Lipoplex Nanoparticle Encapsulation Protocol

The pGluc vector (5764 bp) or pBMP2 vector (3486 bp) (100 μg) was complexed with 500 μg of Poly L-Lysine hydrobromide (PLL) (1000–5000 Da, Sigma, United Kingdom) by sequentially adding 10 μg of pDNA dropwise to 50 μg of PLL using a P200 pipette tip under vortexing. The working concentration was 40 and 200 μg/ml in nuclease-free water for pDNA and PLL, respectively. The mixture of pDNA-PLL NPs in a volume of 5 ml was then concentrated to 250 μl using Amicon^® Ultra Centrifugal Filter units. This volume of the mixture of pDNA-PLL NPs was encapsulated in the PLGA NPs by double emulsion as below. The hydrodynamic size and surface charge of pDNA-PLL NPs were assessed by Malvern Zetasizer Nano ZS. NP morphology was determined by Transmission Electron Microscopy (FEI Tecnai BioTwin-12 TEM) as previously (Osman et al., 2018 (link)).