Microfluidic manufacturing of surface-functionalized graphene oxide nanoflakes for gene delivery


Authors: R Di Santo, L Digiacomo, S Palchetti, V Palmieri, G Perini, D Pozzi, M Papi, G Caracciolo

Journal: Nanoscale

DOI: 10.1039/C8NR09245A

Publication - Summary

January 06, 2019

Summary

Scientists at Sapienza University in Rome report using the NanoAssemblr Benchtop to make Graphene oxide/Cationic lipid (GOCL) hybrid particles for gene delivery. They compared plasmid delivery to standard DOTAP lipoplexes in HEK and HeLa cells and found that transfection efficiency was similar by the hybrid graphene oxide particles had much higher cell viability. They attribute this to the physical properties of the GOCL particles, noting that the GOCL particles were “much more numerous, regular in size, and homogeneously distributed  than DOTAP/DNA ones thus splitting their gene payload over the entire cell population”. 

The group made their GOCL particles by suspending nanosized graphene oxide flakes in an aqueous buffer and mixed that 1:1 with ethanol containing the cationic lipid DOTAP using the NanoAssemblr Benchtop. This is in contrast to how DOTAP/DNA lipoplexes are made: by bulk-mixing preformed vesicles with DNA, which generally results in a heterogeneous population of multilamellar lipoplexes. This is an example of how NanoAssemblr technology is being used to form high quality, homogeneous formulations that including innovative new materials to push boundaries in gene delivery formulations.

Abstract

Graphene oxide (GO) is a single-atomic-layered material made of a sheet of oxidized carbon atoms arranged in a honeycomb structure. Thanks to the notable physical and chemical properties of GO, GO-based nanomaterials have applications in many fields of research, including gene delivery. It has been reported that pristine GO can absorb single-stranded DNA and RNA through π–π stacking, which cannot be used as a gene carrier because it is hard to load double-stranded DNA (dsDNA). To tackle this issue, this work was aimed at developing a hybrid nanoparticle (NP) system made of GO coated with cationic lipids (hereafter referred to as GOCL) with suitable physical–chemical properties for gene delivery applications. To this end, nanosized GO flakes (nGO) were coated with the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) by microfluidic mixing. Comprehensive characterization of GOCL NPs was performed by a combination of dynamic light scattering (DLS), micro-electrophoresis and atom force microscopy (AFM). Our results show that GOCL NPs exhibit adequate size (<150 nm) and surface charge (ξ = +15 mV) for gene delivery purposes. Complexes made of GOCL NPs and plasmid DNA (pDNA) were used to transfect human cervical cancer cells (HeLa) and human embryonic kidney (HEK-293) cells. Pristine nGO and DOTAP cationic liposomes were used as a reference. GOCL NPs exhibited a similar TE but a much higher cell viability compared with DOTAP cationic liposomes. Confocal fluorescence microscopy provided a reasonable explanation for the superior performance of GOCL/DNA complexes showing that they are much more numerous, regular in size and homogeneously distributed than DOTAP/DNA complexes, thus splitting their gene payload over the entire cell population. Because of the imperative demand for efficient and safe nanocarriers, this study will contribute to the development of novel surface-functionalized GO-based hybrid gene vectors.

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