| 1. |
- EL Andaloussi, Samir, et al.
(author)
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Cargo-dependent cytotoxicity and delivery efficacy of cell-penetrating peptides : a comparative study
- 2007
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In: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 407:2, s. 285-292
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Journal article (peer-reviewed)abstract
- The use of CPPs (cell-penetrating peptides) as delivery vectors for bioactive molecules has been an emerging field since 1994 when the first CPP, penetratin, was discovered. Since then, several CPPs, including the widely used Tat (transactivator of transcription) peptide, have been developed and utilized to translocate a wide range of compounds across the plasma membrane of cells both in vivo and in vitro. Although the field has emerged as a possible future candidate for drug delivery, little attention has been given to the potential toxic side effects that these peptides might exhibit in cargo delivery. Also, no comprehensive study has been performed to evaluate the relative efficacy of single CPPs to convey different cargos. Therefore we selected three of the major CPPs, penetratin, Tat and transportan 10, and evaluated their ability to deliver commonly used cargos, including fluoresceinyl moiety, double-stranded DNA and proteins (i.e. avidin and streptavidin), and studied their effect on membrane integrity and cell viability. Our results demonstrate the unfeasibility to use the translocation efficacy of fluorescein moiety as a gauge for CPP efficiency, since the delivery properties are dependent on the cargo used. Furthermore, and no less importantly, the toxicity of CPPs depends heavily on peptide concentration, cargo molecule and coupling strategy.
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| 2. |
- EL Andaloussi, Samir, et al.
(author)
-
Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo
- 2011
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In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 39:9, s. 3972-3987
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Journal article (peer-reviewed)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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| 3. |
- Fischer, D., et al.
(author)
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Importance of Thomas single-electron transfer in fast p-He collisions
- 2010
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In: Physical Review A. Atomic, Molecular, and Optical Physics. - 1050-2947 .- 1094-1622. ; 81:1, s. 12714-
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Journal article (peer-reviewed)abstract
- We report experimental angular differential cross sections for nonradiative single-electron capture in p-He collisions (p + He -> H + He+) with a separate peak at the 0.47 mrad Thomas scattering angle for energies in the 1.3-12.5 MeV range. We find that the intensity of this peak scales with the projectile velocity as v(P)(-11). This constitutes the first experimental test of the prediction from 1927 by L. H. Thomas [Proc. R. Soc. 114, 561 (1927)]. At our highest energy, the peak at the Thomas angle contributes with 13.5% to the total integrated nonradiative single-electron capture cross section.
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| 4. |
- Guterstam, Peter, et al.
(author)
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Splice-switching efficiency and specificity for oligonucleotides with locked nucleic acid monomers
- 2008
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In: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 412, s. 307-313
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Journal article (peer-reviewed)abstract
- The use of antisense oligonucleotides to modulate splicing patterns has gained increasing attention as a therapeutic platform and, hence, the mechanisms of splice-switching oligonucleotides are of interest. Cells expressing luciferase pre-mRNA interrupted by an aberrantly spliced beta-globin intron, HeLa pLuc705, were used to monitor the splice-switching activity of modified oligonucleotides by detection of the expression of functional luciferase. It was observed that phosphorothioate 2'-O-methyl RNA oligonucleotides containing locked nucleic acid monomers provide outstanding splice-switching activity. However, similar oligonucleotides with several mismatches do not impede splice-switching activity which indicates a risk for off-target effects. The splice-switching activity is abolished when mismatches are introduced at several positions with locked nucleic acid monomers suggesting that it is the locked nucleic acid monomers that give rise to low mismatch discrimination to target pre-mRNA. The results highlight the importance of rational sequence design to allow for high efficiency with simultaneous high mismatch discrimination for splice-switching oligonucleotides and suggest that splice-switching activity is tunable by utilizing locked nucleic acid monomers.
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| 5. |
- Lundin, Per, et al.
(author)
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Distinct Uptake Routes of Cell-Penetrating Peptide Conjugates
- 2008
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In: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 19:12, s. 2535-2542
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Journal article (peer-reviewed)abstract
- Cell-penetrating peptides (CPPs) are a growing family of peptides that have opened a new avenue in drug delivery, allowing various hydrophilic macromolecules to enter cells. In accordance with most other cationic delivery vectors, CPPs seem to rely mostly on endocytosis for internalization. However, due to conflicting results the exact endocytic pathways for CPP uptake have not yet been resolved. Here, we evaluated the ability of seven CPPs, with different chemical properties, to convey peptide nucleic acids (PNAs) inside cells. Assays based on both splice correction, generating biologically active read-out, and on traditional fluorescence measurements were utilized. The same assays were employed to assess different endocytic pathways and the dependence on extracellular heparan sulfates for internalization. Both highly cationic CPPs (M918, penetratin, and Tat) and amphipathic peptides (transportan, TP10, MAP, and pVEC) were investigated in this study. Conjugate uptake relied on endocytosis for all seven peptides but splice-correcting activity varied greatly for the investigated CPPs. The exact endocytic internalization routes were evaluated through the use of well-known endocytosis inhibitors and tracers. In summary, the different chemical properties of CPPs have little correlation with their ability to efficiently deliver splice-correcting PNA. However, conjugates of polycationic and amphipathic peptides appear to utilize different internalization routes.
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| 6. |
- Misra, Deepankar, et al.
(author)
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Two-Center Double-Capture Interference in Fast He2++H2 Collisions
- 2009
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In: Physical Review Letters. - 0031-9007 .- 1079-7114. ; 102:15, s. 153201-
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Journal article (peer-reviewed)abstract
- We report the first observation of Young-type interference effects in a two-electron transfer process. These effects change strongly as the projectile velocity changes in fast (1.2 and 2.0 MeV) He^{2+}-H_2 collisions as manifested in strong variations of the double-electron capture rates with the H_2 orientation. This is consistent with fully quantum mechanical calculations, which ignore sequential electron transfer, and a simple projectile de Broglie wave picture assuming that two-electron transfer probabilities are higher in collisions where the projectile passes close to either one of the H_2 nuclei.
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| 7. |
- Mäe, Maarja, et al.
(author)
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A stearylated CPP for delivery of splice correcting oligonucleotides using a non-covalent co-incubation strategy
- 2009
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In: Journal of Controlled Release. - : Elsevier BV. - 0168-3659 .- 1873-4995. ; 134:3, s. 221-227
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Journal article (peer-reviewed)abstract
- Aberrations in splicing patterns play a significant role in several diseases, and splice correction, together with other forms of gene regulation, is consequently an emerging therapeutic target. In order to achieve successful oligonucleotide transfection, efficient delivery vectors are generally necessary. In this study we present one such vector, the chemically modified cell-penetrating peptide (CPP) TP10, for efficient delivery of a splice-correcting 2'-OMe RNA oligonucleotide. Utilizing a functional splice correction assay, we assessed the transfection efficiency of non-covalent complexes of oligonucleotides and stearylated or cysteamidated CPPs. Stearylation of the CPPs Arg9 and penetratin, as well as cysteamidation of MPG and TP10, did not improve transfection, whereas the presence of an N-terminal stearyl group on TP10 improved delivery efficiency remarkably compared to the unmodified peptide. The splice correction levels observed with stearyl-TP10 are in fact in parity with the effects seen with the commercially available transfection agent Lipofectamine (TM) 2000. However, the inherent toxicity associated with cationic lipid-based transfections can be completely eliminated when using the stearylated TP10, making this vector highly promising for non-covalent delivery of negatively charged oligonucleotides.
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| 8. |
- Reinhed, Peter, et al.
(author)
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Cryogenic keV ion-beam storage in ConeTrap - a tool for ion-temperature control
- 2010
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In: Nuclear Instruments and Methods in Physics Research Section A. - : Elsevier B.V.. - 0168-9002 .- 1872-9576. ; 621:1-3, s. 83-90
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Journal article (peer-reviewed)abstract
- We have tested the ion-storage capabilities of the compact triple-electrode electrostatic ion-beam trap, ConeTrap, down to cryogenic temperatures. The low-temperature operation of this electrostatic storage device is an important test for the double electrostatic ion-ring experiment, DESIREE, which is presently under construction at Stockholm University. In the present work we measured the pressure dependent storage lifetimes of 2.5 keV He+ and 2.8 keV Ar+ ion beams in ConeTrap at temperatures down to 28 K and pressures down to 1.3·10-10 mbar. The so far longest measured ion storage lifetime using this system is 21.5±3.8 s for Ar+ ions. The present combination of ConeTrap and the cryogenic experimental chamber was recently applied in the first black-body correction-free measurement of the lifetime of the metastable He- ion at 10 K [Phys. Rev. Lett. 103, 213002(2009)].
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| 9. |
- Reinhed, Peter, et al.
(author)
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Precision lifetime measurements of He- in a cryogenic electrostatic ion-beam trap
- 2009
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In: Physical Review Letters. - 0031-9007 .- 1079-7114. ; 103:21, s. 213002-
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Journal article (peer-reviewed)abstract
- We have developed a small purely electrostatic ion-beam trap which may be operated in thermal equilibrium at precisely controlled temperatures down to 10 K. Thus, we avoid magnetic field induced mixing of quantum states and may effectively eliminate any influence from absorption of photons from black-body radiation. We report the first correction free measurements of the lifetimes of the 1s2s2p 4PoJ state of 4He- and the high precision result 359.0±0.7 μs for the J=5/2 level. The lifetimes for the J=3/2 and J=1/2 levels are determined to be 12.3±0.5 and 7.8±1.0 μs, respectively.
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| 10. |
- Schmidt, Henning, et al.
(author)
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DESIREE as a new tool for interstellar ion chemistry
- 2008
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In: International Journal of Astrobiology. - 1473-5504 .- 1475-3006. ; 7:3-4, s. 205-208
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Journal article (peer-reviewed)abstract
- A novel cryogenic electrostatic storage device consisting of two ion-beam storage rings with a common straight section for studies of interactions between oppositely charged ions at low and well-defined relative velocities is under construction at Stockholm University. Here we consider the prospect of using this new tool to measure cross-sections and rate coefficients for mutual neutralization reactions of importance in interstellar ion chemistry in general and specifically in cosmic pre-biotic ion chemistry.
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