| 1. |
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| 2. |
- Thomas, HS, et al.
(författare)
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- 2019
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swepub:Mat__t
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| 3. |
- Dowaidar, Moataz, et al.
(författare)
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Role of autophagy in cell-penetrating peptide transfection model
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
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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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| 4. |
- EL Andaloussi, Samir, et al.
(författare)
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Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo
- 2011
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 39:9, s. 3972-3987
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Tidskriftsartikel (refereegranskat)abstract
- While small interfering RNAs (siRNAs) have been rapidly appreciated to silence genes, efficient and non-toxic vectors for primary cells and for systemic in vivo delivery are lacking. Several siRNA-delivery vehicles, including cell-penetrating peptides (CPPs), have been developed but their utility is often restricted by entrapment following endocytosis. Hence, developing CPPs that promote endosomal escape is a prerequisite for successful siRNA implementation. We here present a novel CPP, PepFect 6 (PF6), comprising the previously reported stearyl-TP10 peptide, having pH titratable trifluoromethylquinoline moieties covalently incorporated to facilitate endosomal release. Stable PF6/siRNA nanoparticles enter entire cell populations and rapidly promote endosomal escape, resulting in robust RNAi responses in various cell types (including primary cells), with minimal associated transcriptomic or proteomic changes. Furthermore, PF6-mediated delivery is independent of cell confluence and, in most cases, not significantly hampered by serum proteins. Finally, these nanoparticles promote strong RNAi responses in different organs following systemic delivery in mice without any associated toxicity. Strikingly, similar knockdown in liver is achieved by PF6/siRNA nanoparticles and siRNA injected by hydrodynamic infusion, a golden standard technique for liver transfection. These results imply that the peptide, in addition to having utility for RNAi screens in vitro, displays therapeutic potential.
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| 5. |
- El-Sayed, R., et al.
(författare)
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Degradation of pristine and oxidized single wall carbon nanotubes by CYP3A4
- 2019
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Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 515:3, s. 487-492
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Tidskriftsartikel (refereegranskat)abstract
- Carbon nanotubes (CNTs) are a class of carbon based nanomaterials which have attracted substantial attention in recent years as they exhibit outstanding physical, mechanical and optical properties. In the last decade many studies have emerged of the underlying mechanisms behind CNT toxicity including malignant transformation, the formation of granulomas, inflammatory responses, oxidative stress, DNA damage and mutation. In the present investigation, we studied the biodegradation of single-walled carbon nanotubes (SWCNTs) by Cytochrome P450 enzymes (CYP3A4) through using Raman spectroscopy. CYP3A4 is known isozyme accountable for metabolizing various endogenous and exogenous xenobiotics. CYP3A4 is expressed dominantly in the liver and other organs including the lungs. Our results suggest that CYP3A4 has a higher affinity for p-SWNTs compared to c-SWNTs. HEK293 cellular viability was not compromised when incubated with SWNT. However, CYP3A4 transfected HEK293 cell line showed no digestion of c-SWNTs after incubation for 96 h. Cellular uptake of c-SWNTs was observed by electron microscopy and localization of c-SWNTs was confirmed in endosomal vesicles and in the cytoplasm. This is the first study CYP3A4 degrading both p-SWNTs and c-SWNTs in an in vitro setup. Interestingly, our results show that CYP3A4 is more proficient in degrading p-SWNTs than c-SWNTs. We also employed computational modeling and docking assessments to develop a further understanding of the molecular interaction mechanism. © 2019 Elsevier Inc.
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| 6. |
- Ezzat, Kariem
(författare)
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CELL PENETRATING PEPTIDES : CHEMICAL MODIFICATION AND FORMULATION DEVELOPMENT
- 2011
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cell penetrating peptides (CPPs) have been extensively studied and exploited as drug delivery vectors for a wide variety of therapeu-tic cargos. However, several issues remain to be addressed regarding the enhancement of their efficiency and stability. In addition, to be available for patients, CPP-based therapeutics have to be formulated into suitable pharmaceutical forms that can be readily manufactured, transported, stored and conveniently used.In this thesis, three chemically modified CPPs are developed having superior delivery properties for several nucleic acid-based the-rapeutic cargoes including: plasmids, small interfering RNA (siRNA) and splice switching oligonucleutides (SSOs), in different in-vitro and in-vivo models. In Paper I, we show that an N-terminally stearic acid-modified version of transportan-10 (TP10) can form stable nanopar-ticles with plasmids that efficiently transfect different cell types and can mediate efficient gene delivery in-vivo when administrated intra muscularly (i.m.) or intradermaly (i.d.). In paper II, stearyl-TP10 is further modified with pH titratable trifluoromethylquinoline moieties to facilitate endosomal release. The new peptide, denoted PepFect 6 (PF6), elicited robust RNAi responses when complexed with siRNA in several cell models and promoted strong RNAi responses in differ-ent organs following systemic delivery in mice without any associated toxicity. In paper III , a new peptide with ornithine modification, PF14, is shown to efficiently deliver SSOs in different cell models including HeLa pLuc705 and mdx mouse myotubes; a cell culture model of Duchenne‟s muscular dystrophy (DMD). Additionally, we have developed a method for incorporating this delivery system into solid formulation that could be suitable for several therapeutic appli-cations. Solid dispersion technique is utilized and the formed solid formulations are as active as the freshly prepared nanocparticles in solution even when stored at elevated temperatures for several weeks.Taken together, these results demonstrate that certain chemical modifications could drastically enhance the activity and stability of CPPs in-vitro and in-vivo. Moreover, we show that CPP-based thera-peutics could be formulated into convenient and manufacturable do-sage forms.
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| 7. |
- Ezzat, Kariem, 1983-
(författare)
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Cell-penetrating peptides; chemical modification, mechanism of uptake and formulation development
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Gene therapy holds the promise of revolutionizing the way we treat diseases. By using recombinant DNA and oligonucleotides (ONs), gene functions can be restored, altered or silenced according to the therapeutic need. However, gene therapy approaches require the delivery of large and charged nucleic acid-based molecules to their intracellular targets across the plasma membrane, which is inherently impermeable to such molecules. In this thesis, two chemically modified cell-penetrating peptides (CPPs) that have superior delivery properties for several nucleic acid-based therapeutics are developed. These CPPs can spontaneously form nanoparticles upon non-covalent complexation with the nucleic acid cargo, and the formed nanoparticles mediate efficient cellular transfection. In paper I, we show that an N-terminally stearic acid-modified version of transportan-10 (PF3) can efficiently transfect different cell types with plasmid DNA and mediates efficient gene delivery in-vivo when administrated intra muscularly (i.m.) or intradermaly (i.d.). In paper II, a new peptide with ornithine modification, PF14, is shown to efficiently deliver splice-switching oligonucleotides (SSOs) in different cell models including mdx mouse myotubes; a cell culture model of Duchenne’s muscular dystrophy (DMD). Additionally, we describe a method for incorporating the PF14-SSO nanoparticles into a solid formulation that is active and stable even when stored at elevated temperatures for several weeks. In paper III, we demonstrate the involvement of class-A scavenger receptor subtypes (SCARA3 & SCARA5) in the uptake of PF14-SSO nanoparticles, which possess negative surface charge, and suggest for the first time that some CPP-based systems function through scavenger receptors. In paper IV, the ability of PF14 to deliver siRNA to different cell lines is shown and their stability in simulated gastric acidic conditions is highlighted. Taken together, these results demonstrate that certain chemical modifications can drastically enhance the activity and stability of CPPs for delivering nucleic acids after spontaneous nanoparticle formation upon non-covalent complexation. Moreover, we show that CPP-based nanoparticles can be formulated into convenient and stable solid formulations that can be suitable for several therapeutic applications. Importantly, the involvement of scavenger receptors in the uptake of such nanoparticles is presented, which could yield novel possibilities to understand and improve the transfection by CPPs and other gene therapy nanoparticles.
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| 8. |
- 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
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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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| 9. |
- Ezzat, Kariem, et al.
(författare)
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Peptide-based matrices as drug delivery vehicles
- 2010
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Ingår i: Current pharmaceutical design. - 1381-6128 .- 1873-4286. ; 16:9, s. 1167-1178
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Forskningsöversikt (refereegranskat)abstract
- Peptides, polypeptides and proteins have been extensively studied for their various structural and functional roles in living organisms. However, breakthrough discoveries in the last decades identified some peptide-based matrices that posses the ability to traverse biological membranes, and many peptides, polypeptides and even complete proteins have been shown to have such properties. Hence, these matrices have been successfully used for the intracellular delivery of many therapeutic cargos including small molecules, proteins, peptides, oligonucleutides, plasmids and nanoparticles both in vitro and in vivo. Being neither toxic nor carcinogenic and meanwhile efficient in delivery, they are recognized as very promising vectors to overcome the shortcomings of the available technologies. The characteristics of these peptide-based matrices and their applications in drug delivery are here briefly illustrated together with current challenges and future prospects.
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| 10. |
- Ezzat, Kariem, et al.
(författare)
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Scavenger receptor-mediated uptake of cell-penetrating peptide nanoparticles with oligonucleotides
- 2012
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Ingår i: The FASEB Journal. - : Wiley. - 0892-6638 .- 1530-6860. ; 26:3, s. 1172-1180
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Tidskriftsartikel (refereegranskat)abstract
- Cell-penetrating peptides (CPPs) are shortcationic peptides that penetrate cells by interacting withthe negatively charged plasma membrane; however, thedetailed uptake mechanism is not clear. In contrary to theconventional mode of action of CPPs, we show here thata CPP, PepFect14 (PF14), forms negatively charged nanocomplexeswith oligonucleotides and their uptake is mediatedby class-A scavenger receptors (SCARAs). Specificinhibitory ligands of SCARAs, such as fucoidin, polyinosinicacid, and dextran sulfate, totally inhibit the activityof PF14-oligonucleotide nanocomplexes in the HeLapLuc705 splice-correction cell model, while nonspecific,chemically related molecules do not. Furthermore, RNAinterference (RNAi) knockdown of SCARA subtypes(SCARA3 and SCARA5) that are expressed in this cell lineled to a significant reduction of the activity to <50%. Inline with this, immunostaining shows prevalent colocalizationof the nanocomplexes with the receptors, and electronmicroscopy images show no binding or internalizationof the nanocomplexes in the presence of theinhibitory ligands. Interestingly, naked oligonucleotidesalso colocalize with SCARAs when used at high concentrations.These results demonstrate the involvement ofSCARA3 and SCARA5 in the uptake of PF14-oligonucleotidenanocomplexes and suggest for the first time thatsome CPP-based systems function through scavenger receptors,which could yield novel possibilities to understandand improve the transfection by CPPs.
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