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Microfluidic assembly of pDNA/cationic liposome lipoplexes with high pDNA loading for gene delivery Tiago Albertini Balbino, Juliana Matos Serafin, Antonio Augusto Malfatti Gasperini, Cristiano Luis Pinto de Oliveira, Leide P. Cavalcanti, Marcelo Bispo de Jesus, and Lucimara Gaziola de La Torre Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b04177 • Publication Date (Web): 27 Jan 2016 Downloaded from http://pubs.acs.org on February 3, 2016
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Langmuir is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Figure 1. Schematic of the microfluidic device for continuous lipoplex assembly. The hydrodynamic flowfocusing device consisted of three inlets and one outlet. Two programmable pumps controlled the fluid flow rates of the syringes. Cationic liposomes (CLs) were introduced via side streams that hydrodynamically focused the central stream containing the plasmid DNA (pDNA). The microfluidic-assisted assembly of pDNA/CL lipoplexes gradually occurred with the diffusion between stream flows along the main channel. The inset shows a fluorescence micrograph of the flow pattern at the cross-junction, obtained using rhodamine in the central stream. The microchannels were 140-µm wide and 100-µm deep. The white dotted lines indicate the borders of the microchannels. The volumetric flow rates used for the side and central streams were 160 µL/min and 88 µL/min, respectively. The scale bar denotes 200 µm. 262x203mm (96 x 96 DPI)
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Figure 2. Physicochemical properties of plasmid DNA (pDNA)/cationic liposome (CL) lipoplexes prepared by bulk-mixing (BM) and microfluidic processes. (a) Number-weighted hydrodynamic diameter, (b) polydispersity index (PdI), and (c) zeta potential (ζ) of pDNA/CL lipoplexes assembled via bulk-mixing and microfluidic processes as a function of the molar charge ratio (R±). The flow rate of the liposomal streams was maintained at 160 µL/min, and the flow rate of the pDNA stream varied from 22 µL/min to 89 µL/min. The PdI above 0.8 was considered as an inappropriate measurement and is separated from the other values by a horizontal dotted line. The error bars correspond to the standard deviation of three independent experiments. 124x201mm (96 x 96 DPI)
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Figure 3. Plasmid DNA (pDNA) incorporation into liposomal structure. (a) Gel retardation assay, (b) resistance to DNase I at R± 1.5, and (c) accessibility to the fluorescence probe for pDNA/cationic liposome lipoplexes assembled by bulk-mixing and microfluidic methods. For the DNase I assay, lipoplexes were prepared at R± 1.5, and free pDNA was used as positive and negative controls via incubation with or without DNase I, respectively. * p
