4 (print)OAI: oai:DiVA.org:uu-242893DiVA: diva2:785349
Public defence
2015-03-20, B21, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2015-02-24 Created: 2015-02-02 Last updated: 2015-03-11
List of papers
1. Effects of Peptide Secondary Structure on the Interaction with Oppositely Charged Microgels
Open this publication in new window or tab >>Effects of Peptide Secondary Structure on the Interaction with Oppositely Charged Microgels
Show others...
2011 (English)In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 12, no 2, 419-424 p.Article in journal (Refereed) Published
Abstract [en]

The importance of peptide secondary structure on the interaction between antimicrobial peptides and oppositely charged poly(acrylic acid-co-acrylamide) microgels of various charge density was investigated for EFKRIVQRIKDFLRNLV (EFK17). Through D-enantiomer (EFK17-d/a; E(dF)KR(dI)VQR(dI)KD(dF)LRNLV) or tryptophan (EFK17-W/a; EWKRWVQRWKDFLRNLV) substitutions, both conformation-dependent and -independent amphiphilicity of this peptide could be precisely controlled. Peptide secondary structure was investigated by circular dichroism, whereas microgel deswelling and reswelling in response to peptide binding and release were studied by micromanipulator-assisted light and fluorescence microscopy, and peptide uptake in the microgels was determined from solution depletion measurements. Results show that peptide binding to the microgel is highly influenced by peptide secondary structure. EFK17-a, characterized by an idealized helix with all polar/charged amino acids located at one side of the helix, and all nonpolar/hydrophobic residues on the other, displays pronounced alpha-helix induction on peptide binding to the microgels. EFK17-d/a, on the other hand, displays no such amphiphilic helix induction. Mirroring this, EFK17-a displays substantially higher binding to the microgels than EFK17-d/a as well as much larger peptide-induced microgel deswelling. For EFK17-W/a, both conformation-dependent and -independent amphiphilicity effects were demonstrated. Overall, the results show that peptide conformational aspects need to be considered in peptide/microgel interactions, for example, in the design of microgel carrier systems for peptide drugs.

National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-149054 (URN)10.1021/bm101165e (DOI)000287175700017 ()21182237 (PubMedID)
Available from: 2011-03-15 Created: 2011-03-15 Last updated: 2017-12-11Bibliographically approved
2. Effects of peptide cyclization on the interaction with oppositely charged microgels
Open this publication in new window or tab >>Effects of peptide cyclization on the interaction with oppositely charged microgels
2011 (English)In: Colloids and Surfaces A: Physicochemical and Engineering Aspects, ISSN 0927-7757, E-ISSN 1873-4359, Vol. 391, no 1-3, 62-68 p.Article in journal (Refereed) Published
Abstract [en]

The effect of peptide cyclization on the interaction between antimicrobial peptides and oppositely charged poly(acrylic acid-co-acrylamide) microgels of various charge density was investigated for linear and cyclic variants of peptide oligomers (C(ARKKAAKA)nC) (n = 1, 1.5, 2, 3). Through this, peptide length could be varied without substantially affecting peptide charge density and mean hydrophobicity. Furthermore, the peptides were demonstrated to display random coil conformation both in aqueous solution and when bound to oppositely charged microgels, allowing effects of cyclization to be monitored without interference from conformational transitions. With increasing peptide length, both cyclic and linear peptide variants displayed increased binding affinity to oppositely charged microgels. For all peptide lengths, however, the difference between cyclic and linear peptide variants was marginal at most, hence cyclization had little or no influence in peptide incorporation to oppositely charged microgels. In parallel, microgel deswelling increased with peptide length for both linear and cyclic peptide variants, while linear and cyclic peptide variants of the same length displayed very similar peptide-induced deswelling. Also electrolyte-induced peptide desorption from the microgels was similar for linear and cyclic peptide variants. Taken together, these findings demonstrate that end-to-end cyclization does not markedly affect peptide incorporation into, and release from, oppositely charged microgels. This opens up opportunities for the use of microgels as carriers for peptides which have been cyclized in order to improve their proteolytic and chemical stability, or in order to achieve other therapeutic advantages compared to the corresponding linear peptide variant.

Keyword
Cyclization, Microgel, Peptide
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-168831 (URN)10.1016/j.colsurfa.2011.01.029 (DOI)000299068100009 ()
Note
18th International Symposium on Surfactants in Solution (SIS), Melbourne Australia, 14-19 November 2010Available from: 2012-02-16 Created: 2012-02-16 Last updated: 2017-12-07Bibliographically approved
3. Peptide-Microgel Interactions in the Strong Coupling Regime
Open this publication in new window or tab >>Peptide-Microgel Interactions in the Strong Coupling Regime
2012 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 116, no 35, 10964-10975 p.Article in journal (Refereed) Published
Abstract [en]

The interaction between lightly cross-linked poly(acrylic acid) microgels and oppositely charged peptides was investigated as a function of peptide length, charge density, pH, and salt concentration, with emphasis on the strong coupling regime at high charge contrast. By micromanipulator-assisted light microscopy, the equilibrium volume response of single microgel particles upon oligolysine and oligo(lysine/alanine) absorption could be monitored in a controlled fashion. Results show that microgel deswelling, caused by peptide binding and network neutralization, increases with peptide length (3 < 5 < 10) and charge density (30% < 50% < 100%). Furthermore, oligomer-induced microgel deswelling was more pronounced at pH 5 than at pH 8, reflecting the lower network charge density in the former case (pK(a) for the isolated acrylic acid approximate to 4.7). In order to describe these highly coupled systems, a model was developed, in which counterion/peptide-mediated electrostatic attraction between the network chains is described using an exponential force law, and the network elasticity by the inverse Langevin theory. The model was used to calculate the composition of microgels in contact with reservoir solutions of peptides and simple electrolytes. At high electrostatic coupling, the calculated swelling curves were found to display first-order phase transition behavior. The model was demonstrated to capture pH- and electrolyte-dependent microgel swelling, as well as effects of peptide length and charge density on microgel deswelling. The analysis demonstrated that the peptide charge (length), rather than the peptide charge density, determines microgel deswelling. Furthermore, a transition between continuous and discrete network collapse was identified, consistent with experimental results in the present investigations, as well as with results from the literature on microgel deswelling caused by multivalent cations.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-182754 (URN)10.1021/jp306121h (DOI)000308339400060 ()
Available from: 2012-10-18 Created: 2012-10-15 Last updated: 2017-12-07Bibliographically approved
4. Factors Affecting Enzymatic Degradation of Microgel-Bound Peptides
Open this publication in new window or tab >>Factors Affecting Enzymatic Degradation of Microgel-Bound Peptides
2013 (English)In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 14, no 7, 2317-2325 p.Article in journal (Refereed) Published
Abstract [en]

Proteolytic degradation and release of microgel-bound peptides was investigated for trypsin, poly(acrylic acid-co-acrylamide) microgels (70-90 mu m in diameter), and oppositely charged polylysine, using a method combination of confocal microscopy and micromanipulator-assisted light microscopy. Results show that trypsin-induced release of polylysine increased with increasing trypsin concentration, decreasing microgel charge density and decreasing peptide molecular weight. While the microgel offered good protection against enzymatic degradation at high microgel charge density, it was also observed that the cationic peptide enabled trypsin to bind throughout the peptide-loaded microgels, even when it did not bind to the peptide-void ones. With the exception of highly charged microgels, proteolytic degradation throughout the peptide-loaded microgel resulted in the generation of short and non-adsorbing peptide stretches, giving rise to the concentration and peptide length dependence observed. A simple random scission model was able to qualitatively capture these experimental findings. collectively, the results demonstrate that microgel charge density, peptide molecular weight, and enzyme concentration greatly influence degradation/release of microgel-bound peptides and need to be considered in the use of microgels, e.g., as carriers for protein and peptide drugs.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-204977 (URN)10.1021/bm400431f (DOI)000321793700021 ()
Available from: 2013-08-16 Created: 2013-08-13 Last updated: 2017-12-06Bibliographically approved
5. Chain and Pore-Blocking Effects on Matrix Degradation in Protein-Loaded Microgels
Open this publication in new window or tab >>Chain and Pore-Blocking Effects on Matrix Degradation in Protein-Loaded Microgels
2014 (English)In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 15, no 10, 3671-3678 p.Article in journal (Refereed) Published
Abstract [en]

Factors affecting matrix degradation in protein-loaded microgels were investigated for dextran-based microgels, the sugar-binding protein Concanavalin A (ConA), and the dextran-degrading enzyme Dextranase. For this system, effects of enzyme, protein, and glucose concentrations, as well as pH, were considered. Microgel network degradation was monitored by micromanipulator-assisted light microscopy, whereas enzyme and protein distributions were monitored by confocal microscopy. Results show that Dextranase-mediated microgel degradation increased with increasing enzyme concentration, whereas an increased ConA loading in the dextran microgels caused a concentration-dependent decrease in microgel degradation. In the presence of glucose, competitive release of microgel-bound ConA restored the microgel degradation observed in the absence of ConA. To clarify effects of mass transport limitations, microgel degradation was compared to that of non-cross-linked dextran, demonstrating that ConA limits enzyme substrate access in dextran microgels primarily through pore blocking and induction of pore shrinkage. The experimentally observed effects were qualitatively captured by a modified Michaelis-Menten approach for spherical symmetry, in which network blocking by ConA was included. Taken together, the results demonstrate that matrix degradation of protein-loaded microgels depends sensitively on a number of factors, which need to be considered in the use of microgels in biomedical applications.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-236549 (URN)10.1021/bm5009525 (DOI)000343026600022 ()25144139 (PubMedID)
Available from: 2014-11-26 Created: 2014-11-19 Last updated: 2017-12-05Bibliographically approved

Open Access in DiVA

fulltext(1537 kB)363 downloads
File information
File name FULLTEXT01.pdfFile size 1537 kBChecksum SHA-512
3f1143f6647edb93b6fded02493955214f1f9853a5be71544ac4dfae3d9d5b1213f4a01dbbad1e2899dcc3ce570c2daf823f5286ae84af7695a5c27d8a30e478
Type fulltextMimetype application/pdf
Buy this publication >>

Authority records BETA

Widenbring, Ronja

Search in DiVA

By author/editor
Widenbring, Ronja
By organisation
Department of Pharmacy
Pharmaceutical SciencesPhysical ChemistryMaterials Engineering

Search outside of DiVA

GoogleGoogle Scholar
Total: 363 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 1469 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf