Nonviral gene vector formation in monodispersed picolitre incubator for consistent gene delivery

Abstract

A novel picolitre incubator based microfluidic system for consistent nonviral gene carrier formulation is presented. A cationic lipid-based carrier is the most attractive nonviral solution for delivering plasmid DNA, shRNA, or drugs for pharmaceutical research and RNAi applications. The size of the cationic lipid and DNA complex (CL-DNA), or the lipoplex, is one of the important variations for consistency of gene transfection. CL-DNA size, in turn, may be controlled by factors such as the cationic lipid and DNA mixing order, mixing rate, and mixture incubation time. The Picolitre Microfluidic Reactor and Incubator (PMRI) system described here is able to control these parameters in order to create homogeneous CL-DNA. Compared with conventional CL-DNA preparation techniques involving hand-shaking or vortexing, the PMRI system demonstrates a greater ability to constantly and uniformly mix cationic lipids and DNA simultaneously. After mixing in the picolitre droplet reactors, the cationic lipid and DNA is incubated within the picolitre incubator to form CL-DNA. The PMRI generates a narrower size distribution band, while also turning the sample loading, mixing and incubation steps into an integrated process enabling the consistent formation of CL-DNA. The coefficient of variation (CV) of transfection efficiency is 0.05 and 0.30 for PMRI-based and conventional methods, respectively. In addition, this paper demonstrates that the gene transfection efficiency of lipoplex created in the PMRI is more reproducible.

Microfluidic Preparation of Polymer-Nucleic Acid Nanocomplexes Improves Nonviral Gene Transfer

“…Top-down nanoimprinting systems produce nanoparticles with defined shape and size that have proven valuable in deconvoluting the mechanistic effects of such characteristics. However, the rapid reaction kinetics, aqueous conditions, and temperature sensitivity of polyplex assembly favor microfluidic approaches, which have included both monophasic laminar flow systems and emulsion-based designs131819202122. The former suffer from flocculation at high concentrations, while the latter have been used successfully in production of both lipoplexes and polyplexes.…”

Mikrotröpfchen in Mikrofluidiksystemen: eine Technik für Entdeckungen in der Chemie und Biologie

“…Bei der Bildung nichtviraler Vektoren in Mikrotröpf-chen lassen sich das Verhältnis und die Polydispersität der Vektoren besser steuern und eine besser reproduzierbare Transfektion erzielen. [153] Da sich nun einzelne Zellen in Tröpfchen elektroporieren, untersuchen und manipulieren lassen, wäre auch eine Mög-lichkeit wünschenswert, die zellhaltigen Tröpfchen über län-gere Zeiträume zu lagern. Das Einfrieren von Tröpfchen mit einzelnen Zellen könnte eine solche Möglichkeit bieten.…”

Sistemas Microfluídicos De Gotas Para Incorporação De Dna Em Lipossomas Catiônicos Para Aplicação Em Terapia Gênica

“…Os sistemas microfluídicos convencionais baseados em uma fase, contínuos e de fluxo laminar possuem baixo número de Reynolds, já os sistemas microfluídicos baseados em gotas são capazes de promover uma mistura rápida, particularmente quando há uma região de mistura em forma de serpentina, gerando lipoplexos de menor tamanho e polidispersidade (Hsieh et al, 2009). A transfecção de células a partir de lipoplexos é particularmente sensível ao tamanho dos lipoplexos (Harris e Giorgio, 2005), pois cada linhagem celular possui um tamanho de lipoplexo ótimo para a transfecção (Hsieh et al, 2009).…”

“…A transfecção de células a partir de lipoplexos é particularmente sensível ao tamanho dos lipoplexos (Harris e Giorgio, 2005), pois cada linhagem celular possui um tamanho de lipoplexo ótimo para a transfecção (Hsieh et al, 2009). Por exemplo, Hsieh et al (2009) compararam um sistema microfluídico de gotas para complexar DNA aos lipossomas catiônicos compostos por DOTMA (cloreto de N-[1,2,3-dioleiloxi)propil]-N,N,Ntrimetilamônio) e DOPE (1:1 m/m) com as técnicas convencionais de preparação de lipoplexos envolvendo agitação manual e por vórtex; e os resultados demonstraram que o sistema microfluídico possuía uma maior capacidade de complexar lipossomas catiônicos e DNA simultaneamente de forma constante e uniforme, gerando lipoplexos com propriedades físico-químicas mais reprodutíveis e com menor polidispersidade.…”