Inhibition of c-Rel expression in myeloid and lymphoid cells with distearoyl -phosphatidylserine (DSPS) liposomal nanoparticles encapsulating therapeutic siRNA

Abstract

c-Rel, a member of the nuclear factor kappa B (NF-κB) family, is preferentially expressed by immune cells and is known to regulate inflammation, autoimmune diseases and cancer. However, there is a lack of therapeutic intervention to specifically inhibit c-Rel in immune cells. Recent success with Pfizer and Moderna mRNA lipid-encapsulated vaccines as well as FDA approved medicines based on siRNA prompted us to test a lipid nanoparticle-based strategy to silence c-Rel in immune cells. Specifically, we encapsulated c-Rel-targeting siRNA into distearoyl-phosphatidylserine (DSPS)-containing nanoparticles. DSPS is a saturated phospholipid that serves as the "eat-me" signal for professional phagocytes such as macrophages and neutrophils of the immune system. We demonstrated here that incorporation of DSPS in liposome nanoparticles (LNP) improved their uptake by immune cells. LNP containing high concentrations of DSPS were highly effective to transfect not only macrophages and neutrophils, but also lymphocytes, with limited toxicity to cells. However, LNP containing low concentrations of DSPS were more effective to transfect myeloid cells than lymphoid cells. Importantly, DSPS-LNP loaded with a c-Rel siRNA were highly effective to inhibit c-Rel expression in several professional phagocytes tested, which lasted for several days. Taken together, our results suggest that DSPS-LNP armed with c-Rel siRNA could be exploited to target immune cells to limit the development of inflammatory diseases or cancer caused by c-Rel upregulation. In addition, this newly developed DSPS-LNP system may be further tested to encapsulate and deliver other small molecule drugs to immune cells, especially macrophages, neutrophils, and lymphocytes for the treatment of diseases.

Fig 1. Representation of liposomal nanoparticles (LNP) manufactured.

Schematic diagram for the synthesis of DSPS-LNP containing Bodipy-FL with or without cholesterol.

Fig 2. Influence of DSPS on the pickup rate of LNP in macrophage cell line RAW264.7.

(A-C) RAW264.7 cells (2 × 10⁶) were treated/nontreated with LNP containing 0% to 95% of DSPS at (A) 10, (B) 25, (C) 50 μM, for 24h. The percentage of positive cells (positive for fluorescent probe bodipy-FL) as well as the mean fluorescence intensity were measured by flow cytometry. (D) Images of RAW264.7 cells obtained by epifluorescence microscopy at 24h of treatment with LNP at 10μM. The results are representative of two experiments. Data were represented as mean ± standard deviation (SD). Statistical analyses were performed by a One-way ANOVA (with multiple comparisons) complemented by Tukey’s honest significance difference test, and annotated as ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig 3. DSPS-LNP can reach the cytoplasm of macrophages without detectable toxicity.

(A) RAW264.7 cells (2 × 10⁶) were treated for 24 h with LNP-S60 at 50 μM, which contain fluorescent probe Bodipy-FL (green). Cells were then fixed and stained with a fluorescent-labeled WGA coupled with Biotium’s CF®640R (magenta) and DAPI (blue). Cells were imaged by confocal microscopy. (B-C) RAW264.7 cells (2 × 10⁶) were treated with different concentrations of LNP-S60 (0 μM, 0.1 μM, 1 μM, 10 μM, 50 μM) or 0.1%Triton X-100 for 72 h, and cell viability was measured by MTT assay (B) or LDH assay (C). mean ± SD were expressed relative to none-treated group (B) or were expressed relative to the total LDH level obtained from cells treated with 0.1% triton X-100 (C). The results are representative of two experiments.

Fig 4. Ability of DSPS-LNP to transfect and deliver fluorescent siRNA into immune cells.

2 × 10⁶ of (A) RAW264.7, HL-60 (B), and EL4 cells (C), or primary macrophages (D) or splenocytes (E), were seeded in 6-well plates in DMEM containing 10% FBS. On the next day, cells were treated/nontreated with 1 ml LNP-S60 (green fluorescent probe Bodipy-FL) at different concentrations containing fluorescent siRNA (red fluorescent probe cyanine-3) at 37°C in DMEM medium with 10% FBS. The percentage of Bodipy-FL and cyanin-3 positive cells were analyzed by flow cytometry. (F) LNP uptake (using 1μM of LNP) was measured in RAW264.7, HL-60, EL4 cells as well as in primary macrophages and splenocytes and compared. Percentage of RAW264.7, HL-60, EL4 cells (G) as well as primary macrophages and splenocytes (H) containing siRNA was also measured. Results are representative of three independent experiments. mean ± SD were represented. *P <0.05; Two-tailed Mann-Whitney test was used for statistical analysis.

Fig 5. Gene silencing of c-Rel in macrophages with DSPS-LNP encapsulating c-Rel siRNA.

(A) 2 × 10⁶ RAW264.7 cells were treated/nontreated with LNP-S60 at 50 μM, which contained murine c-Rel siRNA at 200 nM. 2, 4 or 6 days post treatment, the c-Rel messenger RNA expression levels were determined by qRT-PCR. (B) The expression of murine c-Rel protein in RAW264.7 cells was measured by western blot 2 days after treatment with LNP (at 0 to 120 μM) encapsulated with murine c-Rel siRNA (0 to 1200 nM). (C and D) 2 × 10⁶ HL-60 cells were treated/nontreated with LNP-S60 containing human c-Rel siRNA for 2 days. The human c-Rel messenger RNA (C) and human c-Rel protein expression levels (D) were determined by qRT-PCR and Western blot, respectively. The level of gene silencing was shown (relative to non-treated group) and results shown are representative of two independent experiments. mean ± SD were represented. *P <0.05; **, P <0.01; ns, nonsignificant. two-tailed unpaired t test was used for statistical analysis. 2 × 10⁶ primary macrophages from bone marrow were treated/nontreated with LNP-S60 containing murine c-Rel siRNA (E and F) or scramble siRNA (G), and the expression of c-Rel mRNA (E) as well as the protein (F and G) was determined 48 h post transfection. Percentage of gene silencing levels was indicated. Results shown are representative from at least two independent experiments. Mean ± SD were represented. *P <0.05; **, P <0.01; ns, nonsignificant. Two-tailed Mann-Whitney test was used for statistical analysis of the qRT-PCR data. For the Western blot data, a semi quantitative analysis represented by histogram was performed by densitometry. Histogram data was based on the c-Rel/beta actin ratio, indicating the level of c-Rel expression relative to untreated cells.