Proton-Actuated Membrane-Destabilizing Polyion Complex Micelles

An error occurred in the sequence of the antisense oligonucleotide shown in the supporting information file of this manuscript. New, corrected supporting information can now be found on its Wiley InterScience abstract page. Information was also missing from the caption of Figure 5; the corrected caption is as follows: Figure 5. a) Flow cytometry experiment showing the fluorescence intensity of PC-3 cells incubated with Cy3-labeled AON (400 nM) free, complexed to G5 PAMAM or entrapped in plain or Fab 0-PEG 115-b-P(PrMA 28-co-MAA 53)/G5 PAMAM PICMs (N/(P + COO-) molar ratio of 1.5). b) Confocal microscopy images of PC-3 cells after treatment with Cy3-AON (red, Panel ii) complexed to A) G5 PAMAM or B) Fab 0-PEG 115-b-P(PrMA 28-co-MAA 53)/G5 PAMAM PICMs. Endosomes/lysosomes were stained by LysoTracker (green, Panel i), and the images were merged in Panel iii. c) Bcl-2 gene silencing in PC-3 cells transfected for 5 h with AON (400 nM) or siRNA (25 nM) complexed to G5 PAMAM or entrapped in plain or Fab 0-PEG 115-b-P(PrMA 28-co-MAA 53)/G5 or G3 PAMAM PICMs at N/(P + COO-) of 1.5 (final volume of the transfection medium is 1 mL). Control cells were treated with medium alone. The effect of preincubation with 10% FBS, free CD71 or bafilomycin is indicated on the graph. The inset is representative of immunoreactive Bcl-2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as determined by immunoblotting. The molar ratio of the Fab 0-PEG 145-b-P(PrMA 27-co-MAA 58) versus PEG 115-b-P(PrMA 28-co-MAA 53) is 2% for all the experiments. The results are representative of several independent experiments. The first 3 lanes on the left hand side of the inset represent the control-untreated cells.

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“…These cells also exhibited a decreased green fluorescence suggesting some leakage from acidic organelles (Panel B-iii). [33,34] …”

Section: Flow Cytometry and Intracellular Trafficking By Lscmmentioning

confidence: 99%

Delivery of Nucleic Acids through the Controlled Disassembly of Multifunctional Nanocomplexes

Elsabahy

Wazen

Bayó‐Puxán

et al. 2009

Adv Funct Materials

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“…Most polymers investigated so far for the design of pH-sensitive liposomes are based on poly(alkyl acrylic acid)s (Chen et al, 1999;Jones et al, 2003;Kyriakides et al, 2002), succinylated PEG (Kono et al, 1994;Mizoue et al, 2002), biodegradable polyphosphazenes (CouffinHoarau and and N-isopropylacrylamide (NIPAM) copolymers (Meyer et al, 1998;Roux et al, 2002aRoux et al, , 2003Zignani et al, 2000). For example, poly(alkyl acrylate-co-methacrylic acid)s have been shown to express strong membrane destabilizing properties (Yessine et al, 2003) and have been used to construct pH-responsive lipoplexes (Yessine et al, 2006) and polyion complex micelles (Yessine et al, 2007) that were able to facilitate the transfer of oligonucleotides from the endosomes to the cytoplasm.…”

Section: Introductionmentioning

confidence: 99%

pH-sensitive immunoliposomes specific to the CD33 cell surface antigen of leukemic cells

Simard

Leroux

2009

International Journal of Pharmaceutics

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“…[53] Release from endosomes is a complex process which depends on the physicochemical properties of the endocytosed material. [54] Facilitated endosomal release can be achieved by endosomolytic agents like chloroquine, [55] Ca II , [56] sucrose, [57] and polyion complex micelles [58] or laser illumination [59] and other photochemical internalisation treatments. [60] Once translocated across the cellular membrane and distributed in the cytoplasm, the nuclear delivery of NLS peptides such as the SV40 large T antigen NLS (PKKKRKV) that was used in this study occurs in an energy-dependent way.…”

Section: Discussionmentioning

confidence: 99%

Enhanced Cellular Uptake and Cytotoxicity Studies of Organometallic Bioconjugates of the NLS Peptide in Hep G2 Cells

et al. 2009

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SPACE INVADERS: Organometallic fragments such as the ferrocenyl group (shown in red in the picture) help to enhance cellular entry of NLS peptides. Eventually, these nontoxic conjugates find their way to the cellular nucleus as shown by fluorescence microscopy studies in this work. Intracellular delivery to biomolecular targets is still a major challenge in molecular and cell biology. We recently found that attaching an organometallic group, namely the cobaltocenium cation, to the SV 40 large T antigen nuclear localisation signal (NLS) greatly enhances cellular uptake of the conjugate (Noor et al., Angew. Chem. Int. Ed. 2005, 45, 2429). In addition, nuclear localisation of the conjugate was observed. In this work, we present a thorough investigation of this novel cellular delivery system with respect to the nature of the metal complex and the peptide sequence. A number of ferrocene ((Fe(II)), neutral metal complex) and cobaltocenium ((Co(III)), cationic metal complex) bioconjugates with both the NLS wild-type sequence PKKKRKV and a scrambled sequence (NLS(scr), KKVKPKR) were prepared by solid-phase peptide synthesis (SPPS). Cellular and nuclear uptake of these bioconjugates was studied by fluorescence microscopy on living Hep G2 cells. In addition, cytotoxicity screening on the conjugates was carried out, as the toxic effects of several simple metallocenes have been noted previously. Rapid cellular uptake as well as nuclear localisation was observed for the metal-NLS conjugates, but not for any dipeptide controls, the metal-NLS(scr) conjugates or any metal-free conjugates. It thus appears that the presence of a metallocene, but not its charge, and the correct NLS sequence is essential for cellular uptake. Fluorescence microscopy co-localisation studies did not reveal a significant endosomal entrapment of the conjugates. The metallocene not only provides a hydrophobic handle for membrane translocation but also facilitates the localisation and distribution of the conjugate in the cytoplasm. The NLS peptide on the other hand is responsible for the nuclear localisation of the bioconjugate. Finally, none of the conjugates were found to be toxic up to the highest concentrations that was tested (1 mM) against the Hep G2 cells that were used in this study. In conclusion, this work supports metallocene-NLS bioconjugates, in particular with the very robust cobaltocenium group, as a simple but potent, nontoxic system for cellular uptake and nuclear delivery. Concurrently, our finding is relevant to the still-unresolved question of cytotoxicity of metallocenes because it excludes binding and/or damage to the DNA as a mechanism of metallocene cytotoxicity. This finding is confirmed by a combined yeast cytotoxicity/genotoxicity assay, which also shows very little toxic effects for all organometal-NLS conjugates that were tested.

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Proton-Actuated Membrane-Destabilizing Polyion Complex Micelles

Cited by 28 publications

References 15 publications

Delivery of Nucleic Acids through the Controlled Disassembly of Multifunctional Nanocomplexes

Delivery of Nucleic Acids through the Controlled Disassembly of Multifunctional Nanocomplexes

pH-sensitive immunoliposomes specific to the CD33 cell surface antigen of leukemic cells

Enhanced Cellular Uptake and Cytotoxicity Studies of Organometallic Bioconjugates of the NLS Peptide in Hep G2 Cells

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