Multiscale Simulation as a Framework for the Enhanced Design of Nanodiamond-Polyethylenimine-based Gene Delivery

Abstract

Nanodiamonds (NDs) are emerging carbon platforms with promise as gene/drug delivery vectors for cancer therapy. Specifically, NDs functionalized with the polymer polyethylenimine (PEI) can transfect small interfering RNAs (siRNA) in vitro with high efficiency and low cytotoxicity. Here we present a modeling framework to accurately guide the design of ND-PEI gene platforms and elucidate binding mechanisms between ND, PEI, and siRNA. This is among the first ND simulations to comprehensively account for ND size, charge distribution, surface functionalization, and graphitization. The simulation results are compared with our experimental results both for PEI loading onto NDs and for siRNA (C-myc) loading onto ND-PEI for various mixing ratios. Remarkably, the model is able to predict loading trends and saturation limits for PEI and siRNA, while confirming the essential role of ND surface functionalization in mediating ND-PEI interactions. These results demonstrate that this robust framework can be a powerful tool in ND platform development, with the capacity to realistically treat other nanoparticle systems.

Fig. 1

The surface charge distribution of a DFTB optimized ND (C5795) structure having a diameter of 4.1 nm. The legend on the right breaks down the intervals of charge that each colored atom represents. (A) the bare optimized ND structure. (B) a close-up view of a graphitized (111) surface. (C) a close-up of the 2×1 surface reconstruction in (100) surface. (D) the complete surface charge (q) distribution. Parts (E) and (F) illustrate the regions containing only the most negatively and most positively charged atoms.

Fig. 2

The importance of the ionization of surface functional groups towards mediating PEI loading is demonstrated in parts A and B. A and B show the final structure of a non-functionalized ND and functionalized ND, respectively, with 24 PEIs surrounding the particle after 8 ns of interaction time. The non-functionalized ND is unable to bind with any PEI because the screening of surface electrostatics by the water solvent is too strong to overcome. On the other hand, the functionalized ND is able to load all 24 PEIs successfully. The binding of PEIs to a functionalized ND at two different mixing ratios is shown in parts C-F and the comparison between the loading results of experiments and simulations is shown in part G. Part C shows the initial structure of a functionalized ND with 15 PEIs surrounding the particle, corresponding to a mixing ratio of 1:25 (ND:PEI, by weight). Part D shows the final structure of part C after 8 ns of interaction time, with most PEI's binding to the ND surface. Similarly, parts E and F show the initial and final configuration of a half cut-away functionalized ND with 120 PEIs after 8 ns interaction, corresponding to 1:200 (ND:PEI, by weight) mixing ratio. Part F shows PEI filling in the vacant space found near the ND surface shown in part E. Part G compares loading trends between the experimental and simulation results as a function of mixing ratio. The simulations were able to predict a saturation in PEI loading at a 1:200 mixing ratio, demonstrated by the lack of increasing PEI loading above the 1:200 mixing ratio.

Fig. 3

The importance of PEI functionalization towards mediating siRNA loading is demonstrated in parts AD. Part A shows the initial structure of a ND (with surface functionalization, but no PEI) with 1 siRNA placed nearest to its [111] facet. Part B shows the final structure of part A after 8 ns of interaction time. This demonstrates that a ND is unable to bind with siRNA without the presence of PEI because the ND surface is not cationic enough to bind with the negatively charged phosphate backbone of the siRNA. Part C shows the initial structure of a ND-PEI (1 ND loaded with 57 PEIs, 1:200 mixing ratio) with 4 siRNA's surrounding the particle, corresponding to a mixing ratio of 1:0.10 (ND:siRNA, by weight). Part D shows the final structure of part C after 8 ns of interaction time, with all siRNA binding to the ND-PEI surface. Part E shows the similar percentage of loading between the experimental and simulation results as a function of mixing ratio. The percentage of siRNA loading decreases above the 1:0.10 ratio while the absolute siRNA loading still increases. The simulations were able to predict the dropoff in efficiency of siRNA loading above the 1:0.10 mixing ratio.

Scheme 1

The schematic above visualizes the synthesis of the ND-PEI-siRNA complex. The functionalization of the ND surface using the polymer, PEI, allows the conjugate to mediate strong interactions with siRNA strands. The loading of siRNA onto ND-PEI may be an effective method for gene delivery, which has many therapeutic applications including cancer treatment.