| 1. |
- Aili, Daniel, 1977-, et al.
(författare)
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Aggregation-Induced Folding of a de novo Designed Polypeptide Immobilized on Gold Nanoparticles
- 2006
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Ingår i: Journal of the American Chemical Society. - : ACS Publications. - 0002-7863 .- 1520-5126. ; 128:7, s. 2194 -2195
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Tidskriftsartikel (refereegranskat)abstract
- This communication reports the first steps in the construction of a novel, nanoparticle-based hybrid material for biomimetic and biosensor applications. Gold nanoparticles were modified with synthetic polypeptides to enable control of the particle aggregation state in a switchable manner, and particle aggregation was, in turn, found to induce folding of the immobilized peptides.
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| 2. |
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| 3. |
- Aili, Daniel, 1977-, et al.
(författare)
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Assembly of Polypeptide-Functionalized Gold Nanoparticles through a Heteroassociation- and Folding-Dependent Bridging
- 2008
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Ingår i: Nano letters (Print). - : ACS Publications. - 1530-6984 .- 1530-6992. ; 8:8, s. 2473-2478
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Tidskriftsartikel (refereegranskat)abstract
- Gold nanoparticles were functionalized with a synthetic polypeptide, de novo-designed to associate with a charge complementary linker polypeptide in a folding-dependent manner. A heterotrimeric complex that folds into two disulphide-linked four-helix bundles is formed when the linker polypeptide associates with two of the immobilized peptides. The heterotrimer forms in between separate particles and induces a rapid and extensive aggregation with a well-defined interparticle spacing. The aggregated particles are redispersed when the disulphide bridge in the linker polypeptide is reduced.
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| 4. |
- Aili, Daniel, 1977-, et al.
(författare)
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Colorimetric Protein Sensing by Controlled Assembly of Gold Nanoparticles Functionalized with Synthetic Receptors
- 2009
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Ingår i: Small. - : Wiley. - 1613-6810 .- 1613-6829. ; 5:21, s. 2445-2452
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Tidskriftsartikel (refereegranskat)abstract
- A novel strategy is described for the colorimetric sensing of proteins, based on polypeptide-functionalized gold nanoparticles. Recognition is accomplished using a polypeptide sensor scaffold designed to specifically bind to the model analyte, human carbonic anhydrase II (HCAII). The extent of particle aggregation, induced by the Zn2+-triggered dimerization and folding of a second polypeptide also present on the surface of the gold nanoparticle, gives a readily detectable colorimetric shift that is dependent on the concentration of the target protein. In the absence of HCAII, particle aggregation results in a major redshift of the plasmon peak, whereas analyte binding prevented the formation of dense aggregates, significantly reducing the magnitude of the redshift. The versatility of the technique is demonstrated using a second model system based on the recognition of a peptide sequence from the tobacco mosaic virus coat protein (TMVP) by a recombinant antibody fragment (Fab57P). Concentrations down to approximate to 10 nM and approximate to 25 nM are detected for HCAII and Fab57P, respectively. This strategy is proposed as a generic platform for robust and specific protein analysis that can be further developed to monitor a wide range of target proteins.
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| 5. |
- Aili, Daniel, 1977-, et al.
(författare)
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Controlled Assembly of Gold Nanoparticles using De Novo Designed Polypeptide Scaffolds
- 2008
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Ingår i: Proceedings SPIE, Vol. 6885, Photonic Biosensing and Microoptics. - : SPIE. ; , s. 688506-1-688506-8
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Konferensbidrag (refereegranskat)abstract
- Heterodimerization between designed helix-loop-helix polypeptides was utilized in order to assemble gold nanoparticles on planar substrates. The peptides were designed to fold into four-helix bundles upon dimerization. A Cys-residue in the loop region was used to immobilize one of the complementary peptides on a maleimide containing SAM on planar gold substrates whereas the second peptide was immobilized directly on gold nanoparticles. Introducing the peptide decorated particles over a peptide functionalized surface resulted in particle assembly. Further, citrate stabilized particles were assembled on amino-silane modified glass and silicon substrates. By subsequently introducing peptides and gold nanoparticles, particle-peptide hybrid multi layers could be formed.
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| 6. |
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| 7. |
- Aili, Daniel, 1977-, et al.
(författare)
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Folding Induced Assembly of Polypeptide Decorated Gold Nanoparticles
- 2008
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 130:17, s. 5780-5788
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Tidskriftsartikel (refereegranskat)abstract
- Reversible assembly of gold nanoparticles controlled by the homodimerization and folding of an immobilized de novo designed synthetic polypeptide is described. In solution at neutral pH, the polypeptide folds into a helix-loop-helix four-helix bundle in the presence of zinc ions. When immobilized on gold nanoparticles, the addition of zinc ions induces dimerization and folding between peptide monomers located on separate particles, resulting in rapid particle aggregation. The particles can be completely redispersed by removal of the zinc ions from the peptide upon addition of EDTA. Calcium ions, which do not induce folding in solution, have no effect on the stability of the peptide decorated particles. The contribution from folding on particle assembly was further determined utilizing a reference peptide with the same primary sequence but containing both D and L amino acids. Particles functionalized with the reference peptide do not aggregate, as the peptides are unable to fold. The two peptides, linked to the nanoparticle surface via a cysteine residue located in the loop region, form submonolayers on planar gold with comparable properties regarding surface density, orientation, and ability to interact with zinc ions. These results demonstrate that nanoparticle assembly can be induced, controlled, and to some extent tuned, by exploiting specific molecular interactions involved in polypeptide folding.
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| 8. |
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| 9. |
- Aili, Daniel, 1977-
(författare)
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Polypeptide-Based Nanoscale Materials
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Self-assembly has emerged as a promising technique for fabrication of novel hybrid materials and nanostructures. The work presented in this thesis has been focused on developing nanoscale materials based on synthetic de novo designed polypeptides. The polypeptides have been utilized for the assembly of gold nanoparticles, fibrous nanostructures, and for sensing applications.The 42-residue polypeptides are designed to fold into helix-loop-helix motifs and dimerize to form four-helix bundles. Folding is primarily driven by the formation of a hydrophobic core made up by the hydrophobic faces of the amphiphilic helices. The peptides have either a negative or positive net charge at neutral pH, depending on the relative abundance of Glu and Lys. Charge repulsion thus prevents homodimerization at pH 7 while promoting hetero-dimerization through the formation of stabilising salt bridges. A Cys incorporated in position 22, located in the loop region, allowed for directed, thiol-dependent, immobilization on planar gold surfaces and gold nanoparticles. The negatively charged (Glu-rich) peptide formed homodimers and folded in solution at pH < 6 or in the presence of certain metal ions, such as Zn2+. The folding properties of this peptide were retained when immobilized directly on gold, which enabled reversible assembly of gold nanoparticles resulting in aggregates with well-defined interparticle separations. Particle aggregation was found to induce folding of the immobilized peptides but folding could also be utilized to induce aggregation of the particles by exploiting the highly specific interactions involved in both homodimerization and hetero-association. The possibility to control the assembly of polypeptide-functionalized gold nanoparticles was utilized in a colorimetric protein assay. Analyte binding to immobilized ligands prevented the formation of dense particle aggregates when subjecting the particles to conditions normally causing extensive aggregation. Analyte binding could hence easily be distinguished by the naked eye. Moreover, the peptides were utilized to assemble gold nanoparticles on planar gold and silica substrates.Fibrous nanostructures were realized by linking monomers through a disulphide-bridge. The disulphide-linked peptides were found to spontaneously assemble into long and extremely thin peptide fibres as a result of a propagating association mediated by folding into four-helix bundles.
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| 10. |
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| 11. |
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| 12. |
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| 13. |
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| 14. |
- Berglund, Linn, et al.
(författare)
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Self-Assembly of Nanocellulose Hydrogels Mimicking Bacterial Cellulose for Wound Dressing Applications
- 2023
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Ingår i: Biomacromolecules. - : American Chemical Society (ACS). - 1525-7797 .- 1526-4602. ; 24:5, s. 2264-2277
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Tidskriftsartikel (refereegranskat)abstract
- The self-assembly of nanocellulose in the form of cellulose nanofibers (CNFs) can be accomplished via hydrogen-bonding assistance into completely bio-based hydrogels. This study aimed to use the intrinsic properties of CNFs, such as their ability to form strong networks and high absorption capacity and exploit them in the sustainable development of effective wound dressing materials. First, TEMPO-oxidized CNFs were separated directly from wood (W-CNFs) and compared with CNFs separated from wood pulp (P-CNFs). Second, two approaches were evaluated for hydrogel self-assembly from W-CNFs, where water was removed from the suspensions via evaporation through suspension casting (SC) or vacuum-assisted filtration (VF). Third, the W-CNF-VF hydrogel was compared to commercial bacterial cellulose (BC). The study demonstrates that the self-assembly via VF of nanocellulose hydrogels from wood was the most promising material as wound dressing and displayed comparable properties to that of BC and strength to that of soft tissue.
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| 15. |
- Christoffersson, Jonas, 1986-, et al.
(författare)
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Fabrication of modular hyaluronan-PEG hydrogels to support 3D cultures of hepatocytes in a perfused liver-on-a-chip device
- 2019
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Ingår i: Biofabrication. - : Institute of Physics (IOP). - 1758-5082 .- 1758-5090. ; 11:1, s. 1-13
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Tidskriftsartikel (refereegranskat)abstract
- Liver cell culture models are attractive in both tissue engineering and for development of assays for drug toxicology research. To retain liver specific cell functions, the use of adequate cell types and culture conditions, such as a 3D orientation of the cells and a proper supply of nutrients and oxygen, are critical. In this article, we show how extracellular matrix mimetic hydrogels can support hepatocyte viability and functionality in a perfused liver-on-a-chip device. A modular hydrogel system based on hyaluronan and poly(ethylene glycol) (HA-PEG), modified with cyclooctyne moieties for bioorthogonal strain-promoted alkyne-azide 1, 3-dipolar cycloaddition (SPAAC), was developed, characterized, and compared for cell compatibility to hydrogels based on agarose and alginate. Hepatoma cells (HepG2) formed spheroids with viable cells in all hydrogels with the highest expression of albumin and urea in alginate hydrogels. By including an excess of cyclooctyne in the HA backbone, azide-modified cell adhesion motifs (linear and cyclic RGD peptides) could be introduced in order to enhance viability and functionality of human induced pluripotent stem cell derived hepatocytes (hiPS-HEPs). In the HA-PEG hydrogels modified with cyclic RGD peptides hiPS-HEPs migrated and grew in 3D and showed an increased viability and higher albumin production compared to when cultured in the other hydrogels. This flexible SPAAC crosslinked hydrogel system enabled fabrication of perfused 3D cell culture of hiPS-HEPs and is a promising material for further development and optimization of liver-on-a-chip devices.
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| 16. |
- Enander, Karin, et al.
(författare)
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Alpha-helix-inducing dimerization of synthetic polypeptide scaffolds on gold
- 2005
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Ingår i: Langmuir. - : ACS Publications. - 0743-7463 .- 1520-5827. ; 21:6, s. 2480-2487
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Tidskriftsartikel (refereegranskat)abstract
- Designed, synthetic polypeptides that assemble into four-helix bundles upon dimerization in solution were studied with respect to folding on planar gold surfaces. A model system with controllable dimerization properties was employed, consisting of negatively and positively charged peptides. Circular dichroism spectroscopy and surface plasmon resonance based measurements showed that at neutral pH, the peptides were able to form heterodimers in solution, but unfavorable electrostatic interactions prevented the formation of homodimers. The dimerization propensity was found to be both pH- and buffer-dependent. A series of infrared absorption−reflection spectroscopy experiments of the polypeptides attached to planar gold surfaces revealed that if the negatively charged peptide was immobilized from a loading solution where it was folded, its structure was retained on the surface provided it had a cysteine residue available for anchoring to gold. If it was immobilized as random coil, it remained unstructured on the surface but was able to fold through heterodimerization if subsequently exposed to a positively charged polypeptide. When the positively charged peptide was immobilized as random coil, heterodimerization could not be induced, probably because of high-affinity interactions between the charged primary amine groups and the gold surface. These observations are intended to pave the way for future engineering of functional surfaces based on polypeptide scaffolds where folding is known to be crucial for function.
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| 17. |
- Eskilson, Olof, 1992-
(författare)
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Multifunctional Nanocellulose Composite Materials
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Nanoparticles (NPs) are particles with more than one dimension between 1 and 100 nm. Because of their small size, they typically display different physical and chemical properties than the corresponding bulk materials. NPs have been used in many different applications, such as in electronics, optics, catalysis, and in biomedicine. Due to their colloidal nature, NPs are often immobilized on a solid substrate, such as glass or polymer-based materials, including biopolymers. Nanocellulose is a biopolymerbased nanomaterial that can be obtained from plants or bacterial biofilms. They can be processed into thin and highly hydrated films with high mechanical strength and can serve as a versatile substrate for NPs. Bacterial cellulose (BC) is also an interesting material for generating wound dressings. The combination of NPs and BC results in soft and flexible nanocomposites (BC-NPs) that can demonstrate novel properties and improve the functionality of wound dressings. BC-NP nanocomposites have previously been obtained by impregnating BC with the reactants needed for synthesis of the NPs and allowing the reaction to proceed in situ, inside and on the surface of the BC. This strategy limits the possibilities to control NP geometry and NP concentration and make synthesis of nanocomposites with more sophisticated compositions very challenging. In addition, the synthesis conditions used can potentially have negative effects on the properties of BC. The work presented in this thesis shows the possibility to produce well-defined, tunable BC-NP nanocomposites using self-assembly under very benign conditions that enable functionalization of BC with a wide range of different types of NPs. In addition to exploring the self-assembly process and the physical properties of these new BC-NP composites, several different applications were investigated. The functionalization of BC with gold nanoparticles (AuNPs) of different sizes and geometries was demonstrated. The resulting materials were used for development of a new sensor transduction technology, exploiting the optical response upon mechanical compression to detect biomolecules. BC-AuNP nanocomposites were also developed for monitoring of protease activity of wound pathogens, for catalysis, and for fabrication of ultra-black materials with unique absorption and scattering profiles of light in the visible and near infrared spectral range. In addition, the self-assembly process could be adopted for generating BC-mesoporous silica nanoparticles (MSNs) nanocomposite wound dressings. The resulting high surface area materials could be used as carriers for pH sensitive dyes. The pH-responsive BC-MSNs demonstrated adequate biocompatibility and allowed for monitoring of wound pH and for assessment of wound status. The strategies for functionalization of BC with inorganic NPs that was developed and explored in this thesis are highly versatile and allow for fabrication of a wide range of multifunctional nanocomposite materials.
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| 18. |
- Eskilson, Olof, 1992-, et al.
(författare)
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Nanocellulose composite wound dressings for real-time pH wound monitoring
- 2023
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Ingår i: Materials Today Bio. - : Elsevier. - 2590-0064. ; 19
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Tidskriftsartikel (refereegranskat)abstract
- The skin is the largest organ of the human body. Wounds disrupt the functions of the skin and can have catastrophic consequences for an individual resulting in significant morbidity and mortality. Wound infections are common and can substantially delay healing and can result in non-healing wounds and sepsis. Early diagnosis and treatment of infection reduce risk of complications and support wound healing. Methods for monitoring of wound pH can facilitate early detection of infection. Here we show a novel strategy for integrating pH sensing capabilities in state-of-the-art hydrogel-based wound dressings fabricated from bacterial nanocellulose (BC). A high surface area material was developed by self-assembly of mesoporous silica nanoparticles (MSNs) in BC. By encapsulating a pH-responsive dye in the MSNs, wound dressings for continuous pH sensing with spatiotemporal resolution were developed. The pH responsive BC-based nanocomposites demonstrated excellent wound dressing properties, with respect to conformability, mechanical properties, and water vapor transmission rate. In addition to facilitating rapid colorimetric assessment of wound pH, this strategy for generating functional BC-MSN nanocomposites can be further be adapted for encapsulation and release of bioactive compounds for treatment of hard-to-heal wounds, enabling development of novel wound care materials.
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| 19. |
- Eskilson, Olof, 1992-, et al.
(författare)
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Self-Assembly of Mechanoplasmonic Bacterial Cellulose-Metal Nanoparticle Composites
- 2020
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Ingår i: Advanced Functional Materials. - : Wiley-VCH Verlagsgesellschaft. - 1616-301X .- 1616-3028. ; 30:40
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Tidskriftsartikel (refereegranskat)abstract
- Nanocomposites of metal nanoparticles (NPs) and bacterial nanocellulose (BC) enable fabrication of soft and biocompatible materials for optical, catalytic, electronic, and biomedical applications. Current BC-NP nanocomposites are typically prepared by in situ synthesis of the NPs or electrostatic adsorption of surface functionalized NPs, which limits possibilities to control and tune NP size, shape, concentration, and surface chemistry and influences the properties and performance of the materials. Here a self-assembly strategy is described for fabrication of complex and well-defined BC-NP composites using colloidal gold and silver NPs of different sizes, shapes, and concentrations. The self-assembly process results in nanocomposites with distinct biophysical and optical properties. In addition to antibacterial materials and materials with excellent senor performance, materials with unique mechanoplasmonic properties are developed. The homogenous incorporation of plasmonic gold NPs in the BC enables extensive modulation of the optical properties by mechanical stimuli. Compression gives rise to near-field coupling between adsorbed NPs, resulting in tunable spectral variations and enhanced broadband absorption that amplify both nonlinear optical and thermoplasmonic effects and enables novel biosensing strategies.
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| 20. |
- Guell-Grau, Pau, et al.
(författare)
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Elastic Plasmonic-Enhanced Fabry-Perot Cavities with Ultrasensitive Stretching Tunability
- 2022
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Ingår i: Advanced Materials. - : WILEY-V C H VERLAG GMBH. - 0935-9648 .- 1521-4095. ; 34:7
-
Tidskriftsartikel (refereegranskat)abstract
- The emerging stretchable photonics field faces challenges, like the robust integration of optical elements into elastic matrices or the generation of large optomechanical effects. Here, the first stretchable plasmonic-enhanced and wrinkled Fabry-Perot (FP) cavities are demonstrated, which are composed of self-embedded arrays of Au nanostructures at controlled depths into elastomer films. The novel self-embedding process is triggered by the Au nanostructures catalytic activity, which locally increases the polymer curing rate, thereby inducing a mechanical stress that simultaneously pulls the Au nanostructures into the polymer and forms a wrinkled skin layer. This geometry yields unprecedented optomechanical effects produced by the coupling of the broad plasmonic modes of the Au nanostructures and the FP modes, which are modulated by the wrinkled optical cavity. As a result, film stretching induces drastic changes in both the spectral position and intensity of the plasmonic-enhanced FP resonances due to the simultaneous cavity thickness reduction and cavity wrinkle flattening, thus increasing the cavity finesse. These optomechanical effects are exploited to demonstrate new strain-sensing approaches, achieving a strain detection limit of 0.006%, i.e., 16-fold lower than current optical strain-detection schemes.
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| 21. |
- Iversen, Alexandra, 1997-, et al.
(författare)
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Enzymatically Triggered Peptide–Lipid Conjugation of Designed Membrane Active Peptides for Controlled Liposomal Release
- 2024
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Ingår i: ACS Omega. - : American Chemical Society. - 2470-1343. ; 9:17, s. 19613-19619
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Tidskriftsartikel (refereegranskat)abstract
- Possibilities for controlling the release of pharmaceuticals from liposomal drug delivery systems can enhance their efficacy and reduce their side effects. Membrane-active peptides (MAPs) can be tailored to promote liposomal release when conjugated to lipid head groups using thiol-maleimide chemistry. However, the rapid oxidation of thiols hampers the optimization of such conjugation-dependent release strategies. Here, we demonstrate a de novo designed MAP modified with an enzyme-labile Cys-protection group (phenylacetamidomethyl (Phacm)) that prevents oxidation and facilitates in situ peptide lipidation. Before deprotection, the peptide lacks a defined secondary structure and does not interact with maleimide-functionalized vesicles. After deprotection of Cys using penicillin G acylase (PGA), the peptide adopts an α-helical conformation and triggers rapid release of vesicle content. Both the peptide and PGA concentrations significantly influence the conjugation process and, consequently, the release kinetics. At a PGA concentration of 5 μM the conjugation and release kinetics closely mirror those of fully reduced, unprotected peptides. We anticipate that these findings will enable further refinement of MAP conjugation and release processes, facilitating the development of sophisticated bioresponsive MAP-based liposomal drug delivery systems.
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| 22. |
- Jury, Michael, 1984-, et al.
(författare)
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Bioorthogonally Cross‐Linked Hyaluronan–Laminin Hydrogels for 3D Neuronal Cell Culture and Biofabrication
- 2022
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Ingår i: Advanced Healthcare Materials. - : Wiley. - 2192-2640 .- 2192-2659. ; 11:11
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Tidskriftsartikel (refereegranskat)abstract
- Laminins (LNs) are key components in the extracellular matrix of neuronal tissues in the developing brain and neural stem cell niches. LN-presenting hydrogels can provide a biologically relevant matrix for the 3D culture of neurons toward development of advanced tissue models and cell-based therapies for the treatment of neurological disorders. Biologically derived hydrogels are rich in fragmented LN and are poorly defined concerning composition, which hampers clinical translation. Engineered hydrogels require elaborate and often cytotoxic chemistries for cross-linking and LN conjugation and provide limited possibilities to tailor the properties of the materials. Here a modular hydrogel system for neural 3D cell cultures, based on hyaluronan and poly(ethylene glycol), that is cross-linked and functionalized with human recombinant LN-521 using bioorthogonal copper-free click chemistry, is shown. Encapsulated human neuroblastoma cells demonstrate high viability and grow into spheroids. Long-term neuroepithelial stem cells (lt-NES) cultured in the hydrogels can undergo spontaneous differentiation to neural fate and demonstrate significantly higher viability than cells cultured without LN. The hydrogels further support the structural integrity of 3D bioprinted structures and maintain high viability of bioprinted and syringe extruded lt-NES, which can facilitate biofabrication and development of cell-based therapies.
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| 23. |
- Jury, Michael, 1984-
(författare)
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Modular Hyaluronan-Based Hydrogels for 3D Cell Culture and Bioprinting
- 2022
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Three-dimensional (3D) cell culture facilitates development of biological relevant assays for drug screening and toxicity testing. Compared to conventional 2D cell culture, cells cultured in 3D can more accurately mimic human tissues and organs and thus provide ex vivo data with potentially better predictive value for cancer research, pharmacology, and toxicology, reducing the need for animal models, improving experimental reproducibility, and reducing time and costs in drug development. The most widely used options for scaffold-based 3D cell culture are, however, based on poorly defined biologically derived extracellular matrix (ECM) with limited possibilities to tailor material properties and that are difficult to combine with state-of-the art biofabrication techniques. The overall aim this thesis was to design and explore modular hyaluronan (HA) based ECM-mimicking hydrogels with tuneable physiochemical properties and biofunctionalities, for development of advanced 3D cell models and biofabrication. The thesis work is presented in five papers. In paper I, we used copper free click chemistry for both hydrogel cross-linking and functionalization with fibronectin derived peptide sequences for culture of human induced pluripotent-derived hepatocytes in a perfused microfluidic system. The tuneable and bioorthogonal cross-linking enabled both retention of high cell viabilities and fabrication of a functional liver-on-chip solution. In paper II, we combined the developed HA-based hydrogel system with homo- and heterodimerizing helix-loop-helix peptides for modulation of both cross-linking density and biofunctionalization. We further demonstrated the possibilities to use these hydrogels as bioinks for 3D bioprinting where both the molecular composition and the physical properties of the printed structures could be dynamically altered, providing new avenues for four-dimensional (4D) bioprinting. In paper III we investigated the possibilities to chemically conjugate full size recombinant human laminin-521 (LN521) in the HA-based hydrogels system using copper-free click chemistry, with the aim to enable 3D culture and 3D bioprinting of neurons. We quantified the impact of using different linkers to tether LN521 and the influence of LN-functionalization on the structural and mechanical properties of the hydrogels. We show that both differentiated and non-differentiated neuroblastoma cells and long-term self-renewing neuroepithelial stem cells (lt-NES) remained viable in the hydrogels. The hydrogels also had a protected effect on lt-NES during syringe ejection and bioprinting. In paper IV, we used HA-based hydrogels modified with peptides sequences derived from fibronectin and laminin for culture of fetal primary astrocytes (FPA). We explored both the interactions between the hydrogels and FPA and possibilities to 3D bioprint FPAs. Finally, in paper V, we developed HA-nanocellulose composite hydrogels with the aim to increase printing fidelity and enable fabrication of multi-layered bioprinted structures without the use of a support bath. In addition to HA, we used wood-fibre derived nanocellulose (NC) to increase the viscosity of the bioink during the printing process. The developed biorthogonal and modular hydrogel systems provide a large degree of flexibility that allows for encapsulation and culture of different cell types and processing using different techniques, which can contribute to further exploration of fabrication of biologically relevant tissue and disease models.
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| 24. |
- Mak, Wing Cheung, et al.
(författare)
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Probing Zinc-Protein-Chelant Interactions using Gold Nanoparticles Functionalized with Zinc-Responsive Polypeptides
- 2014
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Ingår i: Particle & particle systems characterization. - : Wiley-VCH Verlagsgesellschaft. - 0934-0866 .- 1521-4117. ; 31:11, s. 1127-1133
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Tidskriftsartikel (refereegranskat)abstract
- The coordination of zinc by proteins and various other organic molecules is essential for numerous biological processes, such as in enzymatic catalysis, metabolism and signal transduction. Presence of small molecular chelants can have a profound effect on the bioavailability of zinc and affect critical Zn2+-protein interactions. Zn2+ chelators are also emerging therapeutics for Alzheimer’s diseases because of their preventive effect on zinc promoted amyloid formation. Despite the importance of zinc-protein-chelant interactions in biology and medicine, probing such interactions is challenging. Here, we introduce an innovative approach for real-time characterization of zinc-protein-chelant interactions using gold nanoparticles (AuNPs) functionalized with a zinc-responsive protein mimetic polypeptide. The peptide functionalized AuNPs aggregate extensively in the presence of Zn2+, triggered by specific Zn2+-mediated polypeptide dimerization and folding, causing a massive red shift of the plasmon band. Chelants affects the Zn2+- polypeptide interaction and thus the aggregation differently depending on their concentrations, zincbinding affinities and coordination numbers, which affect the position of the plasmon band. This system is a simple and powerful tool that provides extensive information about the interactions of chelants in the formation of Zn2+ coordination complexes and is an interesting platform for development of bioanalytical techniques and characterization of chelation-based therapeutics.
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| 25. |
- Naeimipour, Sajjad, 1987-, et al.
(författare)
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Enzymatically Triggered Deprotection and Cross-Linking of Thiolated Alginate-Based Bioinks
- 2022
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Ingår i: Chemistry of Materials. - : American Chemical Society (ACS). - 0897-4756 .- 1520-5002. ; 34:21, s. 9536-9545
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Tidskriftsartikel (refereegranskat)abstract
- Thiolated polymers are widely used in hydrogels for drug delivery, tissue engineering, and biofabrication. The oxidation of thiols is spontaneous, resulting in the formation of disulfide bridges and cross linking of polymers. The cross-linking process is, however, difficult to control and is initiated directly when the thiolated components are exposed to ambient conditions, which significantly complicates handling of the materials. Here, we show a fully bioorthogonal enzyme-mediated thiol-based chemistry for dynamic covalent cross-linking of carbohydrate-based hydrogels that circumvents the problems with uncontrolled thiol oxidation. Alginate was modified with cysteine residues, protected by an enzyme-labile thiol-protecting group (Phacm). Releasing the Phacm group by penicillin G acylase generates free thiols that oxidize under physiological conditions, resulting in a reversible cross-linking and formation of hydrogels with tunable stiffness. Prior to deprotection, the components can be exposed to ambient conditions. The enzyme-triggered deprotection and subsequent gelation allows for encapsulation of cells and 3D bioprinting of cell-laden hydrogel structures. Remaining deprotected thiols enabled postprinting modifications and hydrogel self-healing. The proposed hydrogel synthesis strategy significantly increases the versatility of thiol-based cross-linking chemistries and provides new possibilities to generate dynamic covalent hydrogels for a broad range of biomedical applications.
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| 26. |
- Naeimipour, Sajjad, 1987-
(författare)
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Modular Enzyme-Responsive Polysaccharide-Based Hydrogels for Biofabrication
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Engineered human tissue and disease models can decrease the cost and time of developing new drugs and treatments, facilitate personalized medicine, and eliminate the need for animal models that poorly represent the human body and are ethically problematic. However, the current conventional cell expansion methods using 2D culture flasks cannot enable the development of such complex multi-cellular 3D models. In general, hydrogels are considered promising materials that can make the biofabrication of tissue models possible. Hydrogels are highly hydrated materials comprised of either synthetic or naturally derived polymers, or a combination of both, and can form an environment mimicking the biomacromolecular network surrounding cells in the body. This network of biopolymers, known as extracellular matrix (ECM), is comprised of proteins such as collagen, laminin, fibronectin, and polysaccharides such as hyaluronan (HA), heparan, keratan, and chondroitin sulfate. The design of hydrogels representing the physical and biochemical properties of the ECM and which can be used for biofabrication is challenging but of increasing interest due to the rapid progress in the development of 3D and 4D bioprinting techniques. As the ECM properties differ between various tissues and disease conditions and change over time, a dynamic modular hydrogel system is needed to that can be optimized for each cell and tissue type. This thesis aims to develop modular enzyme-responsive polysaccharide-based hydrogels for 3D cell culture and biofabrication. The natural polysaccharides, hyaluronic acid (HA) and alginate (Alg) were used as the main backbone in the hydrogels developed in this thesis. HA was modified by conjugating bicyclo[6.1.0]non-4-yne (BCN) to the backbone to form HA-BCN-based hydrogels by a bioorthogonal strain-promoted alkyne-azide cycloaddition. The click reaction between BCN and azide groups allowed for modulating the biochemical and mechanical properties of the HA-BCN hydrogels. HA-BCN was further decorated with peptides to explore peptide folding and dimerization-mediated dynamic cross-linking and biofunctionalization. This system was further used to explore possibilities to dynamically alter the properties of 3D bioprinted structures, mimicking the biomineralization process in bone tissue. In a different study, a tumor model comprising fibroblast and breast cancer cells (MCF7) was bioprinted using HA-BCN cross-linked by matrix metalloporotease (MMP) cleavable and PEG-diazide MMP-resistant cross-linkers, demonstrating the synergistic relationship between hydrogel degradability and cancer cell growth, intensified by the presence of fibroblasts. The possibility of incorporating a conductive module into this hydrogel system was explored using the enzyme-assisted polymerization of ETE-S to form an interpenetrating conductive network inside HA-BCN hydrogel. The in situ and user-triggered polymerization of conductive ETE-S was demonstrated after 3D printing HA-BCN bioink containing ETE-S monomers into a lattice shape structure. We also demonstrated that cellulose nanofibrils (CNF) improved the printability of HA-BCN bioinks, and this hybrid bioink was used for printing self-standing cell-laden 3D structures. Besides these studies, a novel enzymatically triggered thiol-based chemistry was developed to address the unwanted oxidation of thiol-containing hydrogels and decrease the off-target thiol reactions during hydrogel synthesis and formation. Alginate containing sulfhydryl moieties, protected by an enzyme-labile Phacm group (AlgCP), was treated with penicillin G acylase and subsequently formed a disulfide cross-linked hydrogel. We studied the gelation kinetics and rheological properties of AlgCP and different modes of cross-linking by reversible disulfide bonds, a thiol-maleimide Micheale-type addition reaction, and ionic interactions between alginate and Ca2+ ions. MCF7 breast cancer cells cultured in the AlgCP hydrogels formed spheroids that could be harvested by GSH dissolution of the hydrogels. Finally, this novel chemistry enabled bioprinting of multi-material 3D structures with control over the printed structure's physiochemical properties, including the type and density of cross-linkers. Bioprinted fibroblasts formed extended morphology, and MCF7 cells formed spheroids in the bioprinted lattice structures. The hydrogel systems developed and explored in this thesis are modular and exhibit dynamic and tunable properties, and are applicable for a wide range of 3D cell culture and bioprinting applications. The hydrogels were either formed in response to the activity of an enzyme or remodeled by enzymes. Both enzyme-responsive HA-BCN and AlgCP hydrogel systems are promising bioinks for generating more elaborate and spatially defined cell-laden 3D structures whose features can be altered post-printing by cell-secreted and extrinsic reagents. These hydrogel-based toolkits can play a vital role in developing tissue and disease models that can make the drug discovery process faster, cheaper, and animal-free.
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| 27. |
- Nayeri, Fariba, 1958-, et al.
(författare)
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Autocrine production of biologically active hepatocyte growth factor (HGF) by injured human skin
- 2006
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Ingår i: Journal of dermatological science (Amsterdam). - : Elsevier BV. - 0923-1811 .- 1873-569X. ; 43:1, s. 49-56
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundHepatocyte growth factor (HGF) is a potent regenerative factor involved in wound healing. Previous studies have shown that mesenchymal cells produce HGF, stimulating epithelial cells in a paracrine fashion.ObjectiveTo examine whether autocrine HGF production by keratinocytes can occur upon skin injury.MethodsA 31-year-old male patient sustained a burn affecting 80% of his total body surface area. Biopsies were taken from intact skin near the injured area, and skin keratinocytes were separated and cultured. Conditioned medium from keratinocytes was analyzed for HGF by ELISA, surface plasmon resonance (SPR), and dot blotting. Binding of HGF from conditioned medium to its receptor, c-Met, was compared with recombinant HGF by SPR. Finally, we examined the motogenic effect on mouse transformed skin epithelial cells (CCL-53.1) of HGF from conditioned medium.ResultsHGF was detected in the cultured keratinocyte medium. Similar to recombinant HGF, HGF from conditioned medium had a high affinity for dextran sulfate and albumin, and the same epitopes were engaged by the interaction of HGF with the c-Met receptor. The conditioned medium from keratinocytes obtained from the burn patient, but not medium from keratinocytes obtained from healthy volunteers, accelerated the motogenesis of CCL-53.1 cells. Unexpectedly, anti-HGF antibodies did not prevent this effect. However, anti-c-Met antibodies completely inhibited the motogenic effect.ConclusionUpon injury, human skin keratinocytes might produce biologically active HGF in an autocrine fashion. This HGF might have different structural and/or biological properties from HGF produced by mesenchymal cells.
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| 28. |
- Nayeri, Fariba, 1958-, et al.
(författare)
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Clinical impact of real-time evaluation of the biological activity and degradation of hepatocyte growth factor
- 2008
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Ingår i: Growth Factors. - : Informa UK Limited. - 0897-7194 .- 1029-2292. ; 26:3, s. 163-171
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Tidskriftsartikel (refereegranskat)abstract
- Hepatocyte growth factor (HGF) is essential for injury repair. Despite high HGF levels in chronic ulcers, up-regulation of HGF receptor in ulcer tissue and decreased biological activity of HGF in ulcer secretions have been observed. With a surface plasmon resonance-based method, we assessed the binding of HGF to antibodies, receptors, and the basement membrane and identified binding interactions that are indispensable for the biological activity of HGF. Recombinant HGF (rHGF) lots were tested for activity, structural integrity, and degradation, and the results were verified in an in vitro model of cell injury. Biologically active rHGF, as well as plasma from healthy volunteers, bound to heparan sulphate proteoglycan (HSPG) and to anti-HGF antibodies. Decreased binding to HSPG was the first event in rHGF degradation. This study established the feasibility of identifying patients with chronic inflammation who need exogenous HGF and of using ligand-binding assessment to evaluate rHGF lots for biological activity.
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| 29. |
- Nayeri, Fariba, 1958-, et al.
(författare)
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Hepatocyte growth factor (HGF) in fecal samples : rapid detection by surface plasmon resonance
- 2005
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Ingår i: BMC Gastroenterology. - 1471-230X. ; 5:13
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundThe development of biosensors, based on surface plasmon resonance (SPR) technology, enables monitoring of a variety of biospecific interactions without the need for chemical-, biological- or radiological-labelled reagents.MethodWe utilised SPR to detect hepatocyte growth factor (HGF) in reconstituted faecal samples and studied samples from patients with infectious gastroenteritis (n = 20) and normal controls (n = 10). Mouse anti-human HGF monoclonal antibodies and recombinant human HGF receptor (c-Met)/Fc chimera were immobilised in flow cells of a CM5 biosensor chip.ResultsWe found that infectious gastroenteritis produced a higher signal response compared to controls, due to binding of HGF to monoclonal anti-HGF antibody as well as binding of HGF to c-Met receptor (p < 0.01). The SPR signal response correlated with results from ELISA (r = 72%, p > 0.001). The signal response decreased significantly (p < 0.05) when samples were diluted with dextran, because of reduction in both specific as well as unspecific binding of HGF to dextran. The decrease in the specific response might imply that the dextran- binding site for HGF overlaps with the antibody binding epitope, or that dextran binding induces a conformational change of the HGF molecule. Bands corresponding to HGF were found by gel electrophoresis of purified faeces in an affinity chromatography column immobilised by HGF ligands.ConclusionDetermination of HGF by SPR might be beneficial in diagnosis of acute situations that present with symptoms of gastroenteritis and may, possibly, guide appropriate medical treatments. This is to our knowledge the first report on the use of SPR for detection of HGF in faeces samples.
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| 30. |
- Omer, Abubakr A. M., 1982-, et al.
(författare)
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Plantaricin NC8 αβ rapidly and efficiently inhibits flaviviruses and SARS-CoV-2 by disrupting their envelopes
- 2022
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Ingår i: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 17:11
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Tidskriftsartikel (refereegranskat)abstract
- Potent broad-spectrum antiviral agents are urgently needed to combat existing and emerging viral infections. This is particularly important considering that vaccine development is a costly and time consuming process and that viruses constantly mutate and render the vaccine ineffective. Antimicrobial peptides (AMP), such as bacteriocins, are attractive candidates as antiviral agents against enveloped viruses. One of these bacteriocins is PLNC8 αβ, which consists of amphipathic peptides with positive net charges that display high affinity for negatively charged pathogen membrane structures, including phosphatidylserine rich lipid membranes of viral envelopes. Due to the morphological and physiological differences between viral envelopes and host cell plasma membranes, PLNC8 αβ is thought to have high safety profile by specifically targeting viral envelopes without effecting host cell membranes. In this study, we have tested the antiviral effects of PLNC8 αβ against the flaviviruses Langat and Kunjin, coronavirus SARS-CoV-2, influenza A virus (IAV), and human immunodeficiency virus-1 (HIV-1). The concentration of PLNC8 αβ that is required to eliminate all the infective virus particles is in the range of nanomolar (nM) to micromolar (μM), which is surprisingly efficient considering the high content of cholesterol (8–35%) in their lipid envelopes. We found that viruses replicating in the endoplasmic reticulum (ER)/Golgi complex, e.g. SARS-CoV-2 and flaviviruses, are considerably more susceptible to PLNC8 αβ, compared to viruses that acquire their lipid envelope from the plasma membrane, such as IAV and HIV-1. Development of novel broad-spectrum antiviral agents can significantly benefit human health by rapidly and efficiently eliminating infectious virions and thereby limit virus dissemination and spreading between individuals. PLNC8 αβ can potentially be developed into an effective and safe antiviral agent that targets the lipid compartments of viral envelopes of extracellular virions, more or less independent of virus antigenic mutations, which faces many antiviral drugs and vaccines.
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| 31. |
- Selegård, Robert, et al.
(författare)
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Generic Phosphatase Activity Detection using Zinc Mediated Aggregation Modulation of Polypeptide-Modified Gold Nanoparticles
- 2014
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Ingår i: Nanoscale. - : Royal Society of Chemistry. - 2040-3364 .- 2040-3372. ; 6:23, s. 14204-14212
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Tidskriftsartikel (refereegranskat)abstract
- A challenge in the design of plasmonic nanoparticle-based colorimetric assays is that the change in colloidal stability, which generates the colorimetric response, is often directly linked to the biomolecular recognition event. New assay strategies are hence required for every type of substrate and enzyme of interest. Here, a generic strategy for monitoring of phosphatase activity is presented where substrate recognition is completely decoupled from the nanoparticle stability modulation mechanism, which enables detection of a wide range of enzymes using different natural substrates with a single simple detection scheme. Phosphatase activity generates inorganic phosphate that forms an insoluble complex with Zn2+. In a sample containing a preset concentration of Zn2+, phosphatase activity will markedly reduce the concentration of dissolved Zn2+ from the original value, which in turn affects the aggregation of gold nanoparticles functionalized with a designed Zn2+ responsive polypeptide. The change in nanoparticle stability thus provides a rapid and sensitive readout of the phosphatase activity. The assay is not limited to a particular enzyme or enzyme substrate, which is demonstrated using three completely different phosphatases and five different substrates, and thus constitutes a highly interesting system for drug screening and diagnostics.
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| 32. |
- Skog, Mårten, 1982-, et al.
(författare)
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The Effect of Enzymatic Digestion on Cultured Epithelial Autografts
- 2019
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Ingår i: Cell Transplantation. - : Sage Publications. - 0963-6897 .- 1555-3892. ; 28:5, s. 638-644
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Tidskriftsartikel (refereegranskat)abstract
- Severe burns are often treated by means of autologous skin grafts, preferably following early excision of the burnt tissue. In the case of, for example, a large surface trauma, autologous skin cells can be expanded in vitro prior to transplantation to facilitate the treatment when insufficient uninjured skin is a limitation. In this study we have analyzed the impact of the enzyme (trypsin or accutase) used for cell dissociation and the incubation time on cell viability and expansion potential, as well as expression of cell surface markers indicative of stemness. Skin was collected from five individuals undergoing abdominal reduction surgery and the epidermal compartment was digested in either trypsin or accutase. Trypsin generally generated more cells than accutase and with higher viability; however, after 7 days of subsequent culture, accutase-digested samples tended to have a higher cell count than trypsin, although the differences were not significant. No significant difference was found between the enzymes in median fluorescence intensity of the analyzed stem cell markers; however, accutase digestion generated significantly higher levels of CD117- and CD49f-positive cells, but only in the 5 h digestion group. In conclusion, digestion time appeared to affect the isolated cells more than the choice of enzyme.
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| 33. |
- Skyttner, Camilla, 1985-
(författare)
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Peptide-Liposome Model Systems for Triggered Release
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Liposomes are widely used in drug delivery to improve drug efficacy and to reduce side effects. For liposome-encapsulated drugs to become bioavailable and provide a therapeutic effect they must be released, which typically is a slow process that primarily relies on passive diffusion, liposome rupture or endocytotic uptake. Achieving drug concentrations within the therapeutic window can thus be challenging, resulting in poor efficacy and higher risks drug resistance. Finding means to modulate lipid membrane integrity and to trigger rapid and efficient release of liposomal cargo is thus critical to improve current and future liposomal drug delivery systems. The possibilities to tailor lipid composition and surface functionalization is vital for drug delivery applications but also make liposomes attractive model systems for studies of membrane active biomolecules.The overall aim of this thesis work has been to develop new strategies for triggering and controlling changes in lipid membrane integrity and to study the interactions of membrane active peptides with model lipid membranes using both de novo designed and biologically derived synthetic amphipathic cationic peptides. Two different sets of designed peptides have been explored that can fold and heterodimerize into a coiled coil and helix-loop-helix fourhelix bundle, respectively. Conjugation of the cationic lysine rich peptides to liposomes triggered a rapid and concentration dependent release. The additions of their corresponding glutamic acid-rich complementary peptides inhibited the release of liposomal cargo. Possibilities to reduce the inhibitory effect by both proteolytic digestion of the inhibitory peptide and by means of heterodimer exchange have been investigated. Moreover, the effects of peptide size and composition and ability to fold have been studied in order to elucidate the factors that influence the membrane permeabilizing effects of the peptides.In addition, the membrane activity of a the two-peptide bacteriocin PLNC8α and PLNC8β has been explored using liposomes as a model system. PLNC8αβ are expressed by Lactobacillus plantarum and were shown to display pronounced membrane-partition folding coupling, leading to rapid release of liposome encapsulated carboxyfluorescein. PLNC8αβ also kill and suppressed growth of the gram-negative bacteria Porphyromonas gingivalis by efficiently damaging the bacterial membrane.Although membrane active peptides are highly efficient in perturbing lipid membrane integrity, possibilities to trigger release using external stimuli are also of large interest for therapeutic applications. Light-induced heating of liposome encapsulated gold nanoparticles (AuNPs) has been shown by others as a potential strategy to trigger drug release. To facilitate fabrication of thermoplasmonic liposome systems we developed a simple method for synthesis of small AuNPs inside liposomes, using the liposomes as nanoscale reaction vessels.The work presented in this thesis provides new knowledge and techniques for future development of liposome-based drug delivery systems, peptide-based therapeutics and increase our understanding of peptide-lipid interactions.
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| 34. |
- Tran, Thuy, 1980-, et al.
(författare)
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Nanoplasmonic Avidity-Based Detection and Quantification of IgG Aggregates
- 2022
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Ingår i: Analytical Chemistry. - : AMER CHEMICAL SOC. - 0003-2700 .- 1520-6882. ; 94:45, s. 15754-15762
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Tidskriftsartikel (refereegranskat)abstract
- Production of therapeutic monoclonal antibodies (mAbs) is a complex process that requires extensive analytical and bioanalytical characterization to ensure high and consistent product quality. Aggregation of mAbs is common and very problematic and can result in products with altered pharmacodynamics and pharmacokinetics and potentially increased immunogenicity. Rapid detection of aggregates, however, remains very challenging using existing analytical techniques. Here, we show a real-time and label-free fiber optical nanoplasmonic biosensor system for specific detection and quantification of immunoglobulin G (IgG) aggregates exploiting Protein A mediated avidity effects. Compared to monomers, IgG aggregates were found to have substantially higher apparent affinity when binding to Protein Afunctionalized sensor chips in a specific pH range (pH 3.8-4.0). Under these conditions, aggregates and monomers showed significantly different binding and dissociation kinetics. Reliable and rapid aggregate quantification was demonstrated with a limit of detection (LOD) and limit of quantification (LOQ) of about 9 and 30 mu g/mL, respectively. Using neural network-based curve fitting, it was further possible to simultaneously quantify monomers and aggregates for aggregate concentrations lower than 30 mu g/mL. Our work demonstrates a unique avidity-based biosensor approach for fast aggregate analysis that can be used for rapid at-line quality control, including lot/batch release testing. This technology can also likely be further optimized for real-time in-line monitoring of product titers and quality, facilitating process intensification and automation.
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| 35. |
- Tran, Thuy, et al.
(författare)
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Process integrated biosensors for real-time monitoring of antibodies for automated affinity purification
- 2022
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Ingår i: Analytical Methods. - : Royal Society of Chemistry. - 1759-9660 .- 1759-9679. ; 14:44, s. 4555-4562
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Tidskriftsartikel (refereegranskat)abstract
- Therapeutic monoclonal antibodies (mAbs) provide new means for treatments of a wide range of diseases and comprise a large fraction of all new approved drugs. Production of mAbs is expensive compared to conventional drug production, primarily due to the complex processes involved. The affinity purification step is dominating the cost of goods in mAb manufacturing. Process intensification and automation could reduce costs, but the lack of real-time process analytical technologies (PAT) complicates this development. We show a specific and robust fiber optical localized surface plasmon resonance (LSPR) sensor technology that is optimized for in-line product detection in the effluent in affinity capture steps. The sensor system comprises a flow cell and a replaceable sensor chip functionalized with biorecognition elements for specific analyte detection. The high selectivity of the sensor enable detection of mAbs in complex sample matrices at concentrations below 2.5 mu g mL(-1). In place regeneration of the sensor chips allowed for continuous monitoring of multiple consecutive chromatographic separation cycles. Excellent performance was obtained at different purification scales with flow rates up to 200 mL min(-1). This sensor technology facilitates efficient column loading, optimization, and control of chromatography systems, which can pave the way for continuous operation and automation of protein purification steps.
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| 36. |
- Utterström, Johanna, 1993-
(författare)
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Design and Optimization of Membrane Active Peptides and Lipid Vesicles for Triggered Release
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Liposomes can reduce toxic side effects and improve the efficacy of drugs and several liposome-based drug formulations are approved for clinical use. The therapeutic effect is dependent on the bioavailability of the drug and a slow drug release from liposomes can reduce their efficacy. Multiple strategies have been proposed to control the release of drugs from liposomes using both external stimuli such as light, heat and ultrasound, and endogenous factors such as changes in pH or enzymatic activity. However, because of the difficulties to efficiently modulate lipid membrane permeability and the challenges to trigger drug release in the target tissue, no stimuli responsive liposomes have so far been approved. There is consequently a great need for new means to tune lipid membrane integrity for liposome cargo release to improve the development of new advanced drug delivery systems for better and safer treatment of patients. The aim of this thesis was to design and explore synthetic membrane active peptides for triggered release from liposomes and to expand the knowledge on how peptide-lipid conjugation strategies and lipid properties affect the membrane activity of the peptides. This work was based on two different de novo designed cationic and amphipathic, conjugation-dependent membrane active peptides (CKV4 and JR2KC). Both peptides fold and adopt α-helical structures upon conjugation to liposomes, triggering lipid membrane destabilization. Addition of cholesterol in the lipid membrane greatly enhanced the release efficiency of JR2KC due to a peptide-triggered lipid phase separation, resulting in domains with high local peptide concentrations. Additionally, both peptide surface concentrations and lipid net charge were found to be important factors for efficient release. However, when the zeta potential decreased below -75 mV, conjugation-independent release mechanisms were triggered. Liposome size was shown to only have minor effects on the release kinetics for both sets of peptides while a mixture of saturated and unsaturated lipids was beneficial for the peptide-triggered membrane destabilization, possibly due to increased propensity for lipid phase separation. In addition to changing lipid properties, peptide-lipid conjugation strategies proved to highly affect the release kinetics, where the Michael addition reaction between a cysteine in the peptide and maleimide-lipids was much more efficient in causing peptide-triggered membrane destabilization than strain-promoted alkyne azide cycloaddition reactions using azide-modified peptides and DBCO-functionalized lipids. However, thiols tend to oxidize under ambient conditions which complicates peptide-lipid conjugation. This was addressed by synthesizing a peptide with a cysteine modified with an enzyme labile thiol protection group. Enzymatic deprotection allowed efficient peptide-lipid conjugation, reducing the risk of peptide oxidation. To further find means to tailor peptide-lipid interactions, we explored the effect of a competing peptide heterodimerization process on lipid membrane destabilization. Addition of a charge complementary peptide to CKV4 resulted in heterodimerization and folding into a coiled coil, which inhibited its membrane activity. However, when the two peptides were synthesized as a single sequence, the membrane activity was altered, most likely due to the induced preorganization increasing membrane affinity. The results presented in this thesis provide new understandings of the complex peptide-lipid interactions that govern peptide-induced release from liposomes and will facilitate further optimization in peptide design for the future development of advanced liposome-based drug delivery systems.
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| 37. |
- Wiman, Emanuel, 1985-, et al.
(författare)
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Development of novel broad-spectrum antimicrobial lipopeptides derived from plantaricin NC8 β
- 2023
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial resistance towards antibiotics is a major global health issue. Very few novel antimicrobial agents and therapies have been made available for clinical use during the past decades, despite an increasing need. Antimicrobial peptides have been intensely studied, many of which have shown great promise in vitro. We have previously demonstrated that the bacteriocin Plantaricin NC8 αβ (PLNC8 αβ) from Lactobacillus plantarum effectively inhibits Staphylococcus spp., and shows little to no cytotoxicity towards human keratinocytes. However, due to its limitations in inhibiting gram-negative species, the aim of the present study was to identify novel antimicrobial peptidomimetic compounds with an enhanced spectrum of activity, derived from the β peptide of PLNC8 αβ. We have rationally designed and synthesized a small library of lipopeptides with significantly improved antimicrobial activity towards both gram-positive and gram-negative bacteria, including the ESKAPE pathogens. The lipopeptides consist of 16 amino acids with a terminal fatty acid chain and assemble into micelles that effectively inhibit and kill bacteria by permeabilizing their cell membranes. They demonstrate low hemolytic activity and liposome model systems further confirm selectivity for bacterial lipid membranes. The combination of lipopeptides with different antibiotics enhanced the effects in a synergistic or additive manner. Our data suggest that the novel lipopeptides are promising as future antimicrobial agents, however additional experiments using relevant animal models are necessary to further validate their in vivo efficacy.
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