(572e) Protein Nanoparticles for Effective Gene Delivery

Introduction: The largest hurdle to clinical application of gene therapies is the lack of a safe and efficient delivery system. To address this, we used electrohydrodynamic (EHD) jetting to develop a highly efficient, less cytotoxic plasmid DNA (pDNA) delivery system using protein nanoparticles (pNPs). pNPs are highly modular allowing numerous targeting ligands and various sized pDNA to be added without compromising ease of production. This system is viable in various cell lines and formulations, representing a promising platform for delivering various pDNA for applications in basic science and medicine.

Materials and Methods: Synthesis and Characterization: pNPs were fabricated using EHD (Figure 1A) then imaged using SEM and TEM (Figure 1B). The required weight ratio (N/P) between the PEI and pDNA was optimized and used in all pNP formulations (Figure 1C). Hydrodynamic diameter (D) and surface charge (Zeta Potential, ZP) of the pNPs were evaluated using a Zetasizer (Figure 1D, 1E). In Vitro Testing: Transfection was done by suspending pNPs in cell media for the duration of each timepoint. Cytotoxicity was measured using CellTiter-Glo (Figure 2A). pNP uptake was confirmed and quantified using confocal images and flow cytometry, respectively. The transfection efficiency (TE) of the system was measured with flow cytometry. TE measurements were taken at various loadings (weight percent of pDNA in the pNP) (Figure 2B, 2C). TE was measured for all GFP-positive pNP formulations with NLS addition, and the difference between corresponding pNPs was quantified. Timepoints were 24, 36, and 48 hrs. post transfection.

Results and Discussion: To generate modular pNPs, we utilized EHD jetting (Figure 1A) of human serum albumin (HSA) and transferrin, with polyethylenimine (PEI) 25kDa to electrostatically complex the pDNA and enable loading to protect against degradation. The versatility of the EHD jetting process lends itself to changing the base material and easily doping in peptides to serve as targeting ligands or NLS. Size, shape, and charge are critical to successful uptake and reducing pNP toxicity to the cells, which affects both transfection and viability of cells. The successful synthesis of spherical particles was confirmed using SEM (Figure 1B), and DLS and ZP were also quantified. Only pNP formulations that fit the criteria (D < 200nm, ZP > -10mV) were used going forward (Figure 1D, 1E).

After establishing a pNP delivery system that met the design criteria, we explored the effect of pDNA loading percentages and NLS peptide addition on system efficacy. The pNP delivery system exhibited both higher cell uptake and higher cell viability than PEI25k at comparable N/P ratios (Figure 2A). A potential reason for the increase in the observed cytotoxicity is the increase in total pNP delivered, resulting in an increase in PEI. This can be tested by delivering various concentrations of pNP at each loading weight percent. pNPs also showed higher or similar TEs at every tested formulations and N/P ratios while using empty pNPs, PBS as negative controls and PEI25k, lipofectamine as positive controls. The TE was further enhanced by addition of the NLS peptide, which was able to enhance TE in non-dividing cells, proving that the NLS facilitates active nuclear import of the pDNA, a large hurdle to the clinical application of non-viral systems. The higher relative cell viability combined with the ability to induce GFP expression shows a system which is more effective and less cytotoxic in all cell lines tested, for a large size pDNA as tested across a wide parameter space.

Conclusions: This work demonstrates that a new platform for pDNA delivery has been developed, demonstrating low cytotoxicity compared to current systems with a high TE and the ability to induce genetic changes in target cells. Further studies will include a study on the path of pDNA within the cell and quantifying transfection efficiencies, and PCR analysis to see genetic changes that result from delivered DNA. Unforeseen issues or sub-optimal efficacy can readily be adjusted due to the highly modular nature of this system, which will be integral to its adaptation for specific disease states.

Acknowledgements: This study was supported by the NIH (F31 HL149249).