Expanding Structural Space for Immunomodulatory Nucleic Acid Nanoparticles (Nanps) via Spatial Arrangement of Their Therapeutic Moieties

Abstract

Different therapeutic nucleic acids (TNAs) can be unified in a single structure by their elongation with short oligonucleotides designed to self-assemble into nucleic acid nanoparticles (NANPs). With this approach, therapeutic cocktails with precisely controlled composition and stoichiometry of active ingredients can be delivered to the same diseased cells for enhancing pharmaceutical action. In this work, an additional nanotechnology-based therapeutic option that enlists a biocompatible NANP-encoded platform for their controlled patient-specific immunorecognition is explored. For this, a set of representative functional NANPs is extensively characterized in vitro, ex vivo, and in vivo and then further analyzed for immunostimulation of human peripheral blood mononuclear cells freshly collected from healthy donor volunteers. The results of the study present the advancement of the current TNA approach toward personalized medicine and offer a new strategy to potentially address top public health challenges related to drug overdose and safety through the biodegradable nature of the functional platform with immunostimulatory regulation.

Keywords: PBMCs; SAXS; immunotherapy; nucleic acid nanoparticles; therapeutic nucleic acids.

Figure 1.

Schematic representation of experimental flow reported in this work. The library of functional NANPs with different numbers and orientations of Dicer Substrate (DS) RNAs was engineered and extensively characterized and tested in vitro, in vivo, and in human peripheral blood mononuclear cells (PBMCs) to determine NANPs immunostimulatory properties.

Figure 2.

Functionalized NANPs characterized by SAXS, AFM, and native polyacrylamide gel electrophoresis (PAGE). A) Plots of SAXS profiles of NANPs over the q range 0.005–0.5 Å⁻¹ with error shown as vertical bars of the same color. B) The Guinier plots of each structure with the fitted regions represented as solid circles with the line of best fit going through them in the same color. Data which are not included in the Guinier fit are shown as unfilled circles. The residual plots for all four Guinier fits are shown below. C) The pair-wise distance distribution for each structure with the same color scheme. D,E) The Chimera-generated density maps of SAXS-based models (with scale bars), where the transparent gray surface is superimposed with the idealized atomic model shown as a ribbon and stick model. F) Seven representative NANPs visualized by AFM and G) assemblies of all tested NANPs confirmed by native-PAGE stained with EtBr.

Figure 3.

Functional and immunostimulatory properties of NANPs. A) Fluorescent microscopy images of a human breast cancer cell line expressing GFP (MDA-MB-231/GFP) 72 h after transfection with functionalized rings (10 × 10⁻⁹ m, schematically shown in left panel). The same population of cells was imaged for GFP and brightfield after transfection with each ring shown. Scale bar = 50 μm. B) Normalized fold induction over the baseline 24 h after the transfection of rings into immune reporter cell lines. All values are normalized to the cells-only control. The positive control (PC) for each cell line is shown as the uncolored bar. 1X assembly buffer (AB), L2K, and DS RNA (10 × 10⁻⁹ m) are additional controls. Each bar represents the mean of N = 5 biological repeats and error bars denote mean ± SEM. The letters “a”, “b”, and “c” indicate statistically significant differences compared to ring 0, corresponding PC, and ring 2C, respectively (one-way ANOVA with Fisher’s LSD post hoc test, p < 0.05).

Figure 4.

In vivo characterization of ring orientations. A) Schematic of in vivo studies. NANPs complexed with the carrier, PgP were delivered to CD1 male and female via retro-orbital injection. B) IVIS images of mice were acquired 4 h following NANP delivery. C) IVIS images of organs were acquired 24 h post-delivery of NANPs. D) Radiant efficiency of organs. The letters “a” and “b” indicate statistically significant differences compared to the PBS control and to ring 6, respectively (two-way ANOVA with Tukey’s post hoc test, p < 0.05). E) Quantitative PCR analysis of immune mediator production in organs. The letters “a”, “b”, “c”, and “d” indicate statistically significant differences compared to the PgP control and to rings 1, 3C, and 6, respectively (two-sample t-test, p < 0.05). All data in (D,E) is expressed as the mean (N = 6 for rings 1, 3C, and 6; N = 2 for PBS; N = 3 for PgP) ± SEM and detailed statistical analysis (two-sample t-test, p < 0.05).

Figure 5.

Multiplex ELISA analysis of serum samples 24 h following NANP delivery. Horizontal line in each treatment group shows the mean value of individual animal responses; error bars show the SEM for the treatment group. Each dot represents a response from an individual animal. Student’s t-test with one-tailed distribution and two-sample unequal variance [CI 90% and CI95%] was used to compare treatment to control. Physiologically significant changes (i.e., twofold or more different from the baseline) were also monitored.

Figure 6.

Results from a multiplex assay for type I and type III IFNs (IFN-α, -β, -ω, and -λ) 20 h after the transfection of PBMCs with rings using L2K. All rings were tested at the final concentration of 10 × 10⁻⁹ m. Each square on the heatmap represents the mean response of three independent samples (N = 3). Negative control (NC) is untreated cells, positive control is ODN2216, and vehicle control is L2K only. Changes in cytokine levels that were two-fold or more different from the baseline were considered physiologically significant; the comparison of treatment groups to the baseline was done for each donor separately due to the known inter-individual variability in the magnitude of the immune response and small cohort size (N = 6 donors).