| 1. |
- Lundberg, Pontus, et al.
(författare)
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Cell membrane translocation of the N-terminal (1-28) part of the prion protein
- 2002
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - 0006-291X .- 1090-2104. ; 299:1, s. 85-90
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Tidskriftsartikel (refereegranskat)abstract
- The N-terminal (1-28) part of the mouse prion protein (PrP) is a cell penetrating peptide, capable of transporting large hydrophilic cargoes through a cell membrane. Confocal fluorescence microscopy shows that it transports the protein avidin (67 kDa) into several cell lines. The (1-28) peptide has a strong tendency for aggregation and P-structure formation, particularly in interaction with negatively charged phospholipid membranes. The findings have implications for how prion proteins with uncleaved signal peptides in the N-termini may enter into cells, which is important for infection. The secondary structure conversion into beta-structure may be relevant as a seed for the conversion into the scrapie (PrPSc) form of the protein and its arnyloidic transformation.
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| 2. |
- Abdelhamid, Hani Nasser, et al.
(författare)
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Gene delivery using cell penetrating peptides-zeolitic imidazolate frameworks
- 2020
-
Ingår i: Microporous and Mesoporous Materials. - : Elsevier BV. - 1387-1811 .- 1873-3093. ; 300
-
Tidskriftsartikel (refereegranskat)abstract
- Cell-penetrating peptides (CPPs), and metal-organic frameworks (MOFs) are promising as next-generation for the delivery of gene-based therapeutic agents. Oligonucleotide (ON)-mediated assembly of nanostructures composed of hierarchical porous zeolitic imidazolate framework (ZIF-8), and nanoparticles such as graphene oxide (GO), and magnetic nanoparticles (MNPs) for gene therapy are reported. Five different types of non-viral vectors (ZIF-8, RhB@ZIF-8, BSA@ZIF-8, MNPs@ZIF-8, and GO@ZIF-8), and three gene therapeutic agents (plasmid, splice correction oligonucleotides (SCO), and small interfering RNA (siRNA)) were investigated. The polyplexes were characterized and applied for gene transfection. The materials show very low toxicity with high efficiency for luciferase transfection. ZIF-8 enhances the transfection of plasmid, SCO, siRNA of CPPs by 2-8 folds. The mechanism of the cell uptakes was also highlighted. Data reveal cell internalization via scavenger class A (SCARA).
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| 3. |
- Arukuusk, Piret, et al.
(författare)
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PepFects and NickFects for the Intracellular Delivery of Nucleic Acids
- 2015
-
Ingår i: Cell-Penetrating Peptides. - New York : Springer. - 9781493928057 - 9781493928064 ; , s. 303-315
-
Bokkapitel (refereegranskat)abstract
- Nucleic acids can be utilized in gene therapy to restore, alter, or silence gene functions. In order to reveal the biological activity nucleic acids have to reach their intracellular targets by passing through the plasma membrane, which is impermeable for these large and negatively charged molecules. Cell-penetrating peptides (CPPs) condense nucleic acids into nanoparticles using non-covalent complexation strategy and mediate their delivery into the cell, whereas the physicochemical parameters of the nanoparticles determine the interactions with the membranes, uptake mechanism, and subsequent intracellular fate. The nanoparticles are mostly internalized by endocytosis that leads to the entrapment of them in endosomal vesicles. Therefore design of new CPPs that are applicable for non-covalent complex formation strategy and harness endosomolytic properties is highly vital. Here we demonstrate that PepFects and NickFects are efficient vectors for the intracellular delivery of various nucleic acids.This chapter describes how to form CPP/pDNA nanoparticles, evaluate stable nanoparticles formation, and assess gene delivery efficacy.
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| 4. |
- Dowaidar, Moataz, et al.
(författare)
-
Chitosan enhances gene delivery of oligonucleotide complexes with magnetic nanoparticles–cell-penetrating peptide
- 2018
-
Ingår i: Journal of biomaterials applications. - : SAGE Publications. - 0885-3282 .- 1530-8022. ; 33:3, s. 392-401
-
Tidskriftsartikel (refereegranskat)abstract
- Gene-based therapies, including the delivery of oligonucleotides, offer promising methods for the treatment of cancer cells. However, they have various limitations including low efficiency. Herein, cell-penetrating peptides (CPPs)-conjugated chitosan-modified iron oxide magnetic nanoparticles (CPPs-CTS@MNPs) with high biocompatibility as well as high efficiency were tested for the delivery of oligonucleotides such as plasmid pGL3, splice correction oligonucleotides, and small-interfering RNA. A biocompatible nanocomposite, in which CTS@MNPs was incorporated in non-covalent complex with CPPs-oligonucleotide, is developed. Modifying the surface of magnetic nanoparticles with cationic chitosan-modified iron oxide improved the performance of magnetic nanoparticles-CPPs for oligonucleotide delivery. CPPs-CTS@MNPs complexes enhance oligonucleotide transfection compared to CPPs@MNPs or CPPs. The hydrophilic character of CTS@MNPs improves complexation with plasmid pGL3, splice correction oligonucleotides, and small-interfering RNA payload, which consequently resulted in not only strengthening the colloidal stability of the constructed complex but also improving their biocompatibility. Transfection using PF14-splice correction oligonucleotides-CTS@MNPs showed sixfold increase of the transfection compared to splice correction oligonucleotides-PF14 that showed higher transfection than the commercially available lipid-based vector Lipofectamine™ 2000. Nanoscaled CPPs-CTS@MNPs comprise a new family of biomaterials that can circumvent some of the limitations of CPPs or magnetic nanoparticles.
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| 5. |
- Dowaidar, Moataz, et al.
(författare)
-
Graphene oxide nanosheets in complex with cell penetrating peptides for oligonucleotides delivery
- 2017
-
Ingår i: Biochimica et Biophysica Acta - General Subjects. - : Elsevier BV. - 0304-4165 .- 1872-8006. ; 1861:9, s. 2334-2341
-
Tidskriftsartikel (refereegranskat)abstract
- A new strategy for gene transfection using the nanocarrier of cell penetrating peptides (CPPs; PepFect14 (PF14) or PepFect14 (PF14) (PF221)) in complex with graphene oxide (GO) is reported. GO complexed with CPPs and plasmid (pGL3), splice correction oligonucleotides (SCO) or small interfering RNA (siRNA) are performed. Data show adsorption of CPPs and oligonucleotides on the top of the graphenic lamellar without any observed change of the particle size of GO. GO mitigates the cytotoxicity of CPPs and improves the material biocompatibility. Complexes of GO-pGL3-CPPs (CPPs; PF14 or PF221) offer 2.1–2.5 fold increase of the cell transfection compared to pGL3-CPPs (CPPs; PF14 or PF221). GO-SCO-PF14 assemblies effectively transfect the cells with an increase of > 10–25 fold compared to the transfection using PF14. The concentration of GO plays a significant role in the material nanotoxicity and the transfection efficiency. The results open a new horizon in the gene treatment using CPPs and offer a simple strategy for further investigations.
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| 6. |
- Dowaidar, Moataz, et al.
(författare)
-
Magnetic Nanoparticle Assisted Self-assembly of Cell Penetrating Peptides-Oligonucleotides Complexes for Gene Delivery
- 2017
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
-
Tidskriftsartikel (refereegranskat)abstract
- Magnetic nanoparticles (MNPs, Fe3O4) incorporated into the complexes of cell penetrating peptides (CPPs)-oligonucleotides (ONs) promoted the cell transfection for plasmid transfection, splice correction, and gene silencing efficiencies. Six types of cell penetrating peptides (CPPs; PeptFect220 (denoted PF220), PF221, PF222, PF223, PF224 and PF14) and three types of gene therapeutic agents (plasmid (pGL3), splicing correcting oligonucleotides (SCO), and small interfering RNA (siRNA) were investigated. Magnetic nanoparticles incorporated into the complexes of CPPs-pGL3, CPPs-SCO, and CPPs-siRNA showed high cell biocompatibility and efficiently transfected the investigated cells with pGL3, SCO, and siRNA, respectively. Gene transfer vectors formed among PF14, SCO, and MNPs (PF14-SCO-MNPs) showed a superior transfection efficiency (up to 4-fold) compared to the noncovalent PF14-SCO complex, which was previously reported with a higher efficiency compared to commercial vector called Lipofectamine™2000. The high transfection efficiency of the new complexes (CPPs-SCO-MNPs) may be attributed to the morphology, low cytotoxicity, and the synergistic effect of MNPs and CPPs. PF14-pDNA-MNPs is an efficient complex for in vivo gene delivery upon systemic administration. The conjugation of CPPs-ONs with inorganic magnetic nanoparticles (Fe3O4) may open new venues for selective and efficient gene therapy.
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| 7. |
- Dowaidar, Moataz, et al.
(författare)
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Refinement of a Quantitative Structure–Activity Relationship Model for Prediction of Cell-Penetrating Peptide Based Transfection Systems
- 2017
-
Ingår i: International Journal of Peptide Research and Therapeutics. - : Springer Science and Business Media LLC. - 1573-3904 .- 1573-3149. ; 23:1, s. 91-100
-
Tidskriftsartikel (refereegranskat)abstract
- Cell-penetrating peptide (CPP) based transfection systems (PBTS) are a promising class of drug delivery vectors. CPPs are short mainly cationic peptides capable of delivering cell non-permeant cargo to the interior of the cell. Some CPPs have the ability to form non-covalent complexes with oligonucleotides for gene therapy applications. In this study, we use quantitative structure–activity relationships (QSAR), a statistical method based on regression data analysis. Here, a fragment QSAR (FQSAR) model is developed to predict new peptides based on standard alpha helical conformers and Assisted Model Building with Energy Refinement molecular mechanics simulations of previous peptides. These new peptides were examined for plasmid transfection efficiency and compared with their predicted biological activity. The best predicted peptides were capable of achieving plasmid transfection with significant improvement compared to the previous generation of peptides. Our results demonstrate that FQSAR model refinement is an efficient method for optimizing PBTS for improved biological activity.
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| 8. |
- Dowaidar, Moataz, et al.
(författare)
-
Role of autophagy in cell-penetrating peptide transfection model
- 2017
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
-
Tidskriftsartikel (refereegranskat)abstract
- Cell-penetrating peptides (CPPs) uptake mechanism is still in need of more clarification to have a better understanding of their action in the mediation of oligonucleotide transfection. In this study, the effect on early events (1 h treatment) in transfection by PepFect14 (PF14), with or without oligonucleotide cargo on gene expression, in HeLa cells, have been investigated. The RNA expression profile was characterized by RNA sequencing and confirmed by qPCR analysis. The gene regulations were then related to the biological processes by the study of signaling pathways that showed the induction of autophagy-related genes in early transfection. A ligand library interfering with the detected intracellular pathways showed concentration-dependent effects on the transfection efficiency of splice correction oligonucleotide complexed with PepFect14, proving that the autophagy process is induced upon the uptake of complexes. Finally, the autophagy induction and colocalization with autophagosomes have been confirmed by confocal microscopy and transmission electron microscopy. We conclude that autophagy, an inherent cellular response process, is triggered by the cellular uptake of CPP-based transfection system. This finding opens novel possibilities to use autophagy modifiers in future gene therapy.
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| 9. |
- Erlandsson, Mikael, et al.
(författare)
-
Metallic zinc reduction of disulfide bonds between cysteine residues in peptides and proteins
- 2005
-
Ingår i: International Journal of Peptide Research and Therapeutics. - : Springer Science and Business Media LLC. - 1573-3149 .- 1573-3904. ; 11:4, s. 261-265
-
Tidskriftsartikel (refereegranskat)abstract
- The use of powdered metallic zinc in acidic solution for the reduction of disulfide bonds in peptides and proteins has been investigated. The method has several advantages over the traditional mereapto based reducing methods currently used; the reducing agent is readily available and inexpensive; reduction can be performed in weakly acidic solutions of water and/or acetonitrile; work up simply consists of a centrifugation step followed by pipeting the supernatant from the metal pellet, thereby greatly diminishing the risk of reoxidation as a more elaborate work up procedure could result in. As no mercapto compounds are added, there is no risk that the reducing agent will interfere in subsequent modification of the thiol functionality. Disulfides in a model peptide are reduced within 5 min in any mixture of water/acetonitrile containing 1% TFA, all disulfides in insulin is reduced within I h in any mixture of water/acetonitrile containing 5% acetic acid. To stress the convenience of the metallic zinc reduction method, the resulting thiol compound was subjected to two commonly employed reactions in peptide chemistry: Cys(Npys) directed disulfide formation (70% yield) and native chemical ligation between the reduced model peptide and Boc-Ala-p-metylthiobenzyl ester (65% yield of the ligation product plus disulfide formation between Cys and p-thiocresol).
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| 10. |
- Ezzat, Kariem, et al.
(författare)
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PepFect 14, a novel cell-penetrating peptide for oligonucleotide delivery in solution and as solid formulation
- 2011
-
Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 39:12, s. 5284-5298
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Tidskriftsartikel (refereegranskat)abstract
- Numerous human genetic diseases are caused by mutations that give rise to aberrant alternative splicing. Recently, several of these debilitating disorders have been shown to be amenable for splice-correcting oligonucleotides (SCOs) that modify splicing patterns and restore the phenotype in experimental models. However, translational approaches are required to transform SCOs into usable drug products. In this study, we present a new cell-penetrating peptide, PepFect14 (PF14), which efficiently delivers SCOs to different cell models including HeLa pLuc705 and mdx mouse myotubes; a cell culture model of Duchenne's muscular dystrophy (DMD). Non-covalent PF14-SCO nanocomplexes induce splice-correction at rates higher than the commercially available lipid-based vector Lipofectamine™ 2000 (LF2000) and remain active in the presence of serum. Furthermore, we demonstrate the feasibility of incorporating this delivery system into solid formulations that could be suitable for several therapeutic applications. Solid dispersion technique is utilized and the formed solid formulations are as active as the freshly prepared nanocomplexes in solution even when stored at an elevated temperatures for several weeks. In contrast, LF2000 drastically loses activity after being subjected to same procedure. This shows that using PF14 is a very promising translational approach for the delivery of SCOs in different pharmaceutical forms.
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| 11. |
- Holm, Tina, et al.
(författare)
-
Uptake of cell-penetrating peptides in yeasts
- 2005
-
Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 579:23, s. 5217-5222
-
Tidskriftsartikel (refereegranskat)abstract
- The uptake of different cell-penetrating peptides (CPPs) in two yeast species, Saccharomyces cerevisiae and Candida albicans, was studied using fluorescence HPLC-analyses of cell content. Comparison of the ability of penetratin, pVEC and (KFF)(3)K to traverse the yeast cell envelope shows that the cellular uptake of the peptides varies widely. Moreover, the intracellular degradation of the CPPs studied varies from complete stability to complete degradation. We show that intracellular degradation into membrane impermeable products can significantly contribute to the fluorescence signal. pVEC displayed highest internalizing capacity, and considering its stability in both yeast species, it is an attractive candidate for further studies.
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| 12. |
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| 13. |
- Hällbrink, Mattias, et al.
(författare)
-
Prediction of cell-penetrating peptides
- 2007. - 2
-
Ingår i: Handbook of cell-penetrating peptides. - Boca Raton : CRC Press. - 9780849350900 - 9781420006087 ; , s. 77-85
-
Bokkapitel (refereegranskat)
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| 14. |
- Hällbrink, Mattias, et al.
(författare)
-
Prediction of Cell-Penetrating Peptides
- 2015
-
Ingår i: Cell-Penetrating Peptides. - New York, NY : Springer-Verlag New York. - 9781493928057 - 9781493928064 ; , s. 39-58
-
Bokkapitel (refereegranskat)abstract
- The in silico methods for the prediction of the cell-penetrating peptides are reviewed. Those include the multivariate statistical methods, machine-learning methods such as the artificial neural networks and support vector machines, and molecular modeling techniques including molecular docking and molecular dynamics.The applicability of the methods is demonstrated on the basis of the exemplary cases from the literature.
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| 15. |
- Järver, Peter, et al.
(författare)
-
Peptide nanoparticle delivery of charge-neutral splice-switching morpholino oligonucleotides.
- 2015
-
Ingår i: Nucleic Acid Therapeutics. - : Mary Ann Liebert Inc. - 2159-3337 .- 2159-3345. ; 25:2, s. 65-77
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Tidskriftsartikel (refereegranskat)abstract
- Oligonucleotide analogs have provided novel therapeutics targeting various disorders. However, their poor cellular uptake remains a major obstacle for their clinical development. Negatively charged oligonucleotides, such as 2'-O-Methyl RNA and locked nucleic acids have in recent years been delivered successfully into cells through complex formation with cationic polymers, peptides, liposomes, or similar nanoparticle delivery systems. However, due to the lack of electrostatic interactions, this promising delivery method has been unsuccessful to date using charge-neutral oligonucleotide analogs. We show here that lipid-functionalized cell-penetrating peptides can be efficiently exploited for cellular transfection of the charge-neutral oligonucleotide analog phosphorodiamidate morpholino. The lipopeptides form complexes with splice-switching phosphorodiamidate morpholino oligonucleotide and can be delivered into clinically relevant cell lines that are otherwise difficult to transfect while retaining biological activity. To our knowledge, this is the first study to show delivery through complex formation of biologically active charge-neutral oligonucleotides by cationic peptides.
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| 16. |
- Lehto, Tõnis, et al.
(författare)
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Saturated Fatty Acid Analogues of Cell-Penetrating Peptide PepFect14: Role of Fatty Acid Modification in Complexation and Delivery of Splice-Correcting Oligonucleotides
- 2017
-
Ingår i: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 28:3, s. 782-792
-
Tidskriftsartikel (refereegranskat)abstract
- Modifying cell-penetrating peptides (CPPs) with fatty acids has long been used to improve peptide-mediated nucleic acid delivery. In this study we have revisited this phenomenon with a systematic approach where we developed a structure activity relationship to describe the role of the acyl chain length in the transfection process. For that we took a well studied CPP, PepFectl4, as the basis and varied its N-terminal acyl chain length from 2 to 22 carbons. To evaluate the delivery efficiency, the peptides were noncovalently complexed with a splice-correcting oligonucleotide (SCO) and tested in HeLa pLuc705 reporter cell line. Our results demonstrate that biological splice-correction activity emerges from acyl chain of 12 carbons and increases linearly with each additional carbon. To assess the underlying factors regarding how the transfection efficacy of these complexes is dependent on hydrophobicity, we used an array of different methods. For the functionally active peptides (C12-22) there was no apparent difference in their physicochemical properties, including complex formation efficiency, hydrodynamic size, and zeta potential. Moreover, membrane activity studies with peptides and their complexes with SCOs confirmed that the toxicity of the complexes at higher molar ratios is mainly caused by the free fraction of the peptide which is not incorporated into the peptide/oligonucleotide complexes. Finally, we show that the increase in splice-correcting activity correlates with the ability of the complexes to associate with the cells. Collectively these studies lay the ground work for how to design highly efficient CPPs and how to optimize their oligonucleotide complexes for lowest toxicity without losing efficiency.
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| 17. |
- Lindgren, Maria, et al.
(författare)
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Overcoming methotrexate resistance in breast cancer tumour cells by the use of a new cell-penetrating peptide
- 2006
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Ingår i: Biochemical Pharmacology. - : Elsevier BV. - 0006-2952 .- 1356-1839. ; 71:4, s. 416-425
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Tidskriftsartikel (refereegranskat)abstract
- Resistance to chemotherapy limits the effectiveness of anti-cancer drug treatment. Here, we present a new approach to overcome the setback of drug resistance by designing a conjugate of a cell-penetrating peptide and the cytostatic agent methotrexate (MTX). Two different peptides, YTA2 and YTA4, were designed and their intracellular delivery efficiency was characterized by fluorescence microscopy and quantified by fluorometry. MTX was conjugated to the transport peptides and the ability of the peptide–MTX conjugates to inhibit dihydrofolate reductase, the target enzyme of MTX, was found to be 15 and 20 times less potent than MTX. In addition, in vitro studies were performed in a drug resistant cell model using the 100-fold MTX resistant breast cancer cells MDA-MB-231. At a concentration of 1 mM, the peptide–MTX conjugates were shown to overcome MTX resistance and kill the cells more efficiently than MTX alone. Estimated EC50’s were determined for MTX, MTXYTA2 and YTA2 to be 18.5, 3.8 and 20 µM, respectively. In summary, cell-penetrating peptide conjugation of MTX is a new way of increasing delivery, and thereby, the potency of already well-characterized therapeutic molecules into drug resistant tumour cells.
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| 18. |
- Lorents, Annely, et al.
(författare)
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Cell-penetrating Peptides Split into Two Groups Based on Modulation of Intracellular Calcium Concentration
- 2012
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 287:20, s. 16880-16889
-
Tidskriftsartikel (refereegranskat)abstract
- Cell-penetrating peptides (CPPs) promote the uptake of different cargo molecules, e.g. therapeutic compounds, making the harnessing of CPPs a promising strategy for drug design and delivery. However, the internalization mechanisms of CPPs are still under discussion, and it is not clear how cells compensate the disturbances induced by peptides in the plasma membrane. In this study, we demonstrate that the uptake of various CPPs enhances the intracellular Ca2+ levels in Jurkat and HeLa cells. The elevated Ca2+ concentration in turn triggers plasma membrane blebbing, lysosomal exocytosis, and membrane repair response. Our results indicate that CPPs split into two major classes: (i) amphipathic CPPs that modulate the plasma membrane integrity inducing influx of Ca2+ and activating downstream responses starting from low concentrations; (ii) non-amphipathic CPPs that do not evoke changes at relevant concentrations. Triggering of the membrane repair response may help cells to replace distorted plasma membrane regions and cells can recover from the influx of Ca2+ if its level is not drastically elevated.
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| 19. |
- Muthukrishnan, Uma, 1984-, et al.
(författare)
-
The exosome membrane localization of histones is independent of DNA and upregulated in response to stress
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Extracellular histones contribute to many acute and chronic diseases but also populate the secretomes of healthy cells and biofluids. However, a secretory pathway for histones has not been described. Here we report that core and linker histones localize to multivesicular bodies and are secreted via exosomes. Histones are tightly associated with the exosome membrane, with N-terminal domains exposed, in a DNA-independent manner. Furthermore, rapid upregulation of exosomal histones occurs following heat stress, accompanied by enhanced vesicle secretion and a shift towards a population of smaller vesicles. Proteomic analyses identified the downregulation of endosomal sorting complex required for transport (ESCRT) complex as a possible mechanism underlying increased histone secretion.We show for the first time that membrane-associated histones are actively secreted from intact cells via the multivesicular body/exosomal pathway. We demonstrate a novel pathway for extracellular histone release that may have a role in both health and disease.
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| 20. |
- Nordin, Joel Z., et al.
(författare)
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Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties
- 2015
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Ingår i: Nanomedicine. - : Elsevier BV. - 1549-9634 .- 1549-9642. ; 11:4, s. 879-883
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Tidskriftsartikel (refereegranskat)abstract
- Extracellular vesicles (EVs) are natural nanoparticles that mediate intercellular transfer of RNA and proteins and are of great medical interest; serving as novel biomarkers and potential therapeutic agents. However, there is little consensus on the most appropriate method to isolate high-yield and high-purity EVs from various biological fluids. Here, we describe a systematic comparison between two protocols for EV purification: ultrafiltration with subsequent liquid chromatography (UF-LC) and differential ultracentrifugation (UC). A significantly higher EV yield resulted from UF-LC as compared to UC, without affecting vesicle protein composition. Importantly, we provide novel evidence that, in contrast to UC-purified EVs, the biophysical properties of UF-LC-purified EVs are preserved, leading toadifferent in vivo biodistribution, with less accumulation in lungs. Finally, we show that UF-LC is scalable and adaptable for EV isolation from complex media types such as stem cell media, which is of huge significance for future clinical applications involving EVs.
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| 21. |
- Padari, Kart, et al.
(författare)
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S4(13)-PV Cell-Penetrating Peptide Forms Nanoparticle-Like Structures to Gain Entry Into Cells
- 2010
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Ingår i: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 21:4, s. 774-783
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Tidskriftsartikel (refereegranskat)abstract
- Despite increasing interest in cell-penetrating peptides (CPPs) as carriers for drugs and in gene therapy, the current understanding of their exact internalization mechanism is still far from complete. The cellular translocation of CPPs and their payloads has been mostly described by fluorescence- and activity-based methods, leaving the more detailed characterization at the ultrastructural level almost out of attention. Herein, we used transmission electron microscopy to characterize the membrane interaction and internalization of a cell-penetrating peptide S4(13)-PV. We demonstrate that S4(13)-PV peptide forms spherical nanoparticle-like regular structures upon association with cell surface glycosaminoglycans on the plasma membrane. Insertion of S4(13)-PV particles into plasma membrane induces disturbances and leads to the vesicular uptake of peptides by cells. We propose that for efficient cellular translocation S4(13)-PV peptides have to assemble into particles of specific size and shape. The spherical peptide particles are not dissociated in intracellular vesicles but often retain their organization and remain associated with the membrane of vesicles, destabilizing them and promoting the escape of peptides into cytosol. Lowering the temperature and inhibition of dynamins' activity reduce the internalization of S4(13)-PV peptides, but do not block it completely. Our results provide an ultrastructural insight into the interaction mode of CPPs with the plasma membrane and the distribution in cells, which might help to better understand how CPPs cross the biological membranes and gain access into cells.
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| 22. |
- Palm-Apergi, Caroline, et al.
(författare)
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A new rapid cell-penetrating peptide based strategy to produce bacterial ghosts for plasmid delivery
- 2008
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Ingår i: Journal of Controlled Release. - : Elsevier BV. - 0168-3659 .- 1873-4995. ; 132:1, s. 49-54
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Tidskriftsartikel (refereegranskat)abstract
- The production of bacterial ghosts involves the lysis gene E plasmid in order to lyse and empty the bacteria of their cytoplasmic contents. After lysis the ghosts can either be loaded with new desired DNA and used for delivery to mammalian cells or used in vaccination. Cell-penetrating peptides have been used as delivery vehicles of drugs and oligonucleotides. Although many of them show low toxicity they have been compared to antimicrobial peptides involved in innate immunity. Recently we showed that cell-penetrating peptides also could be antimicrobial. In this study we take advantage of the antimicrobial effect of one cell-penetrating peptide, namely MAP, which is a model amphipathic peptide and treat bacteria with the peptide to produce bacterial ghosts. This new peptide based strategy is not dependent on the lysis gene E plasmid thus; several tiresome steps are removed in the production of ghosts. In addition the ghosts can be preloaded with a desired plasmid or DNA further removing time consuming reprocessing steps. To our knowledge this is the first study that uses a cell-penetrating peptide based strategy to produce bacterial ghosts to be used in plasmid delivery.
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| 23. |
- Palm Apergi, Caroline, 1978-
(författare)
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Design and evaluation of drug delivery vehicles
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- A crucial aspect of drug delivery is efficient transport to the site of action. Thus, there is a need to design and evaluate new delivery vehicles. In this thesis two delivery vehicles, cell-penetrating peptides and bacterial ghosts, were evaluated. The understanding of the internalization and degradation kinetics of cell-penetrating peptides is important for the practical aspects of cargo delivery since peptides have a notorious reputation of being rapidly degraded. If the cell-penetrating peptide remains intact inside the cellular environment, there is a possibility that the peptide-cargo conjugate leaks back to the extracellular environment. However, if it is degraded outside the cell, the cargo will never be delivered. In order to improve uptake efficiency and to be able to foresee side effects, the translocation mechanism needs to be fully elucidated. Data gathered from the first two papers led to the proposal of a new me-chanism involved in cell-penetrating peptide uptake: the membrane repair response, a resealing mechanism rapidly patching up broken membranes. This mechanism could explain the divergence in perception concerning the uptake pathways. Furthermore a new assay to produce the second delivery vehicle, bacterial ghosts, was developed based on data from the cell-penetrating peptide investigations. Bacterial ghosts are dead bacteria devoid of cytoplasmic contents but still retaining their structural and morphological characteristics, after protein E lysis of the bacterial cell membrane. By using a cell-penetrating peptide with antimicrobial effects, a new rapid peptide-based strategy to produce ghosts was developed and the capability to deliver plasmid DNA into the cell for expression was evaluated.
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| 24. |
- Palm-Apergi, Caroline, et al.
(författare)
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The membrane repair response masks membrane disturbances caused by cell-penetrating peptide uptake
- 2009
-
Ingår i: The FASEB Journal. - : Wiley. - 0892-6638 .- 1530-6860. ; 23:1, s. 214-223
-
Tidskriftsartikel (refereegranskat)abstract
- Although cell-penetrating peptides are able to deliver cargo into cells, their uptake mechanism is still not fully understood and needs to be elucidated to improve their delivery efficiency. Herein, we present evidence of a new mechanism involved in uptake, the membrane repair response. Recent studies have suggested that there might be a direct penetration of peptides in parallel with different forms of endocytosis. The direct penetration of hydrophilic peptides through the hydrophobic plasma membrane, however, is highly controversial. Three proteins involved in target cell apoptosis—perforin, granulysin, and granzymes—share many features common in uptake of cell-penetrating peptides (e.g., they bind proteoglycans). During perforin uptake, the protein activates the membrane repair response, a resealing mechanism triggered in cells with injured plasma membrane, because of extracellular calcium influx. On activation of the membrane repair response, internal vesicles are mobilized to the site of the disrupted plasma membrane, resealing it within seconds. In this study, we have used flow cytometry, fluorescence, and electron microscopy, together with high-performance liquid chromatography and mass spectrometry, to present evidence that the membrane repair response is able to mask damages caused during cell-penetrating peptide uptake, thus preventing leakage of endogenous molecules out of the cell.—Palm-Apergi, C., Lorents, A., Padari, K., Pooga, M., and Hällbrink, M. The membrane repair response masks membrane disturbances caused by cell-penetrating peptide uptake.
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| 25. |
- Palm, Caroline, et al.
(författare)
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Peptide degradation is a critical determinant for cell-penetrating peptide uptake
- 2007
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434 .- 0005-2736. ; 1768:7, s. 1769-1776
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Tidskriftsartikel (refereegranskat)abstract
- Cell-penetrating peptide mediated uptake of labels appears to follow an equilibrium-like process. However, this assumption is only valid if the peptides are stabile. Hence, in this study we investigate intracellular and extracellular peptide degradation kinetics of two fluorescein labeled cell-penetrating peptides, namely MAP and penetratin, in Chinese hamster ovarian cells. The degradation and uptake kinetics were assessed by RP-HPLC equipped with a fluorescence detector. We show that MAP and penetratin are rapidly degraded both extracellularly and intracellularly giving rise to several degradation products. Kinetics indicates that intracellularly, the peptides exist in (at least) two distinct pools: one that is immediately degraded and one that is stabile. Moreover, the degradation could be decreased by treating the peptides with BSA and phenanthroline and the uptake was significantly reduced by cytochalasin B, chloroquine and energy depletion. The results indicate that the extracellular degradation determines the intracellular peptide concentration in this system and therefore the stability of cell-penetrating peptides needs to be evaluated.
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