| 1. |
- Hedegaard, Sofie Fogh, et al.
(författare)
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Fluorophore labeling of a cell-penetrating peptide significantly alters the mode and degree of biomembrane interaction
- 2018
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 8:1
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Tidskriftsartikel (refereegranskat)abstract
- The demand for highly efficient macromolecular drugs, used in the treatment of many severe diseases, is continuously increasing. However, the hydrophilic character and large molecular size of these drugs significantly limit their ability to permeate across cellular membranes and thus impede the drugs in reaching their target sites in the body. Cell-penetrating peptides (CPP) have gained attention as promising drug excipients, since they can facilitate drug permeation across cell membranes constituting a major biological barrier. Fluorophores are frequently covalently conjugated to CPPs to improve detection, however, the ensuing change in physico-chemical properties of the CPPs may alter their biological properties. With complementary biophysical techniques, we show that the mode of biomembrane interaction may change considerably upon labeling of the CPP penetratin (PEN) with a fluorophore. Fluorophore-PEN conjugates display altered modes of membrane interaction with increased insertion into the core of model cell membranes thereby exerting membrane-thinning effects. This is in contrast to PEN, which localizes along the head groups of the lipid bilayer, without affecting the thickness of the lipid tails. Particularly high membrane disturbance is observed for the two most hydrophobic PEN conjugates; rhodamine B or 1-pyrene butyric acid, as compared to the four other tested fluorophore-PEN conjugates.
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| 2. |
- Holdfeldt, André, et al.
(författare)
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The lipidated peptidomimetic Lau-[(S)-Aoc]-(Lys-βNphe)6-NH2 is a novel formyl peptide receptor 2 agonist that activates both human and mouse neutrophil NADPH-oxidase.
- 2016
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Ingår i: The Journal of biological chemistry. - 1083-351X. ; 291, s. 19888-19899
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Tidskriftsartikel (refereegranskat)abstract
- Neutrophils expressing formyl peptide receptor 2 (FPR2) play key roles in host defense, immune regulation, and in resolution of inflammation. Consequently, the search for FPR2-specific modulators has attracted much attention due to its therapeutic potential. Earlier described agonists for this receptor display potent activity for the human receptor (FPR2) but low activity for the mouse receptor orthologue (Fpr2), rendering them inapplicable in murine models of human disease. Here we describe a novel FPR2 agonist, the proteolytically stable α-peptide/β-peptoid hybrid Lau-[(S)-Aoc]-(Lys-βNphe)6-NH2 (F2M2), showing comparable potency in activating human and mouse neutrophils by inducing a rise in intracellular calcium and assembly of the superoxide-generating NADPH-oxidase. The FPR2/Fpr2 agonist contains a headgroup of 2-aminooctanoic acid (Aoc) residue acylated with lauric acid (C12 fatty acid), which is linked to a peptide/peptoid repeat (Lys-βNphe)6-NH2). Both the fatty acid moiety and the (S)-Aoc residue were required for FPR2/Fpr2 activation. This type of proteolytically stable FPR2-specific peptidomimetics may serve as valuable tools for future analysis of FPR2 signaling as well as for development of prophylactic immunomodulatory therapy. This novel class of cross-species FPR2/Fpr2 agonists should enable translation of results obtained with mouse neutrophils (and disease models) into enhanced understanding of human inflammatory and immune diseases.
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| 3. |
- Jing, Xiaona, et al.
(författare)
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Delivery of siRNA Complexed with Palmitoylated alpha-Peptide/beta-Peptoid Cell-Penetrating Peptidomimetics : Membrane Interaction and Structural Characterization of a Lipid-Based Nanocarrier System
- 2016
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Ingår i: Molecular Pharmaceutics. - : American Chemical Society (ACS). - 1543-8384 .- 1543-8392. ; 13:6, s. 1739-1749
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Tidskriftsartikel (refereegranskat)abstract
- Proteolytically stable alpha-peptide/beta-peptoid peptidomimetics constitute promising cell-penetrating carrier candidates exhibiting superior cellular uptake as compared to commonly used cell-penetrating peptides (CPPs). The aim of the present study was to explore the potential of these peptidomimetics for delivery of small interfering RNA (siRNA) to the cytosol by incorporation of a palmitoylated peptidomimetic construct into a cationic lipid-based nanocarrier system. The optimal construct was selected on the basis of the effect of palmitoylation and the influence of the length of the peptidomimetic on the interaction with model membranes and the cellular uptake. Palmitoylation enhanced the peptidomimetic adsorption to supported lipid bilayers as studied by ellipsometry. However, both palmitoylation and increased peptidomimetic chain length were found to be beneficial in the cellular uptake studies using fluorophore-labeled analogues. Thus, the longer palmitoylated peptidomimetic was chosen for further formulation of siRNA in a dioleoylphosphatidylethanolamine/cholesteryl hemisuccinate (DOPE/CHEMS) nanocarrier system, and the resulting nanoparticles were found to mediate efficient gene silencing in vitro. Cryo-transmission electron microscopy (cryo-TEM) revealed multilamellar, onion-like spherical vesicles, and small-angle X-ray scattering (SAXS) analysis confirmed that the majority of the lipids in the nanocarriers were organized in lamellar structures, yet coexisted with a hexagonal phase, which is important for efficient nanocarrier-mediated endosomal escape of siRNA ensuring cytosolic delivery. The present work is a proof-of-concept for the use of alpha-peptides/beta-peptoid peptidomimetics in an efficient delivery system that may be more generally exploited for the intracellular delivery of biomacromolecular drugs.
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| 4. |
- Jing, Xiaona, et al.
(författare)
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Interaction of Peptidomimetics with Bilayer Membranes : Biophysical Characterization and Cellular Uptake
- 2012
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 28:11, s. 5167-5175
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Tidskriftsartikel (refereegranskat)abstract
- Enzymatically stable cell-penetrating alpha-peptide/beta-peptoid peptidomimetics constitute promising drug delivery vehicles for the transport of therapeutic biomacromolecules across membrane barriers. The aim of the present study was to elucidate the mechanism of peptidomimetic-lipid bilayer interactions. A series of peptidomimetics consisting of alternating cationic and hydrophobic residues displaying variation in length and N-terminal end group were applied to fluid-phase, anionic lipid bilayers, and their interaction was investigated using isothermal titration calorimetry (ITC) and ellipsometry. Titration of lipid vesicles into solutions of peptidomimetics resulted in exothermic adsorption processes, and the interaction of all studied peptidomimetics with anionic lipid membranes was found to be enthalpy-driven. The enthalpy and Gibbs free energy (Delta G) proved more favorable with increasing chain length. However, not all charges contribute equally to the interaction, as evidenced by the charge-normalized Delta G being inversely correlated to the sequence length. Ellipsometry data suggested that the hydrophobic residues also played an important role in the interaction process. Furthermore, Delta G extracted from ellipsometry data showed good agreement with that obtained with ITC. To further elucidate their interaction with biological membranes, quantitative uptake and cellular distribution were studied in proliferating HeLa cells by flow cytometry and confocal microscopy. The cellular uptake of carboxyfluorescein-labeled peptidomimetics showed a similar ranking as that obtained from the adsorbed amount, and binding energy to model membranes demonstrated that the initial interaction with the membrane is of key importance for the cellular uptake.
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| 5. |
- Jing, Xiaona, et al.
(författare)
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Membrane adsorption and binding, cellular uptake and cytotoxicity of cell-penetrating peptidomimetics with alpha-peptide/beta-peptoid backbone : Effects of hydrogen bonding and alpha-chirality in the beta-peptoid residues
- 2012
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Ingår i: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 0005-2736 .- 1879-2642. ; 1818:11, s. 2660-2668
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Tidskriftsartikel (refereegranskat)abstract
- Cell-penetrating peptides (CPPs) provide a promising approach for enhancing intracellular delivery of therapeutic biomacromolecules by increasing transport through membrane barriers. Here, proteolytically stable cell-penetrating peptidomimetics with alpha-peptide/beta-peptoid backbone were studied to evaluate the effect of alpha-chirality in the beta-peptoid residues and the presence of guanidinium groups in the alpha-amino acid residues on membrane interaction. The molecular properties of the peptidomimetics in solution (surface and intramolecular hydrogen bonding, aqueous diffusion rate and molecular size) were studied along with their adsorption to lipid bilayers, cellular uptake, and toxicity. The surface hydrogen bonding ability of the peptidomimetics reflected their adsorbed amounts onto lipid bilayers as well as with their cellular uptake, indicating the importance of hydrogen bonding for their membrane interaction and cellular uptake. Ellipsometry studies further demonstrated that the presence of chiral centers in the beta-peptoid residues promotes a higher adsorption to anionic lipid bilayers, whereas circular dichroism results showed that alpha-chirality influences their overall mean residue ellipticity. The presence of guanidinium groups and alpha-chiral beta-peptoid residues was also found to have a significant positive effect on uptake in living cells. Together, the findings provide an improved understanding on the behavior of cell-penetrating peptidomimetics in the presence of lipid bilayers and live cells.
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| 6. |
- Ongun, Melike, et al.
(författare)
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Lipid nanoparticles for local delivery of mRNA to the respiratory tract : Effect of PEG-lipid content and administration route
- 2024
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Ingår i: European Journal of Pharmaceutics and Biopharmaceutics. - 0939-6411. ; 198
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Tidskriftsartikel (refereegranskat)abstract
- Design of inhalable mRNA therapeutics is promising because local administration in the respiratory tract is minimally invasive and induces a local response. However, several challenges related to administration via inhalation and respiratory tract barriers have so far prevented the progress of inhaled mRNA therapeutics. Here, we investigated factors of importance for lipid nanoparticle (LNP)-mediated delivery of mRNA to the respiratory tract. We hypothesized that: (i) the PEG-lipid content is important for providing colloidal stability during aerosolization and for mucosal delivery, (ii) the PEG-lipid content influences the expression of mRNA-encoded protein in the lungs, and (iii) the route of administration (nasal versus pulmonary) affects mRNA delivery in the lungs. In this study, we aimed to optimize the PEG-lipid content for mucosal delivery and to investigate the effect of administration route on the kinetics of protein expression. Our results show that increasing the PEG-lipid content improves the colloidal stability during the aerosolization process, but has a negative impact on the transfection efficiency in vitro. The kinetics of protein expression in vivo is dependent on the route of administration, and we found that pulmonary administration of mRNA-LNPs to mice results in more durable protein expression than nasal administration. These results demonstrate that the design of the delivery system and the route of administration are important for achieving high mRNA transfection efficiency in the respiratory tract.
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| 7. |
- Rodrigues, Letícia, et al.
(författare)
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Immune responses induced by nano-self-assembled lipid adjuvants based on a monomycoloyl glycerol analogue after vaccination with the Chlamydia trachomatis major outer membrane protein.
- 2018
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Ingår i: Journal of Controlled Release. - : Elsevier BV. - 0168-3659 .- 1873-4995. ; 285, s. 12-22
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Tidskriftsartikel (refereegranskat)abstract
- Nanocarriers based on inverse hexagonal liquid crystalline phases (hexosomes) show promising potential as vaccine delivery systems. Their unique internal structure, composed of both lipophilic domains and water-containing channels, renders them capable of accommodating immunopotentiating compounds and antigens. However, their adjuvant properties are poorly understood. We hypothesized that the supramolecular structure of the lyotropic liquid crystalline phase influences the immunostimulatory activity of lipid-based nanocarriers. To test this, hexosomes were designed containing the lipid phytantriol (Phy) and the immunopotentiator monomycoloyl glycerol-1 (MMG-1). Self-assembly of Phy and MMG-1 into nanocarriers featuring an internal hexagonal phase was confirmed by small-angle X-ray scattering and cryogenic transmission electron microscopy. The effect of the nanostructure on the adjuvant activity was studied by comparing the immunogenicity of Phy/MMG-1 hexosomes with MMG-1-containing lamellar liquid crystalline nanoparticles (liposomes, CAF04). The quality and magnitude of the elicited immune responses were determined after vaccination of CB6/F1 mice using the Chlamydia trachomatis major outer membrane protein (MOMP) as antigen. MMG-1-based hexosomes potentiated significantly stronger MOMP-specific humoral responses than CAF04 liposomes. The liposome-based vaccine formulation induced a much stronger MOMP-specific cell-mediated immune response compared to hexosome-adjuvanted MOMP, which elicited minimal MOMP-specific T-cell stimulation after vaccination. Hence, our data demonstrates that hexosomal and liposomal adjuvants activate the immune system via different mechanisms. Our work provides valuable insights into the adjuvant potential of hexosomes and emphasizes that engineering of the supramolecular structure can be used to design adjuvants with customized immunological properties.
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| 8. |
- Skovbakke, Sarah Line, et al.
(författare)
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Combining Elements from Two Antagonists of Formyl Peptide Receptor 2 Generates More Potent Peptidomimetic Antagonists.
- 2017
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Ingår i: Journal of medicinal chemistry. - : American Chemical Society (ACS). - 1520-4804 .- 0022-2623. ; 60:16, s. 6991-6997
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Tidskriftsartikel (refereegranskat)abstract
- Structural optimization of a peptidomimetic antagonist of formyl peptide receptor 2 (FPR2) was explored by an approach involving combination of elements from the two most potent FPR2 antagonists described: a Rhodamine B-conjugated 10-residue gelsonin-derived peptide (i.e., PBP10, RhB-QRLFQVKGRR-OH) and the palmitoylated α-peptide/β-peptoid hybrid Pam-(Lys-βNspe)6-NH2. This generated an array of hybrid compounds from which a new subclass of receptor-selective antagonists was identified. The most potent representatives displayed activity in the low nanomolar range. The resulting stable and potent FPR2-selective antagonists (i.e., RhB-(Lys-βNphe)n-NH2; n = 4-6) are expected to become valuable tools in further elucidation of the physiological role of FPR2 in health and disease.
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| 9. |
- Skovbakke, Sarah Line, et al.
(författare)
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The peptidomimetic Lau-(Lys-βNSpe)6-NH2 antagonizes formyl peptide receptor 2 expressed in mouse neutrophils.
- 2016
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Ingår i: Biochemical pharmacology. - : Elsevier BV. - 1873-2968 .- 0006-2952. ; 119, s. 56-65
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Tidskriftsartikel (refereegranskat)abstract
- The formyl peptide receptor (FPR) gene family has a complex evolutionary history and comprises eight murine members but only three human representatives. To enable translation of results obtained in mouse models of human diseases, more comprehensive knowledge of the pharmacological similarities/differences between the human and murine FPR family members is required. Compared to FPR1 and FPR2 expressed by human neutrophils, very little is known about agonist/antagonist recognition patterns for their murine orthologues, but now we have identified two potent and selective formylated peptide agonists (fMIFL and PSMα2) for Fpr1 and Fpr2, respectively. These peptides were used to determine the inhibition profile of a set of antagonists with known specificities for the two FPRs in relation to the corresponding murine receptors. Some of the most potent and selective antagonists for the human receptors proved to be devoid of effect on their murine orthologues as determined by their inability to inhibit superoxide release from murine neutrophils upon stimulation with receptor-specific agonists. The Boc-FLFLF peptide was found to be a selective antagonist for Fpr1, whereas the lipidated peptidomimetic Lau-(Lys-βNSpe)6-NH2 and the hexapeptide WRW4 were identified as Fpr2-selective antagonists.
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| 10. |
- Skovbakke, Sarah Line, et al.
(författare)
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The proteolytically stable peptidomimetic Pam-(Lys-βNSpe)6-NH2 selectively inhibits human neutrophil activation via formyl peptide receptor 2.
- 2015
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Ingår i: Biochemical pharmacology. - : Elsevier BV. - 1873-2968 .- 0006-2952. ; 93:2, s. 182-195
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Tidskriftsartikel (refereegranskat)abstract
- Immunomodulatory host defense peptides (HDPs) are considered to be lead compounds for novel anti-sepsis and anti-inflammatory agents. However, development of drugs based on HDPs has been hampered by problems with toxicity and low bioavailability due to in vivo proteolysis. Here, a subclass of proteolytically stable HDP mimics consisting of lipidated α-peptide/β-peptoid oligomers was investigated for their effect on neutrophil function. The most promising compound, Pam-(Lys-βNSpe)6-NH2, was shown to inhibit formyl peptide receptor 2 (FPR2) agonist-induced neutrophil granule mobilization and release of reactive oxygen species. The potency of Pam-(Lys-βNSpe)6-NH2 was comparable to that of PBP10, the most potent FPR2-selective inhibitor known. The immunomodulatory effects of structural analogs of Pam-(Lys-βNSpe)6-NH2 emphasized the importance of both the lipid and peptidomimetic parts. By using imaging flow cytometry in primary neutrophils and FPR-transfected cell lines, we found that a fluorescently labeled analog of Pam-(Lys-βNSpe)6-NH2 interacted selectively with FPR2. Furthermore, the interaction between Pam-(Lys-βNSpe)6-NH2 and FPR2 was found to prevent binding of the FPR2-specific activating peptide agonist Cy5-WKYMWM, while the binding of an FPR1-selective agonist was not inhibited. To our knowledge, Pam-(Lys-βNSpe)6-NH2 is the first HDP mimic found to inhibit activation of human neutrophils via direct interaction with FPR2. Hence, we consider Pam-(Lys-βNSpe)6-NH2 to be a convenient tool in the further dissection of the role of FPR2 in inflammation and homeostasis as well as for investigation of the importance of neutrophil stimulation in anti-infective therapy involving HDPs.
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| 11. |
- Xu, You, et al.
(författare)
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Insights into the mechanisms of interaction between inhalable lipid-polymer hybrid nanoparticles and pulmonary surfactant
- 2023
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Ingår i: Journal of Colloid and Interface Science. - : Elsevier BV. - 0021-9797. ; 633, s. 511-525
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Tidskriftsartikel (refereegranskat)abstract
- Pulmonary delivery of small interfering RNA (siRNA) using nanoparticle-based delivery systems is promising for local treatment of respiratory diseases. We designed dry powder inhaler formulations of siRNA-loaded lipid-polymer hybrid nanoparticles (LPNs) with aerosolization properties optimized for inhalation therapy. Interactions between LPNs and pulmonary surfactant (PS) determine the fate of inhaled LPNs, but interaction mechanisms are unknown. Here we used surface-sensitive techniques to study how physicochemical properties and pathological microenvironments influence interactions between siRNA-loaded LPNs and supported PS layers. PS was deposited on SiO2 surfaces as single bilayer or multilayers and characterized using quartz crystal microbalance with dissipation monitoring and Fourier-transform infrared spectroscopy with attenuated total reflection. Immobilization of PS as multilayers, resembling the structural PS organization in the alveolar subphase, effectively reduced the relative importance of interactions between PS and the underlying surface. However, the binding affinity between PS and LPNs was identical in the two models. The physicochemical LPN properties influenced the translocation pathways and retention time of LPNs. Membrane fluidity and electrostatic interactions were decisive for the interaction strength between LPNs and PS. Experimental conditions reflecting pathological microenvironments promoted LPN deposition. Hence, these results shed new light on design criteria for LPN transport through the air–blood barrier.
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