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In this study, we characterized the conventional physicochemical properties of the complexes formed by plasmid DNA (pDNA) and cationic liposomes (CL) composed of egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (50/25/25% molar ratio). We found that these properties are nearly unaffected at the studied ranges when the molar charge ratio (R(±)) between the positive charge from the CL and negative charge from pDNA is not close to the isoneutrality region (R(±) = 1). However, the results from in vitro transfection of HeLa cells showed important differences when R(±) is varied, indicating that the relationships between the physicochemical and biological characteristics were not completely elucidated. To obtain information regarding possible liposome structural modifications, small-angle X-ray scattering (SAXS) experiments were performed as a function of R(±) to obtain correlations between structural, physicochemical, and transfection properties. The SAXS results revealed that pDNA/CL complexes can be described as being composed of single bilayers, double bilayers, and multiple bilayers, depending on the R(±) value. Interestingly, for R(±) = 9, 6, and 3, the system is composed of single and double bilayers, and the fraction of the latter increases with the amount of DNA (or a decreasing R(±)) in the system. This information is used to explain the transfection differences observed at an R(±) = 9 as compared to R(±) = 3 and 6. Close to the isoneutrality region (R(±) = 1.8), there was an excess of pDNA, which induced the formation of a fraction of aggregates with multiple bilayers. These aggregates likely provide additional resistance against the release of pDNA during the transfection phenomenon, reflected as a decrease in the transfection level. The obtained results permitted proper correlation of the physicochemical and structural properties of pDNA/CL complexes with the in vitro transfection of HeLa cells by these complexes, contributing to a better understanding of the gene delivery process.

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This work presents a study of the association between low molecular weight hyaluronic acid (16 kDa HA) and cationic liposomes composed of egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). The cationic liposome/HA complexes were evaluated to determine their mesoscopic structure, average size, zeta potential, and morphology as a function of the amount of HA in the system. Small angle X-ray scattering results revealed that neighboring cationic liposomes either stick together after a partial coating of low concentration HA or disperse completely in excess of HA, but they never assemble as multilamellar vesicles. Cryo-transmission electron microscopy images confirm the existence of unilamellar vesicles and large aggregates of unilamellar vesicles for HA fractions up to 80% (w/w). High concentrations of HA (> 20% w/w) proved to be efficient for coating extruded liposomes, leading to particle complexes with sizes in the nanoscale range and a negative zeta potential.

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.