Dual-Color Fluorescence Fluctuation Spectroscopy To Study the Complexation between Poly-l-lysine and Oligonucleotides

Lucas, Bart; Rompaey, Elsa Van; Smedt, Stefaan C. De; Demeester, Joseph; Oostveldt, Patrick Van

doi:10.1021/ma0202383

Cited by 22 publications

(26 citation statements)

References 25 publications

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“…FFS monitors fluorescence intensity fluctuations in the excitation volume of a confocal microscope and can be used to evaluate the complexation of fluorescently labeled molecules [16,[31][32][33]. The fluorescence fluctuations originate from the movement of (poorly concentrated) fluorescent molecules in and out of the fixed confocal excitation volume.…”

Section: Methodsmentioning

confidence: 99%

Biodegradable Dextran Nanogels for RNA Interference: Focusing on Endosomal Escape and Intracellular siRNA Delivery

Raemdonck

Naeye

Buyens

et al. 2009

Adv Funct Materials

131

143

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The successful therapeutic application of small interfering RNA (siRNA) largely relies on the development of safe and effective delivery systems that are able to guide the siRNA therapeutics to the cytoplasm of the target cell. In this report, biodegradable cationic dextran nanogels are engineered by inverse emulsion photopolymerization and their potential as siRNA carriers is evaluated. The nanogels are able to entrap siRNA with a high loading capacity, based on electrostatic interaction. Confocal microscopy and flow cytometry analysis reveal that large amounts of siRNA‐loaded nanogels can be internalized by HuH‐7 human hepatoma cells without significant cytotoxicity. Following their cellular uptake, it is found that the nanogels are mainly trafficked towards the endolysosomes. The influence of two different strategies to enhance endosomal escape on the extent of gene silencing is investigated. It is found that both the application of photochemical internalization (PCI) and the use of an influenza‐derived fusogenic peptide (diINF‐7) can significantly improve the silencing efficiency of siRNA‐loaded nanogels. Furthermore, it is shown that an efficient gene silencing requires the degradation of the nanogels. As the degradation kinetics of the nanogels can easily be tailored, these particles show potential for intracellular controlled release of short interfering RNA.

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Section: Methodsmentioning

confidence: 99%

Biodegradable Dextran Nanogels for RNA Interference: Focusing on Endosomal Escape and Intracellular siRNA Delivery

Raemdonck

Naeye

Buyens

et al. 2009

Adv Funct Materials

131

143

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“…Particle Size and Zeta-Potential Measurements. Dynamic light scattering (DLS) and zeta-potential (ζ) measurements on the polyplexes were respectively carried out on a Malvern 4700 instrument (Malvern, Worcestershire, UK) and a Malvern Zetasizer 2000 (Malvern, Worcestershire, UK), as previously described 4 Fluorescence fluctuation profiles of Tetraspeck beads as simultaneously registered by the FFS “red detector” (upper panel) and “green detector” (lower panel). …”

Section: Methodsmentioning

confidence: 99%

“…In each FFS experiment, the fluorescence fluctuations were measured for 30 s. “Highly intense fluorescence peaks” (i.e., bursts of high fluorescence intensity) in the fluorescence fluctuation profiles were identified by a statistical approach previously described. , Hereby a threshold level is calculated, above which all fluorescence values are identified as “highly intense fluorescence peaks”. In the absence of highly intense fluorescence peaks (i.e., when no threshold level could be calculated), the fluorescence fluctuations were analyzed by autocorrelation analysis, and a diffusion coefficient could be calculated (as described elsewhere) …”

Section: Methodsmentioning

confidence: 99%

“…Our former work focused on the complexation of ONs with cationic homopolymers (e.g., polylysine), resulting in nonstructured polyplexes. − Especially, we showed that both single and dual color FFS allow studying the association and induced dissociation of oligonucleotides and cationic polymers. Others applied (single color) FFS to study the condensation of plasmid DNA with cationic carriers. , In this paper we investigate whether dual color FFS also allows evaluating the association/dissociation behavior of pegylated DNA complexes.…”

Section: Introductionmentioning

confidence: 99%

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Studying Pegylated DNA Complexes by Dual Color Fluorescence Fluctuation Spectroscopy

et al. 2004

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In this study, dual color fluorescence fluctuation spectroscopy (dual color FFS) was explored to characterize the association of oligonucleotides (ONs) to pegylated cationic block copolymers, both diblock poly(ethylene glycol)-poly(ethylenimine) (pEG-pEI) and multiblock pEG-pEI. The resulting polyelectrolyte complexes consist of a core of polyion strands, surrounded by a shell of pEG chains. To study the association of the ONs to pEG-pEI by dual color FFS, the oligonucleotides and the cationic block copolymers were labeled with spectrally different fluorescent dyes. Two alternative approaches were executed to label the pEG-pEI's: the dye was attached either to the pEG segment or to the cationic pEI segment of pEG-pEI. When the (diblock and multiblock) pEG-pEI's were labeled on the pEG segment, dual color FFS on the doubly labeled FITC-pEG-pEI/Cy5-ONs complexes revealed the simultaneous appearance of peaks of high fluorescence intensity in both detector channels. This indicates that numerous Cy5-ONs and numerous FITC-pEG-pEI chains move together through the detection volume of the FFS instrument, proving the interaction between many Cy5-ONs and many FITC-pEG-pEI strands to form one multimolecular complex. However, when the fluorescent dye was bound to the cationic pEI segment of diblock or multiblock pEG-pEI, these simultaneously occurring peaks were never observed. Gel electrophoresis experiments proved that diblock pEG-pEI labeled at the pEI segment could not complex the ONs anymore. Multiblock pEG-pEI labeled at the pEI segment was still able to fully complex the oligonucleotides (as shown by gel electrophoresis). The absence of peaks of high fluorescence intensity in FFS measurements was attributed to the quenching of the fluorophores in the core of the complexes. From these results, it could be concluded that dual color FFS allows studying the association and dissociation of pEG-pEI/ONs provided that the fluorescent label of the polymer is attached to the pEG segment and not to the pEI segment.

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“…For cytotoxicity, uptake, antisense activity and acceptor photobleaching experiments, the complexes were diluted in Opti-MEM ® instead of degradation buffer before adding the complexes to the cells. The hydrodynamic size and zeta potential of the polyplexes was checked by respectively dynamic light scattering and surface potential measurements (NanoZS, Malvern, Worcestershire, UK), as previously described (13).…”

Section: Preparation Of Pbae1/odn and Pbae2/odn Polyplexesmentioning

confidence: 99%

Efficient delivery of intact phosphodiester oligonucleotides by poly-β-amino esters

Remaut

Symens

Lucas

et al. 2010

Journal of Controlled Release

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Due to their great instability, phosphodiester antisense oligonucleotides (PO-ODNs) are rapidly degraded in the intracellular environment, which limits their biological activity. The release of PO-ODNs during a prolonged period of time could however greatly enhance their antisense effect by creating a pool of intact PO-ODNs at any time point. Poly-β-aminoesters are biodegradable cationic polymers which show potential for the controlled release of short DNA fragments like ODNs and small interfering RNA (siRNA). In this research we evaluated biodegradable poly-β-aminoesters as carriers for PO-ODNs and compared the antisense activity with nuclease stable phosphothioate (PS) ODNs. PBAE1 polymers were not able to generate an antisense effect with PO-or PS-ODNs, most likely due to their poor cellular uptake. When complexed to PBAE2 polymers at N/P ratio 10, both PO-and PS-ODNs downregulated the targeted protein expression with 70%. By confocal imaging we observed a high concentration of released PO-ODNs that formed nuclear bodies in the nucleoplasm. The ODNs in this nuclear bodies were still intact as could be demonstrated by Fluorescence Resonance Energy Transfer (FRET) and acceptor photobleaching. This was in clear contrast to PO-ODNs delivery by cationic liposomes where the ODNs that accumulated in the nucleus were degraded and nuclear bodies were not observed. We conclude that PBAE2 shows potential for the delivery of nuclease sensitive POODNs. This occurs however not through a time controlled release profile, but rather due to the rapid delivery of a high concentration of intact PO-ODNs that form nuclear bodies in the nuclei of the cells. These nuclear bodies can most likely act as a depot of intact PO-ODNs, resulting in efficient antisense activity.keywords : poly β aminoesters, Fluorescence Correlation Spectroscopy, Fluorescence Resonance Energy Transfer, oligonucleotides, nucleases, delivery 2 INTRODUCTIONDelivery of small DNA fragments such as antisense oligonucleotides (ODNs) and small interfering RNA (siRNA) remains an attractive research domain to achieve the therapeutic regulation of gene expression. Antisense oligonucleotides are short single stranded DNA strands which naturally consist of a phosphodiester (PO) backbone. Due to the high instability of this PO backbone, however, the routinely used ODNs consist of the more stabile phosphothioate (PS) backbone (1;2). ODNs can act both in the cytoplasm and the nucleus by a sequence-specific recognition of the complementary mRNA. Through a variety of mechanisms, the complementary mRNA is then destroyed which results in the downregulation of the corresponding protein (3;4).siRNAs are short double-stranded RNA fragments with a 2 nucleotides overhang at the 3' end. They are very potent molecules since they are recognised by intracellular RNA-induced silencing complexes (RISCs) which specifically and repeatedly destroy the targeted mRNA molecules. SiRNA can be used in much lower concentrations and generally achieves a higher knockdown efficiency when comp...

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Dual-Color Fluorescence Fluctuation Spectroscopy To Study the Complexation between Poly-l-lysine and Oligonucleotides

Cited by 22 publications

References 25 publications

Biodegradable Dextran Nanogels for RNA Interference: Focusing on Endosomal Escape and Intracellular siRNA Delivery

Biodegradable Dextran Nanogels for RNA Interference: Focusing on Endosomal Escape and Intracellular siRNA Delivery

Studying Pegylated DNA Complexes by Dual Color Fluorescence Fluctuation Spectroscopy

Efficient delivery of intact phosphodiester oligonucleotides by poly-β-amino esters

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