(T0930-02-09) Polyplex Particle Size Alters Transgene Expression and Cell Viability in a Cell Type-Dependent Manner

Purpose: Gene therapy has demonstrated tremendous therapeutic potential for inherited diseases as well as highly prevalent diseases such as cancer, diabetes, or age-related macular degeneration. To mediate efficient gene delivery into target cells, numerous synthetic nanocarriers have been engineered. Cationic polymers have been extensively studied due to their low cost, availability, and their ability to readily condense bulky anionic payloads such as plasmids (pDNA) to form nanosized assemblies termed polyplexes. Polyplex size is a critical quality attribute that dictates cellular internalization, interactions with immune cells, and toxicity, but is relatively under-investigated compared to polymer composition and architecture.

Methods: We developed Derjaguin and Lev Landau, Evert Verwey, and Theodoor Overbeek (DLVO) models that informed the selection of pH and ionic strength to kinetically control polyplex formation pathways (Figure 1 A, B). We formulated the polyplexes using statistical polycation (DIP50H50) with pDNA in aqueous or phosphate buffers at low ionic strength (pH 5.8,10mM). An increase in ionic concentration (pH 7.4,100mM) triggered polyplex aggregation, followed by quenching in an acidic environment (low pH), resulting in stabilization of polyplex sizes (from nano to micron). Furthermore, we intend to better understand transgene expression and viability in relation to the varying polyplex size distribution across different cell lines [Epithelial, retinal and macrophages]. The schematic representation of methodology is shown in Figure 1C.

Results: Polyplexes formulated in aqueous milieu generated 40 nm (R_h) sized particles with spherical morphology. Slowly varying the aggregation-quenching time points from 0 to 60 min resulted in the formation of size-controlled polyplexes (Rh: 85 nm to 827 nm) comprising lipophilic polycations (DIP50H50) and pDNA. Using dynamic light scattering measurements, we tracked polyplex aggregation dynamics and measured the hydrodynamic radii of polyplexes at various intervals. The polyplex aggregation and stabilized polyplex morphology were monitored by TEM. The average molecular weight (Mw) and pDNA loading per polyplex were estimated using static light scattering measurements. Control polyplex (Rh: 40 nm) showed 17 pDNA copies per polyplex, meanwhile size-controlled polyplex (Rh: 85 nm) exhibited twofold higher the amount of pDNA copies (25). Higher pDNA loading (time point 0 to 60 min) in sodium phosphate buffer clearly advocates that solvent, pH, and ionic concentration play a significant role in pDNA complexation with lipophilic polycations. Among the different size polyplexes, the quenching time point of 10 min (Rh: 827 nm) showed the highest pDNA loading (766) per polyplex. Zimm plot analysis demonstrated that larger polyplexes with higher molar mass (high cationicity) encapsulated more pDNA copies. When comparing the viability and transgene expression outcomes of HEK 293T, ARPE19, and RAW 264.7, it is obvious that polyplex dimensions significantly impact transfection performance and viability. HEK 293T cells & ARPE-19 exhibited the highest transfection for the size-controlled polyplex size (85 to 349 nm). Pareto front (TxV) findings imply that human embryonic and retinal cells are more intrigued by smaller polyplex sizes, whereas macrophage cells prefer larger polyplexes. Size-dependent cellular internalization was observed in retinal and embryonic cells; however, macrophage internalization was size insensitive. Smaller polyplexes tend to be more hemolytic, whereas polyplexes with a size between 85 and 827 nm appeared less hemolytic and size insensitive.

Conclusion: In our study, we employed DLVO modeling to formulate colloidally stable polyplexes at different sizes. Through DLVO interaction energies, we monitored the aggregation of polyplexes by varying the sodium phosphate buffer conditions (pH 5.8 to 7.4) and ionic strength (10 mM to 100 mM). At specific time intervals, acidification of polyplexes using HCl controlled the aggregation. Polyplexes begin to re-protonate and encounter the potential barrier in acidic environments. As a result of this potential energy change and the total interaction energy shift from attractive to repulsive forces, the polyplex sizes were stabilized. We observed that polyplexes sized between 85 to 350 nm performed best in human embryonic kidney cells (HEK293T) and retinal ARPE-19 cells whereas transfection efficiency was less sensitive to polyplex size in RAW macrophages. However, careful polyplex size control was essential to minimize cytotoxicity (60 % viability) and hemolysis (8-10 %), with 85 & 136 nm polyplexes mediating the most-gentle transfection. By applying DLVO modeling to precisely optimize the parameters of the polyplex formulation, our work effectively deploys colloidal science principles to augment transgene expression in cell types recalcitrant to polymer-mediated pDNA delivery and to improve the hemocompatibility of polymeric vectors. In our future work, we anticipate combining DLVO approaches with confined jet mixing techniques to generate kinetically stable lipophilic polycation-pDNA/mRNA/RNP complexes targeting stem cells and other biologically relevant targets.

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Acknowledgements: National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health under award number R21 EB034464-01. National Science Foundation Graduate Research Fellowship Program, Grant No. 2137099. Colorado School of Mines’ Shared Instrumentation Facility (Electron Microscopy RRID: SCR022048).

Figure 1: DLVO modeling for different volume ratios (v/v) of 100 mM pH 7.4 buffer and 10 mM pH 5.8 buffer (A). We selected 15:85 v/v (orange line) based on modeling calculations, and electrical double layers establish energy barriers against aggregation; however, unquenched polyplexes lack (B). Schematic representation of polyplex formation and aggregation quenching resulted in the formation of size-controlled polyplexes (nano to micron), and we evaluated size-dependent transgene expression/viability across three different cell lines (C).