Complexation of Chol-DsiRNA in place of Chol-siRNA greatly increases the duration of mRNA suppression by polyplexes of PLL(30)-PEG(5K) in primary murine syngeneic breast tumors after i.v. administration

Abstract

RNA interference has tremendous potential for cancer therapy but is limited by the insufficient potency of RNAi molecules after i.v. administration. We previously found that complexation with PLL(30)-PEG(5K) greatly increases the potency of 3'-cholesterol-modified siRNA [Chol-siRNA] in primary murine syngeneic 4T1 breast tumors after i.v. administration but mRNA suppression decreases 24 h after the final dose. We hypothesized that complexation of cholesterol-modified Dicer-substrate siRNA (Chol-DsiRNA) in place of Chol-siRNA can increase the potency and duration of suppression by polyplexes of PLL(30)-PEG(5K) in solid tumors. We found that replacing Chol-siRNA with Chol-DsiRNA increased polyplex loading and nuclease protection, suppressed stably expressed luciferase to the same extent in primary murine 4T1-Luc breast tumors under the current dosage regimen, but maintained suppression ~72 h after the final dose. The kinetics of suppression in 4T1-Luc over 72 h, however, were similar between DsiLuc and siLuc after electroporation and between polyplexes of Chol-DsiLuc and Chol-siLuc after transfection, suggesting that Chol-DsiRNA polyplexes increase the duration of mRNA suppression through differences in polyplex activities in vivo. Thus, replacing Chol-siRNA with Chol-DsiRNA may significantly increase the duration of mRNA suppression by polyplexes of PLL(30)-PEG(5K) and possibly other PEGylated polycationic polymers in primary tumors and metastases after i.v. administration.

Keywords: Chol-DsiRNA polymer micelles; Chol-DsiRNA polyplexes; Chol-siRNA polymer micelles; Chol-siRNA polyplexes; Drug delivery; DsiRNA; RNA interference.

Fig.1

Effect of increasing PLL block length and modification with 3’-cholesterol on the protection of complexed DsiRNA and siRNA from nuclease degradation by PLL-PEG(5K) in high concentrations of murine serum. Polyplexes of DsiCtrl, Chol-DsiCtrl, siCtrl, or Chol-siCtrl and PLL-PEG(5K) were prepared as described (Table 1) at the indicated minimum N/P ratio required to form neutral polyplexes, then incubated in buffer or 90% (v/v) murine serum at 37°C for 24 h. Remaining full-length DsiRNA (white bars), Chol-DsiRNA (black bars), siRNA (not detected), or Chol-siRNA (grey bars) were released from the polyplexes by heparin in the presence of a broad-spectrum nuclease inhibitor. Chol-DsiCtrl and Chol-siCtrl were additionally separated from serum proteins with water-soluble cholesterol, then quantitated by agarose gel electrophoresis. Average percent protection ± SD (n=2) after 24 hours was determined by normalizing the signal density from the remaining single band of the indicated RNAi molecule from serum-treated polyplexes to the signal density from the single band of the indicated RNAi molecule from the respective buffer-treated polyplexes and compared by one-way ANOVA with Tukey’s post-test where *P < 0.05 and **P < 0.01. DsiRNA, siRNA, Chol-DsiRNA, siRNA, and Chol-siRNA alone were undetectable under these conditions (not shown). Results are representative of at least three independent experiments. ^aData taken from Ambardekar et al., 2013.

Fig. 2

Kinetics of siLuc and DsiLuc activity in murine 4T1 breast cancer epithelial cells over 72 hours after electroporation. A murine breast cancer epithelial cell line stably expressing firefly luciferase (4T1-Luc) was electroporated alone or with 300 nM anti-luciferase siRNA (siLuc, triangles) or DsiRNA (DsiLuc, circles) then grown at 37°C. Average percent luciferase activity ± SD (n=2 independent electroporation treatments) was calculated as the average radiance from siLuc- or DsiLuc-treated 4T1-Luc normalized to the average radiance from 4T1-Luc (electroporated alone) on the same plate and compared at the indicated time points by two-sided unpaired t-test. Results are representative of at least two independent experiments.

Fig.3

Effect of increasing PLL block length and modifying DsiRNA with 3’-cholesterol on the activity of PLL-PEG(5K) polyplexes of DsiRNA in murine 4T1 breast cancer epithelial cells 24 hours after treatment. Murine 4T1 breast cancer cells stably expressing luciferase (4T1-Luc) were incubated for 4 h with serum-free complete DMEM containing 200 nM of DsiRNA or Chol-DsiRNA alone (Alone) or complexed with PLL30-PEG(5K) or PLL50-PEG(5K) at the indicated N/P ratio. An equal volume of complete DMEM containing 20% FBS was then added to each well and luciferase activity was measured 20 hrs later by bioluminescent imaging. Average percent luciferase activity ± SD (n=2 independent treatment wells) was calculated as the average radiance from treated 4T1-Luc normalized to the average radiance from untreated 4T1-Luc on the same plate and compared by one-way ANOVA with Tukey’s post-test. Results are representative of two independent experiments. Trypan blue exclusion and number of 4T1-Luc in all treatment groups was between 96%–100% of untreated 4T1-Luc by cell counting with trypan blue exclusion (not shown). Results are representative of at least three independent experiments.

Fig. 4

Effect of complexation with PLL(30)-PEG(5K) and modification with 3’-cholesterol on the potency and duration of DsiRNA activity in primary murine 4T1 breast tumors after i.v. administration. Primary syngeneic breast tumors were established by injecting 4T1-Luc cells (10⁶) SQ into the mammary fat pad of female BALB/c mice, growing tumors to between 50 and 100 mm³, then determining a baseline luciferase signal on the first day of treatment (Day 0). (A) Vehicle alone (HEPES/saline, white triangles), Chol-DsiLuc alone (black triangles) or polyplexes of PLL(30)-PEG(5K) and (B) inactive DsiCtrl (white circles), DsiLuc (black circles), (C) inactive Chol-DsiCtrl, (white squares), or Chol-DsiLuc (black squares) at the indicated N/P ratios were then injected i.v. at 2.5 mg RNAi molecule/kg on Days 0, 1, and 2 (black arrows). Average radiances from 4T1 tumors within the same treatment group at each timepoint were normalized to average radiances on the first day of treatment (Day 0) and expressed as percent luminescence ± SEM (n=4 to 5 mice). (Right inset) Representative images of luciferase activity in primary 4T1-Luc tumors on the first day of treatment (D0) or 24 hrs after the final day of treatment (D3). *P <0.05 or **P <0.01 vs. average radiance on Day 0 within the same treatment group by repeated measures nonparametric one-way ANOVA with Dunn’s post-test. Results are representative of at least two independent studies. (Color in pdf, black and white in print).

Fig. 5

Effect of DsiRNA and Chol-DsiRNA polyplexes of PLL(30)-PEG(5K) on 4T1-Luc breast tumor growth and mouse body weight after i.v. administration. Mice were treated as described in Fig. 4 and tumor volumes and body weights were measured daily beginning the first day of treatment (Day 0). (A) Average tumor volumes or (B) body weights ± SEM (n=4 to 5 mice) at each timepoint within the same treatment group were compared to initial average tumor volumes or body weights on Day 0 by one-way ANOVA with Dunnett post-test. Results are representative of at least two independent studies.

Fig. 6

Activities of Chol-DsiRNA and nuclease-resistant Chol-siRNA polyplexes of PLL(30)-PEG(5K) in primary murine 4T1 breast tumors after i.v. administration. Changes in luminescence from primary 4T1-Luc breast tumors in female BALB/c mice after i.v. administration of polyplexes of PLL(30)-PEG(5K) and nuclease-resistant Chol-*siLuc (black inverted triangles, N/P 3) or Chol-DsiLuc (black squares, N/P 1, taken from Fig. 4C) at 2.5 mg RNAi molecule/kg (described in Fig. 4) were plotted together to facilitate comparison. Average percent changes in luminescence from 4T1-Luc tumors treated with Chol-DsiLuc polyplexes or Chol-*siLuc polyplexes at Days 1 through 4 were compared by two-tailed Mann-Whitney nonparametric unpaired t test (*P <0.05). ^aData taken from Ambardekar et al., 2013.