Click-Modified Cyclodextrins as Nonviral Vectors for Neuronal siRNA Delivery

Abstract

RNA interference (RNAi) holds great promise as a strategy to further our understanding of gene function in the central nervous system (CNS) and as a therapeutic approach for neurological and neurodegenerative diseases. However, the potential for its use is hampered by the lack of siRNA delivery vectors which are both safe and highly efficient. Cyclodextrins have been shown to be efficient and low toxicity gene delivery vectors in various cell types in vitro. However, to date, they have not been exploited for delivery of oligonucleotides to neurons. To this end, a modified β-cyclodextrin (CD) vector was synthesized, which complexed siRNA to form cationic nanoparticles of less than 200 nm in size. Furthermore, it conferred stability in serum to the siRNA cargo. The in vitro performance of the CD in both immortalized hypothalamic neurons and primary hippocampal neurons was evaluated. The CD facilitated high levels of intracellular delivery of labeled siRNA, while maintaining at least 80% cell viability. Significant gene knockdown was achieved, with a reduction in luciferase expression of up to 68% and a reduction in endogenous glyceraldehyde phosphate dehydrogenase (GAPDH) expression of up to 40%. To our knowledge, this is the first time that a modified CD has been used as a safe and efficacious vector for siRNA delivery into neuronal cells.

Early-Stage Development of Novel Cyclodextrin-siRNA Nanocomplexes Allows for Successful Postnebulization Transfection of Bronchial Epithelial Cells

“…Analysis of SC12CDClickpropylamine-siRNA nanocomplexes in this current study and previous studies has shown that they are capable of forming compact nanocomplexes between 125 and 200 nm in size and are highly cationic in nature using dH 2 O. (16,17,33,34) In addition, previous gel Table 3. SC12CDClickpropylamine-siRNA Nanocomplex Droplet Sizes Generated electrophoresis analysis by our group has also demonstrated that SC12CDClickpropylamine nanocomplexes efficiently encapsulate siRNA at MR > 10.…”

“…SC12CDClickpropylamine-siRNA Nanocomplex Droplet Sizes Generated electrophoresis analysis by our group has also demonstrated that SC12CDClickpropylamine nanocomplexes efficiently encapsulate siRNA at MR > 10. (17) In contrast, this study found that formulation of siRNA nanocomplexes in PBS resulted in marked increases in particle size. This phenomenon has been previously reported for siRNA nanoparticles and did not correlate to a loss in transfection efficiency.…”

“…This is in line with earlier toxicity studies of these nanocomplexes in Caco-2 colonic epithelial cells and N41 neuronal cell lines as well as primary rat embryonic neuronal cells. (16,17) It should be noted, however, that trends toward toxicity were noted as MR increased with decreases in cell population of up to 34% -12% and increases in cytochrome c, MMP, and PMP all evident as MR increased, with significant increases in PMP at MR = 100. Although it is expected for MMP to drop in apoptotic cells, it has previously been found that some epithelial cells will exhibit an increase in MMP prior to a loss of MMP if the dose is increased.…”

“…1), rendering it a potential siRNA delivery vector. Studies to date have found it to be effective at delivering siRNA to both gastrointestinal-epithelial and neuronal cells (16,17) ; however, its potential for pulmonary delivery of siRNA has not yet been assessed. To date, research into CD delivery to the lungs has focused primarily on enhancing solubility of preexisting small-molecule drugs, (18,19) mitigating their cytotoxic effects, (20) and improving the respirable fraction and absorption of aerosolized drugs.…”

The use of collagen-based scaffolds to simulate prostate cancer bone metastases with potential for evaluating delivery of nanoparticulate gene therapeutics

“…Lipofectamine 2000 ® has been widely investigated for siRNA delivery in PC3 cells [87e89]. Although the toxicity of this cyclodextrin delivery vector has been analysed and reported in other cell lines [35,40,41,85,86], the toxicity in PC3 cells has not been previously investigated and thus an MTT assay was conducted ( Fig. 7(a)).…”