Preliminary Preclinical Study of Chol-DsiRNA Polyplexes Formed with PLL[30]-PEG[5K] for the RNAi-Based Therapy of Breast Cancer

Abstract

RNA interference molecules have tremendous potential for cancer therapy but are limited by insufficient potency after i.v. administration. We previously found that Chol-DsiRNA Polyplexes formed between cholesterol-modified dicer-substrate siRNA (Chol-DsiRNA) and the cationic diblock copolymer PLL[30]-PEG[5K] greatly increase the activity of Chol-DsiRNA against a stably expressed reporter mRNA in primary murine syngeneic breast tumors after daily i.v. dosing. Here, we provide a more thorough preliminary preclinical study of Chol-DsiRNA Polyplexes against the therapeutically relevant target protein, STAT3. We found that Chol-DsiSTAT3 Polyplexes greatly increase plasma exposure, distribution, potency, and therapeutic activity of Chol-DsiSTAT3 in primary murine syngeneic 4T1 breast tumors after i.v. administration. Furthermore, inactive Chol-DsiCTRL Polyplexes are well tolerated by healthy female BALB/c mice after chronic i.v. administration at 50 mg Chol-DsiCTRL/kg over 28 days. Thus, Chol-DsiRNA Polyplexes may be a good candidate for Phase I clinical trials to improve the treatment of breast cancer and other solid tumors.

Graphical Abstract

Chol-DsiRNA Polyplexes formed between cholesterol-modified dicer-substrate siRNA (Chol-DsiRNA) and a cationic block copolymer of 30 poly-L-lysine residues and 5 kDa polyethylene glycol (PLL[30]-PEG[5K]) significantly increase the potency and activity of complexed Chol-DsiSTAT3 in primary murine syngeneic breast tumors and are well tolerated by healthy female BALB/c mice after chronic i.v. administration at 50 mg/kg over 28 days

1. Introduction

RNA interference (RNAi) is a natural, intracellular process that selectively decreases the expression of any specific protein at the mRNA level through the complementary binding of gene-specific dsRNA molecules including microRNA (miRNA), small, interfering RNA (siRNA), or longer dicer-substrate siRNA (DsiRNA) (“RNAi molecules”). ¹ Several proteins have been identified in tumor or tumor-associated cells where suppression can produce a therapeutic effect and/or increase the efficacy of current cancer treatments. ² Thus, RNAi molecules have tremendous potential in the treatment of cancer.

Many cancer therapy applications require i.v. administration to achieve a therapeutic effect. The potencies of RNAi molecules after i.v. administration, however, are extremely low or undetectable. ¹ Several nanoscale dosage forms have been developed to improve the potency of RNAi molecules after i.v. administration using a wide range of nanomaterials and/or modified RNAi molecules. ^{3, 4} Identifying nanomaterials and/or RNAi molecule modifications that increase the potency of mRNA suppression in primary tumors and metastases and are suitable for clinical application, however, remains a critical barrier to developing nanoscale dosage forms for RNAi-based therapies.

Conjugating 3’-cholesterol to nuclease-resistant siRNA (Chol-*siRNA) increases siRNA suppression of an endogenous mRNA in the liver and jejunum after i.v. administration but with relatively low potency (50 mg Chol-*siRNA/kg). ⁵ We previously found that polymer complexes (polyplexes) formed with a block copolymer of 10 poly-L-lysine residues and 5 kDa polyethylene glycol (PLL[10]-PEG[5K]) increase Chol-siRNA suppression of a native mRNA (CYPB) in murine mammary MVEC (~90% suppression) to a much greater extent than polyplexes formed with a block copolymer of 50 poly-L-lysine residues and 5 kDa polyethylene glycol (PLL[50]-PEG[5K]) (30% suppression), biodegradable nanogels (40% suppression), or a graft copolymer of 2 kDa branched PEI and 10 kDa PEG (30% suppression) without affecting cell viability. ⁶ Given that PLL block length affects Chol-siRNA Polyplex activity and nuclease protection in vitro, we next directly compared Chol-*siRNA polyplexes formed with PLL[10]-PEG[5K], PLL[30]-PEG[5K], or PLL[50]-PEG[5K] and found that polyplexes formed with PLL[30]-PEG[5K] increase the potency of Chol-*siRNA to the greatest extent against a stably expressed reporter gene (luciferase) in primary murine syngeneic 4T1 breast tumors (4T1-Luc) after daily i.v. administration at 2.5 mg Chol-*siLUC/kg over three days without affecting body weight. ⁷ Similar results with PEGylated diblock copolymers and hydrophobically-modified siRNA were later independently reported in primary ectopic human xenogeneic HeLa cervical tumors with targeted, core-crosslinked “Chol-siRNA micelles” composed of conventional Chol-siRNA (5’-cholesterol conjugated to the antisense strand) and integrin-targeted PLL[45]-b-PEG[12K]-cRGD diblock copolymers ⁸ and in primary human xenogeneic MDA-MB-231 breast tumors with polyplexes composed of palmitic acid-modified siRNA (PA-siRNA) and more hydrophobic p(DMAEMA-co-BMA)-b-PEG[5K] diblock copolymers. ⁹ Thus, nanoscale dosage forms composed of hydrophobically-modified RNAi molecules complexed with PEGylated diblock copolymers have the potential to increase the potency of RNAi molecules in solid tumors and metastases after i.v. administration.

Although Chol-siRNA Polyplexes formed with PLL[30]-PEG[5K] increase the potency of Chol-**siLUC in primary murine 4T1-Luc breast tumors after i.v. administration, LUC mRNA suppression decreases 24 hours after the last treatment. ⁷ Given that polyplexes of dicer substrate siRNA (DsiRNA) and Lipofectamine® 2000 have 100-fold higher potency and longer duration of mRNA suppression in HEK293 cells in vitro than polyplexes of siRNA and Lipofectamine® 2000 ¹⁰, we next compared the activities of Chol-*siRNA Polyplexes and Chol-DsiRNA Polyplexes formed with PLL[30]-PEG[5K] (Figure1) ¹¹ and found that Chol-DsiLUC Polyplexes increase the duration of LUC mRNA suppression in primary murine 4T1-Luc breast tumors after i.v. administration ~48 hours longer than Chol-*siLUC Polyplexes under the same dosage regimen. These differences, however, are likely due to differences in the activities of Chol-DsiRNA Polyplexes and Chol-siRNA Polyplexes in primary 4T1-Luc tumors after i.v. administration and not differences in the activities of DsiRNA and siRNA in 4T1 cells because electroporation of 4T1-Luc cells with equimolar amounts of DsiLUC or siLUC suppresses luciferase activity to the same extent over 72 hours. ¹¹ Chol-DsiRNA Polyplexes also have higher loading (50 wt% Chol-DsiRNA vs. 25 wt% Chol-siRNA) and almost fully protect Chol-DsiRNA from degradation in 90% murine serum for 24 h at 37°C without expensive nuclease-resistance modifications to DsiRNA. ¹¹ These results suggest Chol-DsiRNA Polyplexes are a more promising lead formulation than Chol-siRNA Polyplexes to increase the potency and sustain the activity of RNAi molecules in solid breast tumors after i.v. administration.

Figure 1. Idealized self-assembly of Chol-DsiRNA Polyplexes formed with PLL[30]-PEG[5K].

A solution of DsiRNA (red) modified with 3’-cholesterol (brown) on the sense strand (Chol-DsiRNA) is added to a solution of diblock copolymers composed of 30 poly-L-lysine residue blocks (blue) and 5 kDa polyethylene glycol blocks (black) (PLL[30]-PEG[5K]) at an N/P molar charge ratio of 1 (moles of positively-charged primary amines (N) / moles of negatively charged phosphates). The negatively-charged phosphate backbones of Chol-DsiRNA then electrostatically bind and neutralize the positively-charged PLL[30] blocks, converting PLL[30]-PEG[5K] unimers into amphiphilic diblock copolymers that spontaneously self-assemble into Chol-DsiRNA polymer complexes (Polyplexes) that are further stabilized by hydrophobic interactions between 3’-cholesterol groups. A similar idealized self-assembly at slightly higher N/P ratio is envisioned for Chol-siRNA Polyplexes formed with PLL[30]-PEG[5K].

In this study, we provide a more thorough preliminary preclinical assessment of Chol-DsiRNA Polyplexes formed with PLL[30]-PEG[5K] against the therapeutically relevant target gene STAT3 in primary murine syngeneic breast tumors after i.v. administration by determining (i.) Chol-DsiSTAT3 pharmacokinetics, distribution, ED₅₀, and kinetics of STAT3 mRNA suppression in primary 4T1 breast tumors (ii.) rate-based T/C ratio of Chol-DsiSTAT3 Polyplex activity against primary 4T1 breast tumor growth, and (iii.) toxicity of inactive Chol-DsiCTRL Polyplexes in healthy male and female BALB/c mice after dose escalation to 50 mg Chol-DsiCTRL/kg and in healthy female BALB/c mice after chronic administration at 50 mg Chol-DsiCTRL/kg over 28 days.

2. Methods

2.1. Polymer

Methoxy-poly(ethylene glycol)-b-poly(L-lysine hydrochloride) with a 5 kDa polyethylene glycol block (PEG[5K]) and a 30 poly-L-lysine block (PLL[30]) [PLL[30]-PEG[5K]; avg. MW 9900 Da] was obtained from Alamanda Polymers (Huntsville, AL). The number of Lys residues within the PLL[30] block was ±10% (PLL[27] to PLL[33]) and the polydispersity index of the entire polymer was between 1 and 1.1.

2.2. RNAi Molecules

DsiRNA were HPLC-purified, asymmetric, 25-mer dsRNA with 2-nucleotide UU overhangs on the 3’ end of the antisense strand: (1) DsiCTRL (GE Dharmacon) sense: 5’ – CGU UAA UCG CGU AUA AUA CGC GUA U – 3’, antisense: 5’ – AUA CGC GUA UUA UAC GCG AUU AAC G(UU) – 3’; MW: 16,523.02 g/mol; sequence based on scrambled siRNA (IDT). (2) DsiSTAT3 (GE Dharmacon) sense: 5’ – GGU CAA AUU UCC UGA GUU GAA UUA U – 3’, antisense: 5’ – AUA AUU CAA CUC AGG AAA UUU GAC C(UU) – 3’; MW: 16,493.0 g/mol; sequence based on siGENOME Mouse STAT3 (GE Dharmacon). (3) Chol-DsiCTRL (IDT) DsiCTRL with 3’-cholesterol conjugated to the sense strand; MW: 17,278.9 g/mol. (4) Chol-DsiSTAT3 (GE Dharmacon) DsiSTAT3 modified with 3’-cholesterol as described for Chol-DsiCTRL; MW: 17,197.92 g/mol.

For in vitro studies, lyophilized RNAi molecules were resuspended in sterile, nuclease-free ddH₂0 [100 μM] as directed (GE Dharmacon) and stored in aliquots (10 μL) at −80°C. For in vivo studies, lyophilized RNAi molecules were resuspended in the indicated buffer on the day of injection.

2.3. Cell Culture

Murine 4T1 breast cancer epithelial cells (CRL-2539, ATCC) were cultured in Complete RPMI-1640 Media composed of RPMI 1640 / L-glutamine [1.1481 mM] (GE Healthcare Life Sciences) containing heat-inactivated FBS [10% v/v; endotoxin <0.3 EU scale] (Atlanta Biologicals, Atlanta, GA), sodium pyruvate [1 mM] (Gibco), 1X MEM non-essential amino acids (Hyclone), 1X Vitamins (Hyclone), and penicillin G [100 U/mL] / streptomycin sulfate [100 μg/mL] (Gibco). Cells were grown in TPP plates (MIDSCI, St. Louis, MO) at 37°C / 5% CO₂ and detached using Accutase (Innovative Cell Technologies, San Diego, CA) as directed.

2.4. Electroporation of Murine 4T1 Cells

Murine 4T1 cells [10 × 10⁶ cells/mL] were electroporated (Nucleofector, Lonza AG, Bazel, Switzerland) with the indicated DsiRNA [300 nM] using Amaxa Cell Line Kit V for Mouse Epithelial Cells (VCA-1003, Lonza) as directed (Setting T-024) and cultured in 6-well plates [1 × 10⁶ cells / well]. Total RNA was isolated (RNeasy Micro kit, Qiagen) and eluted with sterile, nuclease-free deionized H₂O (20 μL), quantitated by A₂₆₀ (NanoDrop 2000™ Spectrophotometer, Thermo Scientific) (3 μL), and stored at −80°C. Average STAT3 mRNA copies / ng total RNA ± SD (n=2 replicates from two independent electroporations) were determined by RT-ddPCR (Section 2.9), normalized to electroporated 4T1 cells alone at the indicated time points, and compared by two-tailed, unpaired t-test.

2.5. Formation of Chol-DsiRNA Polyplexes

The concentration of a 2X Polymer Complexation Solution at an N/P charge molar ratio of one mole positively-charged primary amines (N) from the PLL[30] block of PLL[30]-PEG[5K] to one mole negatively-charged phosphates (P) from the phosphate backbone of Chol-DsiRNA (N/P 1) ¹¹ was calculated based on the concentration of the 2X Chol-DsiRNA Complexation Solution

$$2X\mathit{Polymer}\mathit{Complexation}\mathit{So}{\mathit{l}}^{\prime }\mathit{n}\left(\frac{g}{\mathit{mL}}\right)=\frac{g\mathit{RNAi}}{\mathit{mL}}\times \frac{\mathit{mol}\mathit{RNAi}}{\mathit{MW}\mathit{RNAi}}\times \frac{\mathit{mol}\mathit{RNAi}P}{\mathit{mol}\mathit{RNAi}}\times \frac{\mathit{mol}\mathit{Polymer}N}{\mathit{mol}\mathit{RNAi}\mathit{P}}\times \frac{\mathit{mol}\mathit{Polymer}}{\mathit{mol}\mathit{Polymer}N}\times \frac{\mathit{MW}\mathit{Polymer}}{\mathit{mol}\mathit{Polymer}}$$

where g RNAi/mL = Concentration of 2X Chol-DsiRNA Complexation Solution (Double the desired concentration of Chol-DsiRNA in the final Chol-DsiRNA Polyplex solution), MW RNAi = MW of Chol-DsiRNA, mol RNAiP / mol RNAi = 52 moles phosphate per mole Chol-DsiRNA, mol Polymer N / mole RNAi P = 1 (N/P 1 charge molar ratio), mol Polymer / mol Polymer N = 1 mole of PLL[30]-PEG[5K] / 30 moles PLL primary amines, MW Polymer / mol Polymer = MW of PLL[30]-PEG[5K] = 9900 Da.

For transfection studies, a Concentrated Stock Solution of Chol-DsiRNA Polyplexes at N/P 1 was prepared by (i.) sterilizing PLL[30]-PEG[5K] in open, sterile 2 mL centrifuge tubes under vacuum for 2 h in a desiccator containing a glass dish of 99% ethanol (ii.) preparing a 2X Chol-DsiRNA Complexation Solution plus 10 μL for experimental loss by diluting a thawed frozen stock of Chol-DsiRNA with sterilized (0.2 μm filter) HEPES Buffer [0.1 M HEPES in deionized H2O, pH 7.4] to twice the desired concentration of Chol-DsiRNA in the final Concentrated Stock Solution (iii.) preparing a 2X Polymer Complexation Solution plus 10 μL for experimental loss by dissolving sterilized PLL[30]-PEG[5K] in HEPES Buffer at 1 mg Polymer/mL, incubating at r.t. for 30 min, and diluting with HEPES Buffer to the calculated 2X concentration (iv.) forming a Concentrated Stock Solution of Chol-DsiRNA Polyplexes by adding the 2X Chol-DsiRNA Complexation Solution dropwise to the 2X Polymer Complexation Solution at 1/1 (v/v), mixing the solution by pipette aspiration / dispensation (30 sec), and incubating (RT, 30 min.).

For in vivo studies, endotoxin-free solutions/reagents were used where possible. A Concentrated Stock Solution of Chol-DsiRNA alone or Chol-DsiRNA Polyplexes at N/P 1 was prepared on the day of injection by (i.) determining the total volume of Concentrated Chol-DsiRNA Stock Solution (Chol-DsiRNA alone and Chol-DsiRNA Polyplexes) required for all mice in the study based on the injected dose (mg/kg), individual mass of each mouse, and total prepared i.v. injection volume (125 μL for dose response / kinetics studies or 225 μL for toxicity studies per mouse) plus 50 μL for experimental loss (ii.) preparing a 2X Chol-DsiRNA Complexation Solution (1/2 total required volume of Concentrated Stock Solution of Chol-DsiRNA) plus 25 μL for experimental loss by resuspending lyophilized Chol-DsiRNA as directed by the manufacturer in sterilized HEPES Buffer to twice the desired concentration of Chol-DsiRNA in the Concentrated Stock Solution (iii.) sterilizing PLL[30]-PEG[5K] and preparing 2X Polymer Complexation Solution (same volume as 2X Chol-DsiRNA Complexation Solution) as described for transfection studies above (iv.) preparing the required volume of Concentrated Stock Solution of Chol-DsiRNA Polyplexes as described above and (v.) preparing the required volume of Concentrated Stock Solution of Chol-DsiRNA alone by diluting the 2X Chol-DsiRNA Complexation Solution 1/1 (v/v) with sterilized HEPES Buffer. The required i.v. injection volume (100 μL or 200 μL) of Chol-DsiRNA alone or Chol-DsiRNA Polyplexes plus 25 μL for each mouse was then prepared by combining (i.) volume of Concentrated Chol-DsiRNA or Chol-DsiRNA Polyplex Stock Solution adjusted to deliver the required dose of Chol-DsiRNA based on mouse bodyweight (ii.) volume of sterilized (0.2 μm filter) HEPES Buffer plus 1.5 M NaCl at 1/10 dilution [0.15 M NaCl final conc.], and (iii.) volume of HEPES Buffer adjusted to final volume.

2.6. Hydrodynamic diameter and zeta potential of Chol-DsiCTRL polyplexes

The average hydrodynamic diameter of Chol-DsiCTRL Polyplexes (N/P 1) was determined by nanoparticle tracking analysis (NanoSight LM10 and NTA 2.3 analytical software, Malvern Instruments, UK). Chol-DsiRNA Polyplexes [0.25 mg Chol-DsiSTAT3/mL] were prepared as described for Transfection (Section 2.5) and recorded (Shutter speed: 500, Gain: 680) for video analysis (screen gain: 6; solution temperature: 22°C; viscosity: 0.95 cP [viscosity of water at 22°C]); remaining parameters: auto). The average estimated concentration of Chol-DsiRNA Polyplexes for each 1 nm bin from three independent analyses was normalized as a percentage of the total average estimated concentration of Chol-DsiRNA Polyplexes. A plot of accumulated percent of total Chol-DsiRNA Polyplexes at each diameter (y-axis) vs. ln diameter (x-axis) was then fit against a cumulative Gaussian (percent) model using GraphPad Prism 9 to determine a best-fit mean and standard deviation from the lognormal curve.

The average zeta potential of Chol-DsiCTRL Polyplexes (N/P 1) at 25°C in 0.1 M HEPES, pH 7.4 [1.5 mg Chol-DsiCTRL/mL] was determined (n=3 independent samples from the same batch with 12 Zeta runs per sample) using a ZetaSizer Nano ZA (Malvern Instruments, Malvern, UK) equipped with a He-Ne laser (λ = 633 nm) as the incident beam.

2.7. Endotoxin levels

Chol-DsiCTRL endotoxin levels were quantitated by the manufacturer (IDT). PLL[30]-PEG[5K] [0.2 mg/mL] and Chol-DsiRNA Polyplexes [0.05 mg Chol-DsiSTAT3/mL] endotoxin levels were quantitated using an Endochrome-K Limulus Amebocyte Lysate Kit (Charles River Labs) as directed with endotoxin-free reagents and disposable labware under aseptic conditions.

2.8. Transfection of Murine 4T1 Cells

Murine 4T1 cells were seeded in 6-well plates [0.5 × 10⁶ cells/well] in RPMI-1640 Media (3 mL) and incubated 14 to 16 h before transfection. On the day of transfection, a concentrated Stock Solution of Chol-DsiRNA Polyplexes [2 μM Chol-DsiRNA] was prepared as described (Section 2.5) then diluted to 200 nM Chol-DsiRNA in Complete RPMI-1640 lacking FBS and antibiotics. Old growth media was aspirated and diluted polyplexes or Complete RPMI-1640 lacking FBS and antibiotics (1.5 mL) were added for 4 h, then an equal volume of 20% FBS Complete RPMI-1640 (1.5 mL) was added before further incubation for 20 h (24 h from the start of transfection). Average murine STAT3 mRNA copy numbers per ng total RNA ± propagated SD (n= 3 replicates from two independent treatments) were then determined by RT-ddPCR (Section 2.9), normalized to untreated cells, and compared by two-tailed, unpaired t-test.

2.9. Quantitation of STAT3 mRNA and Chol-DsiSTAT3 by Reverse Transcription-Droplet Digital™ PCR (RT-ddPCR)

For mRNA, total purified RNA was diluted in sterile, nuclease-free ddH₂0 and converted to cDNA [~1 μg total] (Superscript™ VILO™ cDNA Synthesis Kit, Life Technologies). The number of STAT3 mRNA copies was determined by droplet-digital PCR (ddPCR) (QX200 Droplet Digital PCR System, BioRad) from 10 ng of template cDNA using PrimeTime® Taqman assays (IDT, Coralville, USA) for murine STAT3 mRNA and BioRad ddPCR reagents / consumables as directed. STAT3 mRNA copies were then normalized to the mass of total RNA (template cDNA) [ng] in the ddPCR reaction (assumes 1:1 RT conversion of total RNA to cDNA).

For Chol-DsiRNA, total purified RNA was diluted in sterile, nuclease-free ddH₂0 and converted to cDNA [~1 μg total] (TaqMan™ MicroRNA Reverse Transcription Kit, Invitrogen™, ThermoFisher). Copy numbers of Chol-DsiSTAT3 were quantitated by ddPCR using two different amounts of template cDNA [tumor: 6.67, 33.5 ng; lungs: 6.67, 33.35 ng (Chol-DsiSTAT3 alone), 1.334, 13.4 ng (Chol-DsiSTAT3 Polyplexes); liver: 1.334, 13.34 ng (Chol-DsiSTAT3 alone), 0.1334, 1.334 (Chol-DsiRNA Polyplexes); spleen, kidneys: 0.2668, 1.334 ng; heart, brain: 13.34, 133.4 ng], stem-loop quantitative Custom TaqMan Small RNA Assays (Invitrogen™, ThermoFisher) for the antisense strand of Chol-DsiSTAT3, and BioRad ddPCR reagents / consumables as directed.

Chol-DsiSTAT3 antisense copies / μL / ng template cDNA was calculated as the slope of Chol-DsiSTAT3 antisense strand copies / μL (y-axis) vs. mass of template cDNA (x-axis) (2 amounts). Mass of Chol-DsiSTAT3 (ng) / mg tissue was then calculated by multiplying Chol-DsiSTAT3 antisense copies / μL / ng template cDNA with the slope from the standard curve of ng Chol-DsiSTAT3 (y-axis) vs. Chol-DsiSTAT3 antisense copies / μL (x-axis) and dividing by the volume-normalized mass of the tissue sample (10 mg).

2.10. Tumor growth delay of primary murine 4T1 breast tumors

All procedures were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee. Primary tumors were prepared in female BALB/c mice as described ⁷ but hair was first removed from the region above the tumor cell inoculation site (mammary fat pad #4) by shaving / applying Nair cream (removed with warm water and gauze after 30 sec then rinsed with 70% ethanol) and 10-fold fewer 4T1 cells [5 × 10⁵ 4T1 cells total] were injected SQ to facilitate tumor volume measurements by 3D surface scanning (TumorImager, Biopticon). Tumor volumes and body weights were measured every other day before treatment, then daily until 48 h after the last treatment.

When tumor volumes reached 30 to 50 mm³ (Day 0), vehicle alone [0.1M HEPES, 0.15M NaCl, pH 7.4, filter-sterilized] or vehicle containing the indicated Chol-DsiRNA Polyplexes was injected (0.1 mL) into the tail-vein [2.5 mg Chol-DsiRNA/kg] on days 0, 2, 4, and 6. Average daily tumor volumes ±SD (n=5 mice) were compared at each time point by Multiple t-tests. A rate-based T/C ratio and associated P value ¹² were calculated as directed using the author-provided excel spreadsheet. On Day 8 (48 h after final dose), tumors were isolated, stored at −80°C, and Stat3 protein levels were determined by Western Blot (Section 2.11).

2.11. Quantitation of Stat3 protein in primary 4T1 tumors by Western Blot

Total protein was extracted from thawed tumors using Cell Extraction Buffer (Invitrogen) containing 1 mM PMSF, 1X protease inhibitor cocktail, and 1X EDTA (Thermo Scientific) and quantified using pierce BCA protein assay (Thermo Scientific) as directed. Protein lysates [40 μg] were resolved by SDS-PAGE (Mini-PROTEAN TGX Precast Protein Gels, Bio-Rad) and transferred (Mini Trans-Blot® Cell, BioRad) to PVDF membranes as directed. PVDF membranes were incubated with rocking in Blocking Buffer [5% (w/v) nonfat dry milk in TBST Buffer] (r.t., 1 h), rinsed with TBST (r.t., 5 sec), incubated with primary antibodies (β-actin [sc-47778] and Stat3 [sc-8019], Santa Cruz Biotechnology) [1:200 in TBST] (4°C, overnight), rinsed 3X with TBST (r.t., 5 min.), and incubated with secondary antibody (horseradish peroxidase-conjugated mouse IgGκ binding protein; m-IgGκ BP-HRP [sc-516102], Santa Cruz Biotechnology) [1:5000 in TBST (r.t., 1 h), and rinsed 3X with TBST as above. The blot was incubated with HRP substrate (Luminata Classico Western HRP, Sigma) as directed, visualized (MyECL imager, Thermo Scientific), and the average ratios of Stat3 / β-Actin protein band intensities ±SD (n=2 measurements) were determined by imaging densitometry (ImageJ software).

2.12. Pharmacokinetics of Chol-DsiSTAT3 after i.v. administration

All procedures were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee including guidelines for the humane treatment of animals. Chol-DsiSTAT3 alone and Chol-DsiSTAT3 Polyplexes were prepared for in vivo studies (Section 2.5) and injected (0.1 mL) into the tail vein of female BALB/c mice (n=5 mice) at 2.5 mg Chol-DsiSTAT3 / kg (Section 2.10). Blood (~0.1 mL) from treated and untreated mice (plasma background) was collected into Li-heparinized tubes (0.3 mL Microvette Tubes, Sarstedt) from a submandibular bleed (5 mm Goldenrod lancet, Braintree Scientific) and plasma supernatants (2500 RCF, 15 min) (~50 μL) were stored at −80°C. A standard curve for Chol-DsiSTAT3 was prepared by spiking Chol-DiSTAT3 or Chol-DsiSTAT3 Polyplexes [450, 225, 112.5, 56.3, 28.1, and 14.1 ng Chol-DsiSTAT3] into untreated plasma (5 μL) (n=2 per standard).

Total RNA was isolated from plasma (miRNeasy Mini, Qiagen) with modifications by adding QIAzol Lysis Reagent at 3:1 plasma (v:v), vortexing, incubating (r.t., 5 min.), adding water-soluble cholesterol (Cholesterol-Water Soluble, Sigma) [1 mg/mL in deionized H₂O] at 1:2 plasma (v/v) then SDS [10%] at 3:1 plasma (v/v), heating (95°C, 5 min.) and cooling (2 min.) (4 cycles heating/cooling), adding chloroform at 10:1 plasma (v/v), vortexing, incubating (r.t., 2 min.), pelleting (13,000 RCF, 15 min., 2 times), adding 100% EtOH at 5:1 supernatant (v/v), and column purification as described (MiRNeasy Mini, Qiagen) then quantitated by ABS₂₆₀ (NanoDrop spectrophotometer, ThermoFisher Scientific).

Total RNA isolated from plasma (2 μL) and Kit Diluent (98 μL) (n=3 wells) were pipetted into a 96-well black (clear bottom) plate. Fluorophore (Quant-iT™ RiboGreen® RNA Reagent and Kit, Invitrogen) [1/1000 Dilution in Kit Diluent] (100 μL) was added to each well, the plate was incubated (r.t., 2–5 min), and fluorescence (Ex₄₈₀/Em₅₂₀) was measured (Molecular Devices SpectraMax ID3, top read). Samples and standards were diluted as necessary to fall within the linear range of fluorescence measurements. The average plasma concentration of Chol-DsiSTAT3 at each time point ± propagated SD (n=5 mice) was then calculated as the difference between the plasma concentration of total RNA in treated vs. untreated plasma. Pharmacokinetic parameters were determined by non-compartmental analysis (Phoenix WinNonlin version 8.2, Certara, Princeton, NJ, USA). Area under the curve (AUC) was calculated using the linear log trapezoidal rule.

2.13. Distribution of Chol-DsiSTAT3 in tumors and organs after i.v. administration

All procedures were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee including guidelines for the humane treatment of animals. Chol-DsiSTAT3 alone and Chol-DsiSTAT3 Polyplexes were prepared for in vivo studies (Section 2.5) and injected (0.1 mL) into female BALB/c mice (n=5 mice) at 2.5 mg Chol-DsiSTAT3 / kg (Section 2.10). After 15 min, mice were euthanized (isoflurane drop jar/cervical dislocation), blood was removed / organs were perfused (sterile PBS) by cardiac puncture, organs and tumors were isolated, weighed, suspended in RNAprotect Tissue Reagent (Qiagen) as directed, and stored at −80°C.

To isolate total RNA, two pieces of isolated organs [lungs ~30 mg; liver ~50 mg; spleen ~30 mg; kidneys ~40 mg; heart ~15 mg; brain ~30 mg] or tumors [~10 mg] were thawed and independently homogenized (Precellys Evolution technology, Bertin Instruments) in QIAzol Lysis Reagent (Qiagen) [10 μL QIAzol Lysis Reagent:1 mg tissue] on “Hard” mode. A standard curve for Chol-DsiSTAT3 and Chol-DsiSTAT3 Polyplexes was prepared for each organ or tumor by spiking in Chol-DsiSTAT3 or Chol-DsiSTAT3 Polyplexes [tumor, lungs, liver, spleen, kidneys: 0.6, 3, 15, 75, 150, 300 ng Chol-DsiSTAT3; heart, brain: 0.15, 0.3, 0.6, 3, 15, 75 ng Chol-DsiSTAT3] into the initial “Hard” mode homogenates of the respective organs or tumors isolated from untreated mice. SDS [5% (w/v) in deionized H₂O] [20 μL:1 mg tissue] and water-soluble cholesterol (Sigma) [10 mg/mL in deionized H2O] [0.2 μL:1 mg tissue] was added to the initial “Hard” mode homogenates and further homogenized on “Soft” mode. A portion of the final homogenate (300 μL; normalizes to 10 mg of tissue sample for RNA extraction) was heated (92°C, 5 min), cooled (r.t., 10 sec), and vortexed (4 cycles of heating, cooling, vortexing) ¹³. Total RNA was extracted by column purification after preparing the final homogenate as directed (miRNeasy Mini Kit, Qiagen, Hilden, Germany) and quantitated by ABS₂₆₀ (NanoDrop spectrophotometer, ThermoFisher Scientific). The average mass of Chol-DsiSTAT3 (ng) / mass of tissue (mg) ± propagated SD (n=2 tissue/tumor pieces from n=5 mice) was quantitated by RT-ddPCR (Section 2.9) and compared in each tissue by nonparametric two-tailed t-tests with Holm-Sidak correction.

2.14. Dose escalation of Chol-DsiCTRL Polyplexes

All procedures were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee including guidelines for the humane treatment of animals. Mice were acclimated to the animal room for approximately 7 days to determine that they were healthy based on observed behavior and weight gain. Mice were randomized into treatment cohorts by weight and sex. Chol-DsiCTRL alone and Chol-DsiCTRL Polyplexes were prepared for in vivo studies as described (Section 2.5). On Day 0, vehicle alone or vehicle containing Chol-DsiCTRL or Chol-DsiCTRL Polyplexes was injected (0.2 mL) biweekly into the tail vein of male (n=6 mice) and female BALB/c mice (n=6 mice) at an initial dose of 3.12 mg Chol-DsiCTRL/kg and bodyweight was measured daily until 4 days after the final dose. The dose for each consecutive injection was increased following a modified Fibonacci scheme (100%, 65%, 52%, 40%, 29%, 33%, 33% increase over previous dose) to a maximum of 50 mg Chol-DsiCTRL/kg (8 doses over 28 days). Mice were euthanized at the end of the study by an overdose of Fatal-Plus® (Vortech Pharmaceuticals) (150 mg/kg body weight). Average daily body weights ±SEM (n=6 mice) within each sex were compared to vehicle by 2-way ANOVA

2.15. Chronic dosing of Chol-DsiCTRL Polyplexes

All procedures were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee including guidelines for the humane treatment of animals. Female BALB/c mice, Chol-DsiCTRL alone, and Chol-DsiCTRL Polyplexes were prepared as described (Section 2.14). On Day 0, vehicle alone or vehicle containing Chol-DsiCTRL or Chol-DsiCTRL Polyplexes was injected (0.2 mL) biweekly into the tail vein of male (n=6 mice) and female BALB/c mice (n=10 mice) at the indicated dose of Chol-DsiCTRL/kg. Bodyweight was measured on each day of injection and 3 days after the final dose (9 doses over 28 days) and euthanized as described (Section 2.14). Toxicity was assessed by weight gain and terminal bodyweight, food/water consumption, histopathological evaluation of the thymus, lungs, liver, spleen, kidneys, and bone marrow, and a complete blood count (CBC) including platelet count, serum liver enzymes, creatinine, and BUN. Average values ±SEM (n=10 mice or less as indicated) of treatment groups were compared to vehicle by 2-way ANOVA where appropriate.

3. Results

3.1. Activity of Chol-DsiSTAT3 Polyplexes in murine syngeneic 4T1 breast cancer epithelial cells

Triple negative breast cancer (TNBC) is a subtype of breast cancer characterized by the absence of receptors for estrogen (ER), progesterone (PR), and human epidermal growth factor (HER2) that has the highest rates of growth, metastasis, and recurrence, and the worst stage-dependent prognosis ¹⁴. Although TNBC represents only 15% of new breast cancer diagnoses, it accounts for ~30% of breast cancer-related deaths (~150,000 deaths worldwide) and has fewer treatment options due to the absence of ER, PR, and HER2. ¹⁵

Several therapeutically relevant proteins have been identified in TNBC where suppression could potentially improve treatment outcomes. ¹⁶ One such protein, Signal Transducer and Activator of Transcription Factor 3 (STAT3), is a receptor tyrosine kinase (RTK)-associated signaling protein that is activated and upregulated by cytokines and growth factors found in the tumor microenvironment. ¹⁷ Receptor activation of STAT3 results in STAT3 dimerization, nuclear translocation, binding to STAT3-specific DNA binding elements, and subsequent transcription of genes involved in the regulation of cell differentiation, proliferation, apoptosis, angiogenesis, metastasis, and immune responses. ¹⁸ STAT3 is one of the main transcription factors involved in the immunomodulation of cancer ¹⁹ and elevated expression of activated STAT3 is associated with poor prognosis for breast cancer and many other solid tumors. ^{18, 20} Thus, given the role of STAT3 in breast and other solid tumors and presence of similar STAT proteins (STAT 1, 2, 4, 5, and 6) ²¹, there is great interest in specifically inhibiting the expression of STAT3.

The syngeneic murine breast cancer epithelial cell line, 4T1, is a good model for TNBC because it lacks ER, PR, and HER2, shares substantial molecular features with human TNBC ²², can be grown in immune competent female BALB/c mice, is poorly immunogenic, has rates of growth and patterns of metastasis that resemble human breast cancer, and the extent of late stage disease is comparable to stage IV breast cancer. ^{23, 24} STAT3 is also important in the 4T1 breast tumor model as inhibition of STAT3 by shRNA inhibits tumor formation and the frequency of spontaneous metastases. ²⁵ Delivery of siSTAT3 to 4T1 cells using membrane-penetrating peptides also inhibits 4T1 invasion and migration in vitro ²⁶ and intratumoral administration of shSTAT3-expressing plasmids inhibits primary tumor growth and spontaneous lung metastases. ²⁷

We previously found that Chol-DsiRNA Polyplexes increase the potency of Chol-DsiLUC against stably expressed luciferase in 4T1-Luc cells in vitro and as part of primary 4T1-Luc breast tumors after i.v. administration. ¹¹ Thus, we hypothesized that Chol-DsiRNA Polyplexes can increase the potency of Chol-DsiRNA against therapeutically relevant genes expressed in 4T1 cells such as STAT3.

To identify an active DsiSTAT3 sequence and an inactive DsiCTRL sequence against murine STAT3 mRNA in murine syngeneic breast cancer epithelial cells, we electroporated 4T1 cells with inactive DsiCTRL or murine DsiSTAT3 based on commercially available siSTAT3 sequences and compared normalized murine STAT3 mRNA copy numbers vs. electroporation-only 4T1 cells 24 hours after treatment by RT-ddPCR (Figure 2A). Electroporation with DsiSTAT3 decreased STAT3 mRNA copy numbers 81% below electroporation-only 4T1 cells (Figure 2A, grey bar), whereas electroporation with DsiCTRL had no effect (Figure 2A, white bar). Thus, the DsiSTAT3 sequence is sufficiently active and the DsiCTRL sequence is sufficiently inactive against murine STAT3 mRNA expression in murine syngeneic breast cancer epithelial cells.

Figure 2. Suppression of murine STAT3 mRNA by DsiSTAT3 and Chol-DsiSTAT3 Polyplexes in murine 4T1 breast cancer epithelial cells 24 hours after treatment.

4T1 cells were (A) electroporated alone or in the presence of inactive DsiCTRL (white bar) or active DsiSTAT3 (grey bar) [300 nM] then incubated at 37°C for 24 h or (B) incubated 4 h with serum-free Media alone or containing inactive Chol-DsiCTRL (white bar) or Chol-DsiSTAT3 (black bar) [200 nM] complexed with PLL[30]-PEG[5K] at an N/P ratio of 1 (50 wt% Chol-DsiRNA) before adding media containing 20% FBS at 1/1 (v/v) and incubating at 37°C for 20 h. Average murine STAT3 mRNA copy numbers per ng total RNA ± propagated SD (n= (A) 2 or (B) 3 replicates from two independent treatments) were then determined by RT-ddPCR, normalized to (A) electroporation-only 4T1 cells or (B) untreated 4T1 cells, respectively, and compared by two-tailed, unpaired t-test. Results are representative of at least two independent experiments.

To determine if Chol-DsiRNA Polyplexes increase the potency of Chol-DsiRNA against a therapeutically relevant target gene in murine syngeneic breast cancer epithelial cells, we treated 4T1 cells with Chol-DsiCTRL or Chol-DsiSTAT3 complexed with PLL[30]-PEG[5K] at N/P 1 (50 wt% Chol-DsiRNA) and compared normalized murine STAT3 mRNA copy numbers to untreated 4T1 cells 24 hours after treatment by RT-ddPCR (Figure 2B). Transfection with Chol-DsiSTAT3 Polyplexes decreased STAT3 mRNA copy numbers 64% below untreated 4T1 cells (Figure 2B, black bar), whereas transfection with Chol-DsiCTRL Polyplexes decreased STAT3 mRNA copy numbers by 6% (Figure 2B, white bar). These results indicate that STAT3 suppression in 4T1 cells is due primarily to complexed Chol-DsiSTAT3 and not potential non-specific activity of Chol-DsiRNA Polyplexes. Thus, Chol-DsiRNA Polyplexes increase the potency of Chol-DsiRNA against a therapeutically relevant target gene in murine syngeneic breast cancer epithelial cells.

3.2. Potency of Chol-DsiSTAT3 Polyplex activity in primary murine syngeneic 4T1 breast tumors after i.v. administration

We previously found that Chol-DsiLUC Polyplexes formed with PLL[30]-PEG[5K] at N/P 1 (50 wt% Chol-DsiLUC) suppress the luminescence of stably expressed luciferase in early-stage primary murine syngeneic 4T1-Luc breast tumors by ~78% after daily i.v. injections of 2.5 mg Chol-DsiLuc/kg over three days, whereas uncomplexed Chol-DsiLuc under the same dosage regimen has no effect. ¹¹ Daily injections at a constant dose, however, precluded determining the actual potency of Chol-DsiRNA Polyplexes in primary murine syngeneic breast tumors.

To determine the potency of Chol-DsiRNA Polyplexes against a therapeutically relevant target gene in primary murine syngeneic breast tumors after i.v. administration, we intravenously administered increasing doses of Chol-DsiSTAT3 (up to 5 mg/kg) or a single high dose of inactive Chol-DsiCTRL (5 mg/kg) complexed with PLL[30]-PEG[5K] at N/P 1 (50 wt% Chol-DsiRNA) and compared normalized murine STAT3 mRNA copy numbers in early-stage primary 4T1 breast tumors to treatment with vehicle alone by RT-ddPCR (Figure 3A). We chose a 48-hour timepoint given that (i.) Chol-DsiLUC Polyplexes maximally suppress the luminescence of stably expressed luciferase in primary murine 4T1 breast tumors 48 hours after the first daily i.v. injection of a three-day regimen ¹¹ and (ii.) luciferase activity closely matches luciferase mRNA and protein levels due the short intracellular half-life of the luciferase protein (~2 hours). ²⁸

Figure 3. Dose response and kinetics of STAT3 mRNA suppression in primary murine 4T1 breast tumors after i.v. administration of Chol-DsiSTAT3 Polyplexes.

(A) Vehicle alone (HEPES/0.15 M NaCl; 0.1 mL) or vehicle containing increasing doses of Chol-DsiSTAT3 (black circles) or a high dose of inactive Chol-DsiCTRL (white circle) complexed with PLL[30]-PEG[5K] at N/P ratio 1 (~50 wt% Chol-DsiRNA) was injected into the tail veins of female BALB/c mice bearing a single subcutaneous 4T1 tumor (~30 to 50 mm³) in the mammary fat pad. After 48 hours, average ratios of murine STAT3 to murine HPRT1 mRNA copies in primary 4T1 breast tumors ± propagated SD (n=5 mice) were determined by RT-ddPCR then normalized and compared to vehicle alone (0 mg Chol-DsiSTAT3/kg) by nonparametric Kruskal-Wallis One-Way ANOVA with Dunn’s post-test. A half maximal ED₅₀ was calculated from a nonlinear fit of the data. (B) Vehicle alone (HEPES/0.15 M NaCl; 0.1 mL) or vehicle containing Chol-DsiSTAT3 (0.5 mg/kg) complexed with PLL[30]-PEG[5K] at N/P ratio 1 (~50 wt% Chol-DsiRNA) was injected into the tail veins of female BALB/c mice bearing a single subcutaneous 4T1 tumor (~30 to 50 mm³) in the mammary fat pad. At the indicated time point after injection, average ratios of murine STAT3 mRNA copies to murine HPRT1 mRNA copies in primary 4T1 breast tumors ± propagated SD (n=5 mice) were determined by RT-ddPCR then normalized and compared to vehicle alone at the same time point by nonparametric Mann-Whitney Two-Tailed t-test. Results are representative of at least two independent experiments.

Chol-DsiSTAT3 Polyplexes (Figure 3A, black circles) suppressed STAT3 mRNA copy numbers in primary 4T1 breast tumors to a maximum of 54% vs. vehicle alone at the highest dose (5 mg Chol-DsiSTAT3/kg) with a half-maximal ED₅₀ of 0.3 ± 0.1 mg/kg, whereas inactive Chol-DsiCTRL Polyplexes had no effect at the highest dose of Chol-DsiSTAT3 Polyplexes (5 mg Chol-DsiCTRL/kg) (Figure 3A, white circle). These results indicate that the suppression of STAT3 mRNA expression in primary 4T1 tumors is due to the activity of complexed Chol-DsiSTAT3 and not potential non-specific effects of the Chol-DsiRNA Polyplexes. Thus, Chol-DsiRNA Polyplexes greatly increase the potency of Chol-DsiRNA against a therapeutically relevant target gene in primary murine syngeneic breast tumors after i.v. administration.

3.3. Kinetics of Chol-DsiSTAT3 Polyplex activity in primary murine syngeneic breast tumors after i.v. administration

We previously found that Chol-DsiLUC Polyplexes maximally suppress the luminescence of stably expressed luciferase in primary murine syngeneic 4T1-Luc breast tumors 48 hours after the first daily i.v. injection of a three-day regimen at 2.5 mg Chol-DsiLuc/kg and maintain near-maximal suppression at least 48 hours after the last injection. ¹¹ Multiple daily i.v. injections and the possibility of dose depot effects in the primary tumor by potential tumor dose saturation at 2.5 mg Chol-DsiLuc/kg, however, precluded determining the actual kinetics of Chol-DsiRNA Polyplex activity in primary murine syngeneic breast tumors.

To determine the kinetics of Chol-DsiSTAT3 Polyplex activity in primary murine syngeneic breast tumors after i.v. administration, we intravenously injected Chol-DsiSTAT3 complexed with PLL[30]-PEG[5K] at N/P 1 (50 wt% Chol-DsiSTAT3) and compared normalized STAT3 mRNA copy numbers in early-stage primary murine 4T1 breast tumors to vehicle alone every 24 hours over 96 hours (four days) by RT-ddPCR (Figure 3B). We chose an i.v. dose of Chol-DsiSTAT3 Polyplexes that did not maximally suppress STAT3 mRNA expression in primary 4T1 breast tumors (0.5 mg Chol-DsiSTAT3/kg) (Figure 3A, black circles) to exclude possible tumor dose saturation effects on the kinetics of Chol-DsiSTAT3 activity in the primary tumor.

Chol-DsiSTAT3 Polyplexes (Figure 3B, black circles) suppressed STAT3 mRNA copy numbers in primary 4T1 breast tumors to a maximum of 43% vs. vehicle alone after 48 hours but suppression decreased to 11% after 24 hours (72 hours post-injection). Thus, similar to Chol-DsiLuc Polyplex activity against the luminescence of stably expressed luciferase in primary 4T1-Luc breast tumors after the last daily i.v. injection of 2.5 mg Chol-DsiRNA/kg ¹¹, a single dose of Chol-DsiSTAT3 Polyplexes that is unlikely to saturate the primary tumor is maximally active in primary murine breast tumors 48 hours after i.v. administration but maintains maximal mRNA suppression less than 24 hours.

3.5. Activity of Chol-DsiSTAT3 Polyplexes against the growth of primary murine syngeneic breast tumors after i.v. administration

STAT3 is critical for the growth of 4T1 murine breast tumors. ^{25, 27, 29} Thus, given that Chol-DsiSTAT3 Polyplexes suppressed the expression of STAT3 mRNA in primary 4T1 tumors after i.v. administration (Figure 3), we expected that Chol-DsiSTAT3 Polyplexes would also inhibit 4T1 tumor growth.

To determine if Chol-DsiSTAT3 Polyplexes are therapeutically active against primary murine syngeneic breast tumors after i.v. administration, we intravenously injected Chol-DsiSTAT3 or inactive Chol-DsiCTRL complexed with PLL[30]-PEG[5K] at N/P 1 (50 wt% Chol-DsiRNA) [2.5 mg Chol-DsiRNA/kg] every other day to early-stage 4T1 breast tumor-bearing mice and compared tumor volumes to vehicle alone over 8 days by 3D surface scanning (Figure 4A, Figure S2). We chose 2.5 mg Chol-DsiRNA/kg because it was the lowest dose of Chol-DsiSTAT3 Polyplexes that provided maximal suppression of STAT3 mRNA expression in primary 4T1 breast tumors (Figure 3A, black circles).

Figure 4. Effect of Chol-DsiSTAT3 Polyplexes on primary murine 4T1 breast tumor growth and murine STAT3 protein expression after multiple i.v. treatments.

(A) Vehicle alone (white triangles) or vehicle containing Chol-DsiSTAT3 (black circles) or inactive Chol-DsiCTRL (Figure S2, white circles) complexed with PLL[30]-PEG[5K] at N/P ratio 1 (50 wt% Chol-DsiRNA) was injected into the tail veins of female BALB/c mice (black arrows) bearing a single subcutaneous 4T1 breast tumor. Average daily tumor volumes ±SD (n=5 mice) were then determined by 3D surface scanning and compared at each time point by Multiple t-tests. A rate-based T/C ratio and associated P value [11] were calculated as described (B) On Day 8 (48 h after final i.v. injection), steady-state murine Stat3 protein levels normalized to steady-state murine β-Actin protein levels in primary 4T1 tumors were determined by Western Blot and (C) average ratios of Stat3/β-Actin protein band intensities ±SD (n=2 measurements) were determined by imaging densitometry. Protein bands from images of the same Western blot were used to generate (B). Results are representative of at least two independent experiments.

Chol-DsiSTAT3 Polyplexes (Figure 4A, black circles) inhibited the growth of primary murine 4T1 breast tumors at a rate-based T/C ratio of 8.6% ¹² vs. vehicle alone (Figure 4A, white triangles), whereas inactive Chol-DsiCTRL Polyplexes (Figure S1, white circles) had no effect on the growth of primary murine 4T1 breast tumors vs. vehicle alone (Figure S1, white triangles) under the same dosage regimen. Consistent with STAT3 mRNA suppression (Figure 3A) and the inhibition of primary 4T1 breast tumor growth (Figure 4A, black circles), Chol-DsiSTAT3 Polyplexes suppressed 50% of total murine Stat3 protein in primary 4T1 breast tumors vs. vehicle alone 48 hours after the final i.v. dose (Day 8) (Figure 4B&C). Furthermore, average body weights of mice from all treatment groups gradually increased over the course of the study (not shown), indicating that (i.) tumor growth inhibition is due to the activity of complexed Chol-DsiSTAT3 and not animal weight loss and (ii.) Chol-DsiSTAT3 Polyplexes are not toxic under the current dosage regimen. Thus, given that the National Cancer Institute at NIH defines high anti-tumor activity as T/C ratios ≤10% at non-toxic doses ¹², Chol-DsiSTAT3 Polyplexes have high anti-tumor activity against primary murine syngeneic breast tumors after i.v. administration.

3.6. Pharmacokinetics of Chol-DsiSTAT3 and Chol-DsiSTAT3 Polyplexes in healthy female BALB/c mice after i.v. administration

DsiRNA must enter the cytosol of target cells to be cleaved by Dicer into siRNA and incorporated into a RISC complex before suppressing complementary mRNA. ³⁰ Thus, Chol-DsiRNA Polyplexes are expected to increase the potency of Chol-DsiRNA in primary syngeneic breast tumors after i.v. administration, in part, by affecting the pharmacokinetics and distribution of Chol-DsiRNA.

To first determine if Chol-DsiRNA Polyplexes formed with PLL[30]-PEG[5K] affect the pharmacokinetics of Chol-DsiSTAT3, we intravenously injected Chol-DsiSTAT3 alone or complexed with PLL[30]-PEG[5K] at N/P 1 (50 wt% Chol-DsiSTAT3) [2.5 mg Chol-DsiSTAT3/kg] into the tail veins of healthy female BALB/c mice. We then indirectly determined plasma concentrations of Chol-DsiSTAT3 over time as an increase in total RNA extracted from the plasma of treated vs. untreated mice (Figure 5) given that total RNA in plasma from untreated mice is relatively constant (not shown). The high variability of plasma concentrations and limited number of time points (Figure 5A&B) prevented complete and accurate compartmental analysis of the PK profiles. Despite the limited analysis, Chol-DsiRNA Polyplexes increased the AUC of Chol-DsiRNA 13-fold [39 ± 13 (SD) vs. 3 ± 1 h*μg/mL] (Table 1) and decreased clearance 8.3-fold [0.0012 ± 0.0003 (SD) vs. 0.01 ± 0.01 L/min/kg] (not shown). Thus, Chol-DsiRNA Polyplexes may increase the potency of Chol-DsiSTAT3 in primary syngeneic breast tumors after i.v. administration, in part, by increasing plasma exposure to Chol-DsiSTAT3.

Figure 5. Effect of Chol-DsiRNA Polyplexes on the pharmacokinetics of Chol-DsiSTAT3 healthy female BALB/c mice after i.v. administration.

(A) Chol-DsiSTAT3 alone or (B) complexed with PLL[30]-PEG[5K] at N/P ratio 1 (50 wt% Chol-DsiSTAT3) [2.5 mg Chol-DsiSTAT3/kg] was injected into the tail veins of healthy female BALB/c mice and average plasma concentrations of Chol-DsiSTAT3 ± SD (n = 5 mice) were determined indirectly at each time point as differences in total extracted RNA from the plasma of treated vs. untreated mice by fluorescence assay. Error bars are present in (A) but indistinguishable at the current scale.

Table 1.

Pharmacokinetic parameters of Chol-DsiSTAT3 and Chol-DsiSTAT3 polyplexes in healthy female BALB/c mice after IV administration.

PK Parameter	Chol-DsiSTAT3 (±SD)	Chol-DsiSTAT3 polyplexes (±SD)
C0	56 (64)	110 (7)
AUC0−∞ (h*μg/mL)	3 (1)	39 (13)
MRT0−∞ (h)	0.8 (0.9)	0.9 (0.4)

Average plasma concentration at time point = 0 (C₀) and mean residence time (MRT_0-∞) of Chol-DsiSTAT3 were determined by non-compartmental analysis (Phoenix WinNonlin) and average area under the curve (AUC_0-∞) of Chol-DsiSTAT3 (±SD from 5 mice) was determined by the linear log trapezoidal rule of the respective PK profiles (Figure 5).

3.7. Distribution of Chol-DsiSTAT3 and Chol-DsiSTAT3 Polyplexes in 4T1 breast tumor-bearing female BALB/c mice

To next determine if Chol-DsiSTAT3 Polyplexes affect the distribution of Chol-DsiSTAT3 to primary murine syngeneic breast tumors and normal tissues after i.v. administration, we intravenously injected Chol-DsiSTAT3 alone or complexed with PLL[30]-PEG[5K] at N/P 1 (50 wt% Chol-DsiSTAT3) [2.5 mg Chol-DsiSTAT3/kg] into the tail vein of primary 4T1 breast tumor-bearing female BALB/c mice and compared the distribution of Chol-DsiSTAT3 in perfused organs and tumors fifteen minutes post-injection by RT-ddPCR of the anti-sense strand of Chol-DsiSTAT3 (Figure 6). Chol-DiSTAT3 Polyplexes (Figure 6, black bars) increased the distribution of Chol-DsiSTAT3 2.7-fold to primary 4T1 tumors vs. uncomplexed Chol-DsiSTAT3 (Figure 6, white bars) [1.6 ± 0.4 (SD) vs. 0.6 ± 0.1 ng Chol-DsiSTAT3/mg tumor]. Consistent with increased tumor distribution, Chol-DsiRNA Polyplexes (Figure 6, black bars) also increased the distribution of Chol-DsiSTAT3 4-fold to the kidneys [4 ± 2 (SD) vs. 1.0 ± 0.4 ng Chol-DsiSTAT3/mg organ], 11.2-fold to the lungs [4.6 ± 0.6 (SD) vs. 0.41 ± 0.05 ng Chol-DsiSTAT3/mg organ], 9-fold to the liver [8.1 ± 0.9 (SD) vs. 0.9 ± 0.2 ng Chol-DsiSTAT3/mg organ], and 4.3-fold to the spleen [10 ± 2 (SD) vs. 2.3 ± 0.4 ng Chol-DsiSTAT3/mg organ] vs. uncomplexed Chol-DsiSTAT3 (Figure 6, white bars). Furthermore, although much lower overall levels were observed than in other tissues, Chol-DsiRNA Polyplexes increased the distribution of Chol-DsiSTAT3 4.6-fold to the brain [0.41 ± 0.05 (SD) vs. 0.088 ± 0.007 ng Chol-DsiSTAT3/mg tumor] and 9.3-fold to the heart [0.27 ± 0.04 (SD) vs. 0.029 ± 0.005 ng Chol-DsiSTAT3/mg tumor]. Thus, Chol-DsiRNA Polyplexes may increase the potency of Chol-DsiSTAT3 in primary syngeneic breast tumors after i.v. administration, in part, by increasing the distribution of Chol-DsiSTAT3 to the primary tumor.

Figure 6. Effect of Chol-DsiRNA Polyplexes on the distribution of Chol-DsiSTAT3 in 4T1 breast tumor-bearing mice after i.v. administration.

Chol-DsiSTAT3 alone [2.5 mg/kg] or complexed with PLL[30]-PEG[5K] at N/P ratio 1 (50 wt% Chol-DsiSTAT3) was injected into the tail veins of female BALB/c mice (black arrows) bearing single, orthotopic (mammary fat pad) 4T1 tumors (~30 to 50 mm³). After 15 minutes, the average ng of Chol-DsiSTAT3/mg tissue ± SD (n=5 mice) was determined by RT-ddPCR of the DsiSTAT3 antisense strand and compared in each tissue by nonparametric two-tailed t-tests with Holm-Sidak correction.

3.8. Dose escalation of Chol-DsiCTRL Polyplexes by i.v. administration

Chol-DsiRNA Polyplexes formed with PLL[30]-PEG[5K] significantly increase the potency of Chol-DsiLUC ¹¹ and Chol-DsiSTAT3 in primary murine syngeneic breast tumors after multiple daily i.v. injections at 2.5 mg/kg without affecting body weight gain over the course of the study. This suggests that Chol-DsiRNA Polyplexes are well tolerated after i.v. administration.

To determine a maximally tolerated dose (MTD) of Chol-DsiRNA Polyplexes in mice after i.v. administration, we intravenously injected healthy male and female BALB/c mice with vehicle alone or vehicle containing increasing doses of Chol-DsiCTRL alone or complexed with PLL[30]-PEG[5K] at N/P 1 (50 wt% Chol-DsiCTRL) up to 50 mg Chol-DsiCTRL/kg two times a week over 28 days (8 injections total) and compared body weights (Figure 7A, Table S1), terminal body and tissue weights (Table S2), the presence of liver cysts (Table S3), and histopathology of the liver, spleen, kidneys, and femurs after decalcification (Table S4). We focused on inactive Chol-DsiCTRL Polyplexes (Table 2) to separate the possibility of additional toxicity by the suppression of STAT3 by Chol-DsiSTAT3 Polyplexes.

Figure 7. Dose escalation and chronic dosing of Chol-DsiCTRL or Chol-DsiCTRL Polyplexes in healthy mice by i.v. administration.

(A) Vehicle alone (endotoxin-free PBS, 0.2 mL) or vehicle containing uncomplexed Chol-DsiCTRL or Chol-DsiCTRL complexed with PLL[30]-PEG[5K] at N/P 1 (50 wt% Chol-DsiCTRL) was injected biweekly into the tail veins of healthy male and female BALB/c mice at the indicated dose (black arrows) and average daily body weights ±SEM (n=6 mice) (Table S1) within each sex were compared to vehicle by 2-way ANOVA. Deaths: ^aVehicle (Day 18, one male, one female); ^bChol-DsiCTRL (Day 8, one male) (B) Vehicle alone (endotoxin-free PBS, 0.2 mL) or vehicle containing uncomplexed Chol-DsiCTRL or Chol-DsiCTRL complexed with PLL[30]-PEG[5K] at N/P 1 (50 wt% Chol-DsiCTRL) were injected into the tail veins of healthy female BALB/c mice biweekly at the indicated dose (black arrows). Average daily body weights ±SEM (n=10 mice) were compared to vehicle by 2-way ANOVA. Deaths: ^aChol-DsiCTRL (Day 14, one female); ^bChol-DsiCTRL Polyplexes [50 mg Chol-DsiCTRL/kg] (Day 3, one female, Day 14 two females, Day 21 one female).

Table 2.

Representative characteristics of Chol-DsiCTRL, PLL[30]-PEG[5K], and Chol-DsiCTRL polyplexes.

Sample	Endotoxin EU/mg (±SD)	Loading Chol-DsiCTRL wt%	Hydrodynamic Diameter nm (±SD)	Zeta potential mV (±SD)
Chol-DsiCTRL	0.104 (n.r.)a	N/A	N/A	N/A
PLL[30]-PEG[5K]	0.0171 (0.0008)b	N/A	N/A	N/A
Chol-DsiCTRL polyplexes (N/P 1)	0.023 (0.002)b	50	33 (2)c	5.2 (0.7)d

^aDetermined by manufacturer (IDT).

^bDetermined by Endochrome-K™ kit.

^cCalculated from a nonlinear fit of the lognormal distribution of Chol-DsiCTRL polyplex diameters in 0.1 M HEPES, pH 7.4 (Figure S2).

^dDetermined in 0.1 M HEPES, pH 7.4.

Male (Figure 7A, black symbols) and female mice (Figure 7A, white symbols) treated with vehicle alone (Figure 7A, triangles), Chol-DsiCTRL alone (Figure 7A, circles), or Chol-DsiCTRL Polyplexes (Figure 7A, circles) continued to gain weight up to four days after the highest dose [50 mg Chol-DsiCTRL/kg] despite deaths with vehicle alone (Day 18: one male and one female) and Chol-DsiCTRL alone (Day 8: one male). Male and female mice from all treatment groups also had similar terminal body weights (Table S2).

Dose escalation with Chol-DsiCTRL increased relative liver weights by 10% in male mice and decreased total and relative liver weights by ~8% in female mice and compared to vehicle alone but was not associated with histopathologic changes. Chol-DsiCTRL Polyplexes also increased total and relative spleen weight by ~37% in male mice and decreased total and relative liver weights by 5 to 7% compared to vehicle alone (Table S2). The liver did not show any histopathologic changes and the spleens only showed differences in blood congestion but without abnormalities in the lymphoid structures.

Chol-DsiCTRL Polyplexes had the highest number of surviving male and female mice with liver cysts (4 out of 6) but was similar to female mice treated with Chol-DsiCTRL (3 out of 6) and both male (3 out of 5) and female mice (2 out of 5) treated with vehicle alone (Table S3). These cysts, however, commonly occur in BALB/c mice and were of similar size and frequency between treatment groups. Furthermore, most liver cysts were microscopic in size and differences in numbers between treatment groups may have been due to sampling. As such, cystic changes in the liver are not considered to be treatment related.

Liver, spleen, kidney, and femur histology was normal in male and female mice from all treatment groups with occasional mice having changes that are common in this strain of mice but without differences between groups, thus not considered treatment related (Table S4). Two deaths occurred with vehicle alone (Figure 7A, Day 18: one male and one female) and one death occurred with Chol-DsiCTRL (Figure 7A, Day 8, one male), whereas no deaths occurred with Chol-DsiCTRL Polyplexes, suggesting that deaths were likely caused by the stress of multiple i.v. injections. Thus, i.v. administration of Chol-DsiCTRL alone or complexed with PLL[30]-PEG[5K] at N/P 1 appears to be well tolerated by healthy male and female mice to at least 50 mg Chol-DsiCTRL/kg.

3.7. Chronic dosing with Chol-DsiCTRL Polyplexes by i.v. administration

To assess the toxicity of Chol-DsiRNA Polyplexes in mice after chronic dosing by i.v. administration, we intravenously injected healthy female BALB/c mice with vehicle alone or vehicle containing uncomplexed Chol-DsiCTRL at 50 mg/kg or Chol-DsiCTRL complexed with PLL[30]-PEG[5K] at N/P 1 (50 wt% Chol-DsiCTRL) at 25 and 50 mg Chol-DsiCTRL/kg biweekly over 28 days (9 injections total) and compared body weights (Figure 7B, Table S5), food and water consumption (Table S6), terminal body and tissue weights (Table S7), histopathology of major organs and tail vein injection sites (Table S8), and hematology (Table S9).

Mice chronically treated with vehicle alone (Figure 7B, white triangles), uncomplexed Chol-DsiCTRL (Figure 7B, white circles), or Chol-DsiCTRL Polyplexes at 25 mg Chol-DsiCTRL/kg (Figure 7B, black/white squares) or 50 mg Chol-DsiCTRL/kg (Figure 7B, black squares) continued to gain weight up to four days after the last dose despite deaths with 50 mg/kg Chol-DsiCTRL alone (Day 14) and 50 mg/kg Chol-DsiCTRL Polyplexes (Days 3, 14 [2 mice], 21). Mice from all treatment groups also had similar rates of food and water consumption (Table S6) and terminal body weights (Table S7).

Chronic i.v. administration of Chol-DsiCTRL Polyplexes increased total and relative spleen weights by 22% at 25 mg Chol-DsiCTRL/kg and 29% at 50 mg/Chol-DsiCTRL/kg and increased total and relative liver weights by 10% at 50 mg Chol-DsiCTRL/kg compared to vehicle alone (Table S7) although no histological abnormalities were observed in the lungs, liver, kidneys, spleen, brain, thymus, or femurs of any treatment group compared to vehicle alone. There were occasional animals in each group with minimal changes commonly seen in BALB/c mice and were, consequently, not considered treatment related. Changes in the tails at the injection sites were similar between groups with changes ranging from mild chronic inflammation to ulceration with acute inflammation, thus related to the repeated injections and not the administered material (Table S8).

WBC, hemoglobin, hematocrit, lymphocytes, and platelets from all treatment groups were also within normal values in BALB/c mice (Charles River Laboratories) (Table S9) and trauma observed around tail injection sites was consistent with damage from the injection itself (not shown). Furthermore, Chol-DsiRNA Polyplex endotoxin levels at 50 mg Chol-DsiCTRL/kg (0.023 EU/mg Chol-DsiCTRL Polyplexes, Table 2) were below maximum FDA limits for i.v. administration to a 20 g mouse per hour (0.1 EU/mg drug) ³¹, indicating that deaths were likely caused by the stress of multiple i.v. injections and not the administered material or levels of endotoxin. Thus, chronic i.v. administration of Chol-DsiRNA alone or complexed with PLL[30]-PEG[5K] is well tolerated by healthy female mice up to at least 50 mg Chol-DsiCTRL/kg.

4. Discussion

Our study provides evidence that Chol-DsiRNA Polyplexes formed with PLL[30]-PEG[5K] are highly potent in solid primary murine syngeneic breast tumors and well tolerated after i.v. administration. We found that Chol-DsiRNA Polyplexes (i.) greatly increase the potency (half-maximal ED₅₀ of 0.3 ± 0.1 mg Chol-DsiRNA/kg) (Figure 3A) and therapeutic activity (rate-based T/C ratio of 8.6%) (Figure 4A) of Chol-DsiRNA against an endogenous, therapeutically relevant target gene (STAT3) in early-stage primary murine 4T1 breast tumors after i.v. administration and (ii.) are well tolerated as an inactive dosage form (i.e., not targeting a specific mRNA) to at least 50 mg Chol-DsiCTRL/kg by healthy male and female BALB/c after i.v. dose escalation over 28 days (8 injections total, 2 per week) (Figure 7A, Tables S1–S4) and by healthy female BALB/c after chronic i.v. administration over 28 days (9 injections total, 2 per week) (Figure 7B, Tables S5–S9).

Our study also provides evidence that Chol-DsiRNA Polyplexes formed with PLL[30]-PEG[5K] may increase the potency of Chol-DsiRNA in primary murine syngeneic tumors, in part, by increasing plasma exposure and subsequent localization to the tumor. We found that Chol-DsiRNA Polyplexes increase the AUC_0−∞ of Chol-DsiSTAT3 in plasma 13-fold (Figure 5, Table 1) and distribution to primary 4T1 breast tumors 2.7-fold (Figure 6) vs. uncomplexed Chol-DsiSTAT3 after i.v. administration.

We previously found that Chol-DsiLUC Polyplexes suppress luciferase activity (indirect measure of LUC mRNA) in primary 4T1-Luc breast tumors to a maximum of 77% after a daily i.v. dose of 2.5 mg Chol-DsiLuc/kg over three days and maintained near-maximal suppression at least 48 hours after the final dose. ¹¹ In contrast, Chol-DsiSTAT3 Polyplexes suppressed STAT3 mRNA in primary 4T1 tumors a maximum of ~54% after a single dose of 5 mg Chol-DsiSTAT3/kg and maintained maximal suppression less than 24 hours after a single dose of 0.5 mg Chol-DsiSTAT3/kg (Figure 3B).

There are several possible reasons that may individually or collectively explain why Chol-DsiRNA Polyplexes suppress STAT3 mRNA to a lesser extent and shorter duration than stably expressed LUC mRNA in 4T1 breast tumors. The first possibility is that multiple dosing with Chol-DsiLUC Polyplexes (a daily dose of Chol-DsiLUC Polyplexes over three days vs. one dose of Chol-DsiSTAT3 Polyplexes) at a higher dose (2.5 mg Chol-DsiLUC/kg vs. 0.5 mg Chol-DsiSTAT3/kg) may saturate primary 4T1 tumors and, consequently, increase the extent and duration of LUC mRNA suppression vs. STAT3 mRNA suppression. The second possibility is that 4T1 cells may be more easily transfected by Chol-DsiRNA Polyplexes than other cells within the primary 4T1 breast tumor given that LUC mRNA is only expressed in stably transfected 4T1-Luc cells within 4T1-Luc tumors, whereas STAT3 is expressed in 4T1 cells as well as tumor-associated cells (e.g., mammary gland tissue) ³² and tumor-infiltrating cells. ¹⁹ Consistent with the second possibility, the third possibility is that steady-state levels of endogenous STAT3 mRNA in 4T1 breast tumors are higher than stably expressed LUC mRNA in 4T1-Luc breast tumors, in part, due to higher transcription rates of STAT3 mRNA caused by cytokine and growth factor signaling within the tumor microenvironment, a longer intracellular half-life ³³, and the additional expression of STAT3 mRNA by tumor-associated and tumor-infiltrating cells. Thus, initial steady-state levels of STAT3 mRNA may be more difficult to overcome, less accessible to Chol-DsiRNA Polyplexes, and persist longer in primary 4T1 tumors compared to steady-state levels of LUC mRNA in primary 4T1-Luc tumors. The fourth possibility is that using fewer 4T1 cells to form primary 4T1 breast tumors (1 × 10⁶ 4T1-Luc cells in the previous study ¹¹ vs. 1 × 10⁵ 4T1 cells in the current study to facilitate 3D surface scanning) decreases tumor vascularization and subsequent accessibility to cells within 4T1 tumors. This difference in the number of 4T1 cells used for tumor inoculation, however, is unlikely to affect tumor vascularization given that similar levels of tumor vascularization and necrosis are observed in primary 4T1 breast tumors after inoculation between 500 to 1 × 10⁶ 4T1 cells. ³⁴ These possibilities will be resolved in the future by directly comparing the activities of Chol-DsiRNA Polyplexes and Chol-siRNA Polyplexes against the same mRNA target in the same tumor model under the same dosage regimen.

In summary, our results indicate that Chol-DsiRNA Polyplexes formed with PLL[30]-PEG[5K] greatly increase the potency of Chol-DsiRNA molecules in primary murine syngeneic breast tumors and are well tolerated after i.v. administration. Thus, Chol-DsiRNA Polyplexes may be a good candidate for Phase I clinical trials of RNAi-mediated therapeutic approaches in breast cancer and other solid tumors.

Abbreviations

4T1

Syngeneic murine breast cancer epithelial cells / TNBC model cells

4T1-Luc

4T1 cells that stably express luciferase

Chol-DsiRNA

DsiRNA modified with 3’-cholesterol (sense strand)

Chol-*siRNA

Nuclease-resistant siRNA modified with 3’-cholesterol (sense strand)

DsiRNA

Dicer-Substrate siRNA

miRNA

MicroRNA

N/P Ratio

Charge molar ratio of positively charged amines from the polymer to negatively charged phosphates from the RNAi molecule

PLL[n]-PEG[5K]

Diblock copolymer of n poly-L-lysine residues and 5 kDa polyethylene glycol

Polyplexes

Polymer complexes

RNAi

RNA interference

siRNA

Small, interfering RNA

STAT3

Signal Transducer and Activator of Transcription Factor 3