Ethylendiamine core PAMAM dendrimers/siRNA complexes as in vitro silencing agents

Perez, Ana Paula; Romero, Eder Lilia; Morilla, María José

doi:10.1016/j.ijpharm.2009.06.035

Cited by 59 publications

(72 citation statements)

References 36 publications

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“…34 Similar results were recently reported by Waite and Roth, who found 40% silencing of the EGFP gene in U87 glioma cells using siRNA/G5 dendriplexes. 35 The delivery of siRNA to the cytoplasm of the cell is a critical step for the RNAinduced silencing assembly complex.…”

Section: Introductionsupporting

confidence: 87%

“…Previously, we have reported lower silencing activity of siRNA dendriplexes in T98G cells than in J774 cells (35% vs 45%). 34 This finding indicated that extent of cellular uptake of dendriplexes was not directly related to silencing. Similarly, O´Neill et al found that despite its higher uptake, cyclodextrinmediated luciferase expression was signifi cantly lower than for Lipofectamine 2000 and comparable with that of polyethylenimine in Caco2 cells.…”

Section: Discussionmentioning

confidence: 97%

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Uptake and intracellular traffic of siRNA dendriplexes in glioblastoma cells and macrophages

Perez¹,

Cosaka²,

Romero³

et al. 2011

IJN

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Background: Gene silencing using small interfering RNA (siRNA) is a promising new therapeutic approach for glioblastoma. The endocytic uptake and delivery of siRNA to intra cellular compartments could be enhanced by complexation with polyamidoamine dendrimers. In the present work, the uptake mechanisms and intracellular traffic of siRNA/generation 7 dendrimer complexes (siRNA dendriplexes) were screened in T98G glioblastoma and J774 macrophages. Methods: The effect of a set of chemical inhibitors of endocytosis on the uptake and silenc ing capacity of dendriplexes was determined by flow cytometry. Colocalization of fluorescent dendriplexes with endocytic markers and occurrence of intracellular dissociation were assessed by confocal laser scanning microscopy. Results: Uptake of siRNA dendriplexes by T98G cells was reduced by methylβcyclodextrin, and genistein, and cytochalasine D, silencing activity was reduced by genistein; dendriplexes colocalized with cholera toxin subunit B. Therefore, caveolindependent endocytosis was involved both in the uptake and silencing activity of siRNA dendriplexes. On the other hand, uptake of siRNA dendriplexes by J774 cells was reduced by methylβcyclodextrin, genistein, chlorpromazine, chloroquine, cytochalasine D, and nocodazole, the silencing activity was not affected by chlorpromazine, genistein or chloroquine, and dendriplexes colocalized with transferrin and cholera toxin subunit B. Thus, both clathrindependent and caveolindependent endocytosis mediated the uptake and silencing activity of the siRNA dendriplexes. SiRNA dendriplexes were internalized at higher rates by T98G but induced lower silencing than in J774 cells. SiRNA dendriplexes showed relatively slow dissociation kinetics, and their escape towards the cytosol was not mediated by acidification independently of the uptake pathway. Conclusion: The extent of cellular uptake of siRNA dendriplexes was inversely related to their silencing activity. The higher silencing activity of siRNA dendriplexes in J774 cells could be ascribed to the contribution of clathrindependent and caveolindependent endocytosis vs only caveolindependent endocytosis in T98G cells.

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Section: Introductionsupporting

confidence: 87%

Section: Discussionmentioning

confidence: 97%

Uptake and intracellular traffic of siRNA dendriplexes in glioblastoma cells and macrophages

Perez¹,

Cosaka²,

Romero³

et al. 2011

IJN

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“…Polycationic nonviral gene carriers benefit from greater flexibility, improved safety, and facile manufacturing, 7,8 as they offer advanced therapeutic efficacy with doses of siRNA that are substantially lower and/or delivered less frequently than otherwise. 9 Synthetic cationic polymers which have been used in gene transfection include poly(ethylenimine) (PEI), 10 poly(amidoamine) (PAMAM) dendrimer, 11 poly(histidine), 12 poly(L-lysine), 12,13 and chitosan.…”

Section: Introductionmentioning

confidence: 99%

Synthesis, self-assembly and stimuli responsive properties of cholesterol conjugated polymers

et al. 2012

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Reversible addition-fragmentation chain transfer (RAFT) polymerization was used to generate welldefined pH-responsive biofunctional polymers as potential 'smart' gene delivery systems. A series of five poly(dimethylamino ethyl methacrylate-co-cholesteryl methacrylate) P(DMAEMA-co-CMA) statistical copolymers, with similar molecular weights and varying cholesterol content, were prepared. The syntheses, compositions and molecular weight distributions for P(DMAEMA-co-CMA) were monitored by nuclear magnetic resonance (NMR), solid-state NMR and gel permeation chromatography (GPC) evidencing well-defined polymeric structures with narrow polydispersities. Aqueous solution properties of the copolymers were investigated using turbidimetry and light scattering to determine hydrodynamic diameters and zeta potentials associated with the phase transition behaviour of P(DMAEMA-co-CMA) copolymers. UV-Visible spectroscopy was used to investigate the pH-responsive behaviour of copolymers. Hydrodynamic radii were measured in the range 10-30 nm (pH, temperature dependent) by dynamic light scattering (DLS). Charge studies indicated that P(DMAEMA-co-CMA) polymers have an overall cationic charge, mediated by pH. Potentiometric studies revealed that the buffering capacity and pK a values of polymers were dependent on cholesterol content as well as on cationic charge. The buffering capacity increased with increasing charge ratio, overall demonstrating transitions in the pH endosomal region for all five copolymeric structures. Cell viability assay showed that the copolymers displayed increasing cytotoxicity with decreasing number of cholesterol moieties. These preliminary results show the potential of these welldefined P(DMAEMA-co-CMA) polymers as in vitro siRNA delivery agents.

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“…We found that the silencing caused by the released material was nearly 50% of that produced by siRNA dendriplexes in buffer. In view of the absence of cell internalization and therefore lack of silencing activity of naked siRNA, 28 these results suggested that at least part of the released material was dendriplexes.…”

Section: In Situ-forming Gelsmentioning

confidence: 97%

“…27 We have previously shown that degradation of naked siRNA by RNAses can be reduced and its endocytic uptake increased after electrostatic complexation with cationic dendrimers. 28 We showed that electrostatic complexes between siRNA/poly(amidoamine) generation 7 dendrimers (G7) (siRNA dendriplexes) prepared in low ionic strength medium inhibited the expression of enhanced green fluorescent protein both in human glioblastoma cells and in macrophages in vitro.…”

Section: Introductionmentioning

confidence: 99%

Increased brain radioactivity by intranasal 32P-labeled siRNA dendriplexes within in situ-forming mucoadhesive gels

Perez¹,

Mundiña‐Weilenmann²,

Romero³

et al. 2012

IJN

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Background: Molecules taken up by olfactory and trigeminal nerve neurons directly access the brain by the nose-to-brain pathway. In situ-forming mucoadhesive gels would increase the residence time of intranasal material, favoring the nose-to-brain delivery. In this first approach, brain radioactivity after intranasal administration of 32 P-small interference RNA (siRNA) complexed with poly(amidoamine) G7 dendrimers (siRNA dendriplexes) within in situ-forming mucoadhesive gels, was determined. Materials:32 P-siRNA dendriplexes were incorporated into in situ-forming mucoadhesive gels prepared by blending thermosensitive poloxamer (23% w/w) with mucoadhesive chitosan (1% w/w, PxChi) or carbopol (0.25% w/w, PxBCP). Rheological properties, radiolabel release profile, and local toxicity in rat nasal mucosa were determined. The best-suited formulation was intranasally administered to rats, and blood absorption and brain distribution of radioactivity were measured. Results:The gelation temperature of both formulations was 23°C. The PxChi liquid showed non-Newtonian pseudoplastic behavior of high consistency and difficult manipulation, and the gel retained 100% of radiolabel after 150 minutes. The PxCBP liquid showed a Newtonian behavior of low viscosity and easy manipulation, while in the gel phase showed apparent viscosity similar to that of the mucus but higher than that of aqueous solution. The gel released 35% of radiolabel and the released material showed silencing activity in vitro. Three intranasal doses of dendriplexes in PxCBP gel did not damage the rat nasal mucosa. A combination of 32 P-siRNA complexation with dendrimers, incorporation of the dendriplexes into PxCBP gel, and administration of two intranasal doses was necessary to achieve higher brain radioactivity than that achieved by intravenous dendriplexes or intranasal naked siRNA. Conclusion:The increased radioactivity within the olfactory bulb suggested that the combination above mentioned favored the mediation of a direct brain delivery.

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Ethylendiamine core PAMAM dendrimers/siRNA complexes as in vitro silencing agents

Cited by 59 publications

References 36 publications

Uptake and intracellular traffic of siRNA dendriplexes in glioblastoma cells and macrophages

Uptake and intracellular traffic of siRNA dendriplexes in glioblastoma cells and macrophages

Synthesis, self-assembly and stimuli responsive properties of cholesterol conjugated polymers

Increased brain radioactivity by intranasal 32P-labeled siRNA dendriplexes within in situ-forming mucoadhesive gels

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