Transferrin-mediated PEGylated nanoparticles for delivery of DNA PLL

摘要

The purpose of this work was to determine the stability of pDNA poly(L-lysine) complex (DNA PLL) during microencapsulation, prepare transferrin (TF) conjugated PEGylated nanoparticles (TF-PEG-NP) loading DNA PLL, and assess its physicochemical characteristics and in vitro transfection efficiency. The DNA PLL was prepared by mixing plasmid DNA (pDNA) in deionized water with various amounts of PLL. PEGylated nanoparticles (PEG-NP) loading DNA PLL were prepared by a water-oil-water double emulsion solvent evaporation technique. TF-PEG-NP was prepared by coupling TF with PEG-NP. The physicochemical characteristics of TF-PEG-NP and in vitro transfection efficiency on K562 cells were measured. The results showed that free pDNA reserved its double supercoiled form (dsDNA) for only on average 25.7 percent after sonification, bu over 70 percent of dsDNA was reserved after pDNA was contracted with PLL. The particle size range of TF-PEG-NP loading DNA/PLL was 150-450 nm with entrapment efficiency over 70 percent. TF-PEG-NP exhibited the low burst effect (<10 percent) within 1 day. After the first phase, DNA PLL displayed a sustained release. The amount of cumulated DNA PLL release from TF-PEG-NP with 2 percent polymer over 7 days was 85.4 percent for DNA PLL (1:0.3 mass ratio), 59.8 percent and 43.1 percent for DNA PLL (1:0.6) and DNA PLL (1:1.0), respectively. To TF-PEG-NP loading DNA PLL without chloroquine, the percentage of EGFP expressing cells was 28.9 percent for complexes consisting of DNA PLL (1:0.3), 38.5 percent and 39.7 percent for DNA PLL (1:0.6) and DNA PLL (1:1.0), respectively. In TF-PEG-NP loading DNA PLL with chloroquine, more cells were transfected, the percentage of positive cells was 37.6 percent (DNA PLL, 1:0.3), 47.1 percent (DNA PLL, 1:0.6) and 45.8 percent (DNA PLL, 1:1.0), which meant that the transfection efficiency of pDNA was increased by over 50 times when PLL and TF-PEG-NP were jointly used as a plasmid DNA carrier, in particular, the maximal percentage of positive cells (47.1 percent) from TF-PEG-NP loading DNA PLL (1:0.6) was about 70 times the transfection efficiency of free plasmid DNA. The average cell viability of TF-PEG-NP loading DNA PLL was about 90 percent, which meant that TF-PEG-NP appeared to be safer than PLL alone. As a result, TF-PEG-NP loading DNA PLL could be a more effective non-viral vector for the delivery of pDNA.