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Volume 3
Journal of Neurology and Clinical Neuroscience
Neurology 2019 | Neuropsychology 2019 | Drug Delivery Summit 2019
June 24-25, 2019
June 24-25, 2019 | Rome, Italy
Neurology and Healthcare
3
rd
WorldDrug Delivery and Formulations Summit
Clinical and Experimental Neuropsychology
4
th
International Conference on
International Conference on
&
Formulation of non-ionic surfactant vesicles (NISV) prepared by microfluidics for therapeutic delivery
of siRNA into cancer cells
Mohammad Ali Obeid, Alexander B Mullen
1
, Rothwelle J Tate
1
, Valerie A Ferro
1
Yarmouk University, Jordan
1
University of Strathclyde, UK
Introduction:
RNA interference involves the degradation of a target messenger RNA through the incorporation of short
interfering RNAs (siRNA) [1]. The application of siRNA-based therapeutics is limited by the development of an effective
delivery system.Anovel type of nanoparticles known as Non-Ionic Surfactant Vesicles (NISV) are commonly used for drug
delivery of various therapeutics, are relatively safe and non-expensive, have not been extensively studied for siRNAdelivery
[2]. Therefore, the aim of this study was to investigate the potential of NISV prepared by microfluidics for siRNAdelivery.
Methods:
NISV were prepared by microfluidic mixing which is a recently developed method used to prepare lipid-based
nanoparticles and results in the production of small vesicles with efficient encapsulation of a therapeutic agent. To prepare
NISV, specific volumes from each stock solution of the NISV components were mixed together to prepare the lipid phase.
The lipid phase was injected into the first inlet and the aqueous phase into the second inlet of the microfluidic micromixer,
with the mixing temperature set at 50°C. The Flow Rate Ratios (FRR) between the aqueous and organic phase was set
at 3:1 and the Total Flow Rates (TFR) of both phases was set at 12 ml/min. This allows for fast mixing between the two
phases at high flow rates and at a temperature above the phase transition of the lipids. Dispersions were then collected
from the outlet stream and immediately diluted in order to reduce the final ethanol content in the preparation to 6.25%
(v/v). Cytotoxicity evaluation of NISV were carried out on non-small lung cancer cells (A549) and mouse melanoma cells
(B16-F10-LUC). siRNA targeting Green Fluorescent Protein (GFP) in copGFP-A549 cells, or luciferase in B16-F10-LUC
cells were encapsulated in NISV. Inhibition of GFP expression by anti-GFP siRNA (siGFP) delivered using NISV was
evaluated by flow cytometry, polymerase chain reaction, and Western blotting. Nude BALB/c mice inoculated with B16-
F10-LUC cells that induce melanoma expressing luciferase was used to assess the NISV ability to deliver siRNA in vivo.
Results:
Cytotoxicity studies indicated that NISVwere not toxic at or below 40 µg/ml. NISV formulations had high siRNA
encapsulation efficiency. Fluorescent microscope and flow cytometry studies indicated high cellular uptake by the cells
compared to naked siRNA, whichwas not taken up by the cells. NISVwere able to deliver siGFPto the cells and significantly
suppress GFP expression. These results were confirmed by transfecting the luciferase producing B16-F10-LUC cells with
anti-luciferase siRNA (siLUC). Measuring the level of luciferase expression after siLUC transfections using a luciferase
protein assay system successfully demonstrated the suppression of luciferase expression. NISV were then used in in vivo
experiments using nude BALB/c mice. After intra-tumoural injection, siLUC was delivered to the cells and suppressed
luciferase expression at a significantly higher level than mice treated with naked siLUC. These in vivo results confirm
the ability of NISV to successfully deliver siRNA into the cytoplasm of the target cells and suppress the target protein.
Conclusion:
NISV have been demonstrated extensively and for the first time to have the potential to be used as a delivery
system for siRNA. These results have shown that NISV can be used to overcome the barriers, such as low stability and
poor cellular uptake, in siRNA-based therapeutics.
J Neurol Clin Neurosci, Volume 3