The efficacy of nuclease-resistant Chol-siRNA in primary breast tumors following complexation with PLL-PEG(5K)

Abstract

Modifying the sense strand of nuclease-resistant siRNA with 3'-cholesterol (Chol-*siRNA) increases mRNA suppression after i.v. administration but with relatively low efficacy. We previously found evidence in vitro that suggests complexation of Chol-siRNA with PLL-PEG(5K), a block copolymer of poly-l-lysine and 5 kDa polyethylene glycol, may increase the efficacy of Chol-siRNA in vivo in a PLL block length-dependent manner. In this study, the extent that polyplexes of PLL10-PEG(5K), PLL30-PEG(5K), and PLL50-PEG(5K) protect complexed Chol-siRNA in high concentrations of murine serum and affect the activity of Chol-*siRNA in murine 4T1 breast tumor epithelial cells in vitro and in primary orthotopic tumors of 4T1 was compared. PLL-PEG(5K) required 3'-Chol to protect full-length siRNA from nuclease degradation in 90% (v/v) murine serum and protection was increased by increasing PLL block length and nuclease resistance of Chol-siRNA. Polyplexes of Chol-*siLuc suppressed stably expressed luciferase in 4T1-Luc cells to different levels in vitro where PLL30 > PLL50 > PLL10. In contrast, only polyplexes of Chol-*siLuc and PLL30-PEG(5K) or PLL50-PEG(5K) suppressed high levels of luciferase in primary orthotopic tumors of 4T1-Luc after i.v. administration, whereas polyplexes of Chol-*siLuc and PLL10-PEG(5K), inactive Chol-*siCtrl polyplexes of PLL-PEG(5K), or Chol-*siLuc alone had no detectable activity. As a whole, these results indicate that polyplexes of PLL-PEG(5K) increase the efficacy of nuclease-resistant Chol-siRNA in primary breast tumors after i.v. administration in a PLL block length-dependent manner. Thus, complexation of Chol-siRNA with PLL-PEG(5K) may be a promising approach to increase the efficacy of Chol-siRNA in a wide range of primary tumors, metastases, and other tissues but likely requires a PLL block length that balances polymer-related adverse effects, Chol-siRNA bioavailability, and subsequent activity in the target cell.

Figure 1. Pattern of free siRNA and Chol-siRNA degradation in 90% (v/v) murine serum

(A) siRNA (siCtrl: 5’- UGG UUU ACA UGU CGA CUA A - 3’ with 3’-UU overhangs) was incubated at 37°C for 15 min in HEPES buffer (lane 1), 90% v/v m urine serum (lane 2), or 90% v/v murine serum pretreated with a broad spectrum RNase inhibitor (lane 3) then separated and imaged on a 10% polyacrylamide gel post stained with SYBR Gold. (B) Chol-siRNA (siCtrl modified with 3’-cholesterol on the sense strand) was incubated at 37°C for 15 m in in HEPES buffer (lane 1), 90% v/v murine serum (lane 2), or 90% v/v murine serum pretreated with a broad spectrum RNase inhibitor (lanes 3 & 4). HEPES buffer (lanes 1–3) or water solubilized cholesterol (*Cholesterol) at a 1:1 molar ratio cholesterol:Chol-siRNA (lane 4) was added and Chol-siRNA was separated and imaged on a 10% polyacrylamide gel post stained with SYBR Gold.

Figure 2. Effect of PLL block length, modifying siRNA with 3’-cholesterol, and complexation with nuclease-resistant Chol-siRNA on protection of complexed siRNA from degradation in high serum concentrations by PLL-PEG(5K)

(A) siRNA (siCtrl: 5’- UGG UUU ACA UGU CGA CUA A - 3’ with 3’-UU overhangs), (B) Chol-siRNA (siCtrl modified with 3’-cholesterol on the sense strand), or (C) nuclease-resistant Chol-*siRNA (Chol-siCtrl without 3’-UU overhangs on the sense stand) was incubated with PLL-PEG(5K) in HEPES buffer at room temperature for 30 min at the indicated minimum N/P ratio required for complexation. Polyplexes were then incubated in the presence or absence of 90%v/v murine serum at 37°C for the indicated time, disassembled by heparin, and resolved with or without water solubilized cholesterol (*Cholesterol) as described in Fig.1. The single band for (A) truncated siRNA (open symbols), (B) full-length Chol-siRNA (closed symbols), or (C) full-length Chol-*siRNA (closed symbols) from serum-treated polyplexes was quantified by densitometry and normalized to the respective full-length band from untreated polyplexes at the same N/P ratio. Percent protection ± SD (n=2) is an average of two independent experiments. siRNA and Chol-siRNA were completely degraded within 1 h and Chol-*siRNA was degraded within 1.5 h under the same conditions.

Figure 3. Effect of PLL block length and modifying siRNA with 3’-cholesterol on the displacement of siRNA from polyplexes of PLL-PEG(5K) in high serum concentrations

siRNA (siCtrl: 5’- UGG UUU ACA UGU CGA CUA A - 3’ with 3’-UU overhangs) or Chol-siRNA (siCtrl modified with 3’-cholesterol on the sense strand) was incubated with PLL-PEG(5K) in HEPES buffer at room temperature for 30 min at the minimum N/P ratio required for complexation. Polyplexes were then incubated in the presence or absence of 90%v/v murine serum at 37°C for the indicated time and resolved with or without heparin or water soluble cholesterol (*Cholesterol) as described in Fig.1.

Figure 4. Effect of PLL block length and modifying siRNA with 3’-cholesterol on the suppression of luciferase activity in 4T1-Luc by polyplexes of PLL-PEG(5K)

(A) Electroporation: A murine mammary tumor epithelial cell line stably expressing firefly luciferase (4T1-Luc) was electroporated with 300 nM control siRNA (siCtrl), anti-luciferase siRNA (siLuc), Chol-siCtrl (3’-sense), Chol-siLuc or Chol-*siLuc (3’-sense) and luciferase activity was measured by bioluminescent imaging after 24 h. Radiance from 4T1-Luc electroporated with the indicated siRNA was normalized to radiance from 4T1-Luc electroporated without siRNA on the same plate and expressed as percent luciferase activity. (B) Transfection: Serum- and antibiotic-free complete DMEM containing 200 nM of the indicated siRNA complexed with PLL-PEG(5K) at the indicated N/P ratio or 200 nM Chol-siLuc alone (Alone) was added to 4T1-Luc for 4 h then an equal volume of complete DMEM containing 20% FBS was added and luciferase activity was measured 20 h later by bioluminescent imaging. Average radiance from transfected 4T1-Luc was normalized to average radiance from untreated cells on the same plate and expressed as percent luciferase activity ± SD (n=2, ***P < 0.001 by one way ANOVA and Tukey post-test). Results are representative of two independent experiments. (C) Cytostatic Effects: The viability of 4T1-Luc in all treatment groups relative to untreated 4T1-Luc was between 98%–100% by trypan blue exclusion (data not shown). The average number of live cells from treated 4T1-Luc was normalized to the average number of live cells from untreated 4T1-Luc on the same plate and expressed as percent live cells ± propagated SD (n=2).

Figure 5. Effect of PLL block length on the suppression of luciferase expression in primary 4T1-Luc tumor by polyplexes of nuclease-resistant Chol-siRNA and PLL-PEG(5K)

Primary breast tumors were established by injecting 4T1-Luc cells (1 ×10⁶) SQ into the mammary fat pad of female BALB/c, allowing tumors to grow between 60 and 100 mm³, then determining a baseline luciferase signal. (A) HEPES/saline (open triangles), nuclease resistant Chol-*siLuc alone (closed triangles) or (B) PLL10-PEG, (C) PLL30-PEG or (D) PLL50-PEG complexed with Chol-*siCtrl (open squares) or Chol-*siLuc (closed squares) at the indicated N/P ratio was then intravenously injected at 2.5 mg Chol-siRNA/kg on days 0, 1, and 2. Average radiance from 4T1 tumors within the same cohort was normalized to the average radiance on the first day of treatment (Day 0) and expressed as % luminescence ± propagated SEM (n=3–5 animals). Representative images of luciferase activity in primary 4T1-Luc tumors on the first (D0) and third (D2) day of treatment are shown. *P <0.05 vs. average percent luciferase activity on Day 0 within the same treatment group by Friedman non-parametric repeated measures ANOVA and Dunn’s multiple comparison test.

Figure 6. Effect of treatments on tumor volume of 4T1-Luc and body weight

Mice were treated as described in Fig.5 and (A) tumor volume and (B) body weight were measured beginning the first day of treatment (Day 0). Values are expressed as the mean ± SEM (n=3–5 animals).