Integrated microfluidic devices for the synthesis of nanoscale liposomes and lipoplexes.

In this work, pDNA/cationic liposome (CL) lipoplexes for gene delivery were prepared in one-step using multiple hydrodynamic flow-focusing regions. The microfluidic platform was designed with two distinct regions for the synthesis of liposomes and the subsequent assembly with pDNA, forming lipoplexes. The obtained lipoplexes exhibited appropriate physicochemical characteristics for gene therapy applications under varying conditions of flow rate-ratio (FRR), total volumetric flow rate (QT) and pDNA content (molar charge ratio, R±). The CLs were able to condense and retain the pDNA in the vesicular structures with sizes ranging from 140nm to 250nm. In vitro transfection assays showed that the lipoplexes prepared in one step by the two-stage configuration achieved similar efficiencies as lipoplexes prepared by conventional bulk processes, in which each step comprises a series of manual operations. The integrated microfluidic platform generates lipoplexes with liposome formation combined in-line with lipoplex assembly, significantly reducing the number of steps usually required to form gene carrier systems.

Microfluidic Assembly of pDNA/Cationic Liposome Lipoplexes with High pDNA Loading for Gene Delivery.

Microfluidics offers unique characteristics to control the mixing of liquids under laminar flow. Its use for the assembly of lipoplexes represents an attractive alternative for the translation of gene delivery studies into clinical trials on a sufficient throughput scale. Here, it was shown that the microfluidic assembly of pDNA/cationic liposome (CL) lipoplexes allows the formation of nanocarriers with enhanced transfection efficiencies compared with the conventional bulk-mixing (BM) process under high pDNA loading conditions. Lipoplexes generated by microfluidic devices exhibit smaller and more homogeneous structures at a molar charge ratio (R±) of 1.5, representing the ratio of lipid to pDNA content. Using an optimized model to fit small-angle X-ray scattering (SAXS) curves, it was observed that large amounts of pDNA induces the formation of aggregates with a higher number of stacked bilayers (N ∼ 5) when the BM process was used, whereas microfluidic lipoplexes presented smaller structures with a lower number of stacked bilayers (N ∼ 2.5). In vitro studies further confirmed that microfluidic lipoplexes achieved higher in vitro transfection efficiencies in prostate cancer cells at R ± 1.5, employing a reduced amount of cationic lipid. The correlation of mesoscopic characteristics with in vitro performance provides insights for the elucidation of the colloidal arrangement and biological behavior of pDNA/CL lipoplexes obtained by different processes, highlighting the feasibility of applying microfluidics to gene delivery.