Preliminary preclinical study of Chol-DsiRNA polyplexes ...

Nanomedicine: Nanotechnology, Biology, and Medicine33 (2021) 102363

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NANO-0000102363; No of Pages 14

Preliminary preclinical study of Chol-DsiRNA polyplexes formed withPLL[30]-PEG[5K] for the RNAi-based therapy of breast cancer

Zhen Ye, PhDb, Mai Mohamed Abdelmoaty, MScb,c, Vishakha V. Ambardekar, PhDb,1,Stephen M. Curran, MScb, Shetty Ravi Dyavar, PhDd, Lora L. Arnoldf,

Samuel M. Cohen, MD, PhDe, f, Devendra Kumar, PhDb, Yazen Alnouti, PhDb,Don W. Coulter, MDg, Rakesh K. Singh, PhDa, f, Joseph A. Vetro, PhDa,b,⁎

aCenter for Drug Delivery and Nanomedicine, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE, USAbDepartment of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE, USA

cTherapeutic Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, Giza, EgyptdDepartment of Pharmacy Practice, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE, USA

eHavlik-Wall Professor of Oncology, University of Nebraska Medical Center, Omaha, NE, USAfDepartment of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, USA

gDepartment of Pediatrics, Division of Pediatric Hematology/Oncology, Department of Radiation Oncology, J. Bruce Henriksen Cancer ResearchLaboratories, University of Nebraska Medical Center, Omaha, NE, USA

Revised 28 December 2020

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 thecationic diblock copolymer PLL[30]-PEG[5K] greatly increase the activity of Chol-DsiRNA against a stably expressed reporter mRNA inprimary 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 increaseplasma 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 trialsto improve the treatment of breast cancer and other solid tumors.© 2021 Elsevier Inc. All rights reserved.Key words: RNA interference; DsiRNA; Drug delivery; Chol-DsiRNA polymer micelles; Chol-siRNA polymer micelles

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 thepolymer to negatively charged phosphates from the RNAi molecule; PLL[n]-PEG[5K], diblock copolymer of n poly-L-lysine residues and 5 kDapolyethylene glycol; Polyplexes, polymer complexes; RNAi, RNAinterference; siRNA, small, interfering RNA; STAT3, signal transducerand activator of transcription factor 3.Declaration of competing interests: JAV is cofounder of a company thatlicenses the described technology.

⁎ Corresponding author at: 986025 Nebraska Medical Center, WSH3026A, Omaha, Nebraska.E-mail address: [email protected] (J.A. Vetro).

1 Current address: Lupin Ltd., 46/47, A, Village Nande, Taluka MulshiDist. Pune, India.

https://doi.org/10.1016/j.nano.2021.1023631549-9634/© 2021 Elsevier Inc. All rights reserved.

RNA interference (RNAi) is a natural, intracellular processthat selectively decreases the expression of any specific proteinat the mRNA level through the complementary binding of gene-specific dsRNA molecules including microRNA (miRNA);small, interfering RNA (siRNA); or longer dicer-substratesiRNA (DsiRNA) (“RNAi molecules”).1 Several proteins havebeen identified in tumor or tumor-associated cells wheresuppression can produce a therapeutic effect and/or increasethe efficacy of current cancer treatments.2 Thus, RNAimolecules have tremendous potential in the treatment of cancer.

Many cancer therapy applications require i.v. administrationto achieve a therapeutic effect. The potencies of RNAi moleculesafter i.v. administration, however, are extremely low orundetectable.1 Several nanoscale dosage forms have beendeveloped to improve the potency of RNAi molecules after i.v.administration using a wide range of nanomaterials and/or

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2 Z. Ye et al / Nanomedicine: Nanotechnology, Biology, and Medicine 33 (2021) 102363

modified RNAi molecules.3,4 Identifying nanomaterials and/orRNAi molecule modifications that increase the potency ofmRNA suppression in primary tumors and metastases and aresuitable for clinical application, however, remains a criticalbarrier to developing nanoscale dosage forms for RNAi-basedtherapies.

Conjugating 3′-cholesterol to nuclease-resistant siRNA(Chol-*siRNA) increases siRNA suppression of an endogenousmRNA in the liver and jejunum after i.v. administration but withrelatively low potency (50 mg Chol-*siRNA/kg).5 We previ-ously found that Chol-siRNA polymer complexes (Chol-siRNApolyplexes) formed with a block copolymer of 10 poly-L-lysineresidues and 5 kDa polyethylene glycol (PLL[10]-PEG[5K])increase Chol-siRNA suppression of a native mRNA (CYPB) inmurine mammary MVEC (~90% suppression) to a much greaterextent than Chol-siRNA polyplexes formed with a blockcopolymer of 50 poly-L-lysine residues and 5 kDa polyethyleneglycol (PLL[50]-PEG[5K]) (30% suppression), biodegradablenanogels (40% suppression), or a graft copolymer of 2 kDabranched PEI and 10 kDa PEG (30% suppression) withoutaffecting cell viability.6 Given that PLL block length affectsChol-siRNA polyplex activity and nuclease protection in vitro,we next directly compared Chol-*siRNA polyplexes formedwith 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 extentagainst a stably expressed reporter gene (luciferase) in primarymurine syngeneic 4T1 breast tumors (4T1-Luc) after daily i.v.administration at 2.5 mg Chol-*siLUC/kg over three dayswithout affecting body weight.7 Similar increases in RNAipotency using PEGylated diblock copolymers and hydrophobi-cally modified siRNA were later independently reported inprimary ectopic human xenogeneic HeLa cervical tumors withtargeted, core-crosslinked “Chol-siRNA micelles” composed ofconventional Chol-siRNA (5′-cholesterol conjugated to theantisense strand) and integrin-targeted PLL[45]-b-PEG[12 K]-cRGD diblock copolymers8 and in primary human xenogeneicMDA-MB-231 breast tumors with polyplexes composed ofpalmitic acid-modified siRNA (PA-siRNA) and more hydro-phobic p(DMAEMA-co-BMA)- b -PEG[5K] diblockcopolymers.9 Thus, nanoscale dosage forms composed ofhydrophobically-modified RNAi molecules complexed withPEGylated diblock copolymers have the potential to increasethe potency of RNAi molecules in solid tumors and metastasesafter i.v. administration.

Although Chol-siRNA polyplexes formed with PLL[30]-PEG[5K] increase the potency of Chol-*siLUC in primarymurine 4T1-Luc breast tumors after i.v. administration, LUCmRNA suppression decreases 24 h after the last treatment.7

Given that polyplexes of dicer substrate siRNA (DsiRNA) andLipofectamine® 2000 have 100-fold higher potency and longerduration of mRNA suppression in HEK293 cells in vitro thanpolyplexes of siRNA and Lipofectamine® 200010, we nextcompared 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 theduration of LUC mRNA suppression in primary murine 4T1-Lucbreast tumors after i.v. administration ~48 h longer than Chol-

*siLUC polyplexes under the same dosage regimen. Theincreased duration of RNAi activity, however, is likely due todifferences in the activities of Chol-DsiRNA polyplexes andChol-siRNA polyplexes in primary 4T1-Luc tumors after i.v.administration and not differences in the activities of DsiRNAand siRNA in 4T1 cells as electroporation of 4T1-Luc cells withequimolar amounts of DsiLUC or siLUC suppresses luciferaseactivity to the same extent over 72 h.11 Chol-DsiRNApolyplexes also have higher loading (50 wt% Chol-DsiRNAvs. 25 wt% Chol-siRNA) and almost fully protect Chol-DsiRNAfrom degradation in 90% murine serum for 24 h at 37 °C withoutexpensive nuclease-resistance modifications to DsiRNA.11

These results suggest Chol-DsiRNA polyplexes are a morepromising lead formulation than Chol-siRNA polyplexes toincrease the potency and sustain the activity of RNAi moleculesin solid breast tumors after i.v. administration.

In this study, we provide a more thorough preliminarypreclinical assessment of Chol-DsiRNA polyplexes formed withPLL[30]-PEG[5K] against the therapeutically relevant targetgene STAT3 in primary murine syngeneic breast tumors after i.v.administration by determining (i) Chol-DsiSTAT3 pharmacoki-netics, distribution, half-maximal ED50, and kinetics of STAT3mRNA suppression in primary 4T1 breast tumors; (ii) rate-basedT/C ratio of Chol-DsiSTAT3 polyplex activity against primary4T1 breast tumor growth; and (iii) toxicity of inactive Chol-DsiCTRL polyplexes in healthy male and female BALB/c miceafter dose escalation to 50 mg Chol-DsiCTRL/kg and in healthyfemale BALB/c mice after chronic administration at 50 mgChol-DsiCTRL/kg over 28 days.

Methods

Polymer

Methoxy-poly(ethylene glycol)-b-poly(L-lysine hydrochlo-ride) with a 5 kDa polyethylene glycol block (PEG[5K]) and a30 poly-L-lysine block (PLL [30]) [PLL[30]-PEG[5K]; avg. MW9900 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 theentire polymer was between 1 and 1.1.

RNAi molecules

DsiRNA was HPLC-purified, asymmetric, 25-mer dsRNA with2-nucleotide UU overhangs on the 3′ end of the antisense strand: (1)DsiCTRL (GEDharmacon) sense: 5′-CGUUAAUCGCGUAUAAUA CGC GUA U-3′, antisense: 5′-AUA CGC GUA UUA UACGCG AUU AAC G(UU)-3′; MW: 16,523.02 g/mol; sequencebased 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 siGENOMEMouse 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) DsiSTAT3modified with 3′-cholesterol as described for Chol-DsiCTRL; MW:17,197.92 g/mol.

Figure 1. Idealized electrostatic self-assembly of Chol-DsiRNA polyplexes formed with PLL[30]-PEG[5K]. A solution of negatively-charged DsiRNA (red)modified with 3′-cholesterol (brown) on the sense strand (Chol-DsiRNA) is added to a solution of positively-charged 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-chargedprimary amines (N)/moles of negatively charged phosphates). The negatively-charged phosphate backbones of Chol-DsiRNA then electrostatically bind andneutralize the positively-charged PLL [30] blocks, converting PLL[30]-PEG[5K] unimers into amphiphilic diblock copolymers that spontaneously self-assembleinto Chol-DsiRNA polymer complexes (polyplexes) that are further stabilized by hydrophobic interactions between 3′-cholesterol groups. A similar idealizedself-assembly at slightly higher N/P ratio is envisioned for Chol-siRNA polyplexes formed with PLL[30]-PEG[5K].

3Z. Ye et al / Nanomedicine: Nanotechnology, Biology, and Medicine 33 (2021) 102363

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

Cell culture

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

Electroporation of murine 4T1 cells

Murine 4T1 cells [10 × 106 cells/mL] were electroporated(Nucleofector, Lonza AG, Bazel, Switzerland) with the indicatedDsiRNA [300 nM] using Amaxa Cell Line Kit V for MouseEpithelial Cells (VCA-1003, Lonza) as directed (Setting T-024)and cultured in 6-well plates [1 × 106 cells/well]. Total RNAwas 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 independentelectroporations) were determined by RT-ddPCR (Section 2.9),

normalized to electroporated 4T1 cells alone at the indicated timepoints, and compared by two-tailed, unpaired t test.

Formation of Chol-DsiRNA polyplexes

The concentration of a 2× Polymer Complexation Solution atan N/P charge molar ratio of one mole positively-chargedprimary amines (N) from the PLL [30] block of PLL[30]-PEG[5K] to one mole negatively-charged phosphates (P) from thephosphate backbone of Chol-DsiRNA (N/P 1)11 was calculatedbased on the concentration of the 2× Chol-DsiRNA Complex-ation Solution

2� Polymer Complexation Sol 0ng

mL

� �

¼ g RNAi

mL� mol RNAi

MW RNAi�mol RNAi P

mol RNAi

�mol Polymer N

mol RNAi P� mol Polymer

mol Polymer N�MW Polymer

mol Polymer

where g RNAi/mL = concentration of 2× Chol-DsiRNA Com-plexation Solution (double the desired concentration of Chol-DsiRNA in the final Chol-DsiRNA Polyplex solution), MWRNAi = MW of Chol-DsiRNA, mol RNAi P/mol RNAi = 52 molphosphate per mol Chol-DsiRNA, mol Polymer N/mole RNAiP = 1 (N/P 1 charge molar ratio), mol Polymer/mol PolymerN = 1 mol of PLL[30]-PEG[5K]/30 mol PLL primary amines,MW Polymer/mol Polymer = MW of PLL[30]-PEG[5K] =9900 Da.

For transfection studies, a concentrated stock solution ofChol-DsiRNA polyplexes at N/P 1 was prepared by (i) sterilizingPLL[30]-PEG[5K] in open, sterile 2 mL centrifuge tubes undervacuum for 2 h in a desiccator containing a glass dish of 99%ethanol; (ii) preparing a 2× Chol-DsiRNA Complexation


Solution plus 10 μL for experimental loss by diluting a thawedfrozen stock of Chol-DsiRNA with sterilized (0.2 μm filter)HEPES Buffer [0.1 M HEPES in deionized H2O, pH 7.4] totwice the desired concentration of Chol-DsiRNA in the finalConcentrated Stock Solution; (iii) preparing a 2× PolymerComplexation Solution plus 10 μL for experimental loss bydissolving sterilized PLL[30]-PEG[5K] in HEPES buffer at1 mg polymer/mL, incubating at r.t. for 30 min, and dilutingwith HEPES Buffer to the calculated 2× concentration; (iv)forming a Concentrated Stock Solution of Chol-DsiRNApolyplexes by adding the 2× Chol-DsiRNA ComplexationSolution dropwise to the 2× Polymer Complexation Solution at1/1 (v/v), mixing the solution by pipette aspiration/dispensation(30 s), and incubating (RT, 30 min).

For in vivo studies, endotoxin-free solutions/reagents wereused where possible. A Concentrated Stock Solution of Chol-DsiRNA alone or Chol-DsiRNA polyplexes at N/P 1 wasprepared on the day of injection by (i) determining the totalvolume of Concentrated Chol-DsiRNA Stock Solution (Chol-DsiRNA alone and Chol-DsiRNA polyplexes) required for allmice in the study based on the injected dose (mg/kg), individualmass of each mouse, and total prepared i.v. injection volume(125 μL for dose response/kinetics studies or 225 μL for toxicitystudies per mouse) plus 50 μL for experimental loss; (ii)preparing a 2× Chol-DsiRNA Complexation Solution (1/2 totalrequired volume of Concentrated Stock Solution of Chol-DsiRNA) plus 25 μL for experimental loss by resuspendinglyophilized Chol-DsiRNA as directed by the manufacturer insterilized HEPES buffer to twice the desired concentration ofChol-DsiRNA in the Concentrated Stock Solution; (iii) steriliz-ing PLL[30]-PEG[5K] and preparing 2× Polymer ComplexationSolution (same volume as 2× Chol-DsiRNA ComplexationSolution) as described for transfection studies above; (iv)preparing the required volume of Concentrated Stock Solutionof Chol-DsiRNA polyplexes as described above and (v)preparing the required volume of Concentrated Stock Solutionof Chol-DsiRNA alone by diluting the 2× Chol-DsiRNAComplexation 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 eachmouse was then prepared by combining (i) volume ofConcentrated Chol-DsiRNA or Chol-DsiRNA Polyplex StockSolution adjusted to deliver the required dose of Chol-DsiRNAbased on mouse bodyweight, (ii) volume of sterilized (0.2 μmfilter) HEPES buffer plus 1.5 M NaCl at 1/10 dilution [0.15 MNaCl final conc.], and (iii) volume of HEPES buffer adjusted tofinal volume.

Hydrodynamic diameter and zeta potential of Chol-DsiCTRLpolyplexes

The average hydrodynamic diameter of Chol-DsiCTRLpolyplexes (N/P 1) was determined by nanoparticle trackinganalysis (NanoSight LM10 and NTA 2.3 analytical software,Malvern Instruments, UK). Chol-DsiRNA polyplexes [0.25 mgChol-DsiSTAT3/mL] were prepared as described for Transfec-tion (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]); remainingparameters: auto). The average estimated concentration of Chol-DsiRNA polyplexes for each 1 nm bin from three independentanalyses was normalized as a percentage of the total averageestimated concentration of Chol-DsiRNA polyplexes. A plot ofaccumulated percent of total Chol-DsiRNA polyplexes at eachdiameter (y-axis) vs. ln diameter (x-axis) was then fit against acumulative Gaussian (percent) model using GraphPad Prism 9 todetermine a best-fit mean and standard deviation from thelognormal curve.

The average zeta potential of Chol-DsiCTRL polyplexes (N/P1) 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 samebatch with 12 Zeta runs per sample) using a ZetaSizer Nano ZA(Malvern Instruments, Malvern, UK) equipped with a He-Nelaser (λ = 633 nm) as the incident beam.

Endotoxin levels

Chol-DsiCTRL endotoxin levels were quantitated by themanufacturer (IDT). PLL[30]-PEG[5K] [0.2 mg/mL] and Chol-DsiRNA polyplexes [0.05 mg Chol-DsiSTAT3/mL] endotoxinlevels were quantitated using an Endochrome-K LimulusAmebocyte Lysate Kit (Charles River Labs) as directed withendotoxin-free reagents and disposable labware under asepticconditions.

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 to16 h before transfection. On the day of transfection, aconcentrated 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-1640lacking FBS and antibiotics. Old growth media were aspiratedand diluted polyplexes or Complete RPMI-1640 lacking FBSand antibiotics (1.5 mL) were added for 4 h; then an equalvolume of 20% FBS Complete RPMI-1640 (1.5 mL) was addedbefore further incubation for 20 h (24 h from the start oftransfection). Average murine STAT3 mRNA copy numbers perng total RNA ± propagated SD (n = 3 replicates from twoindependent treatments) were then determined by RT-ddPCR(Section 2.9), normalized to untreated cells, and compared bytwo-tailed, unpaired t test.

Quantitation of STAT3 mRNA and Chol-DsiSTAT3 by ReverseTranscription-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 Technolo-gies). The number of STAT3 mRNA copies was determined bydroplet-digital PCR (ddPCR) (QX200 Droplet Digital PCRSystem, BioRad) from 10 ng of template cDNA usingPrimeTime® Taqman assays (IDT, Coralville, USA) for murineSTAT3 mRNA and BioRad ddPCR reagents/consumables asdirected. STAT3 mRNA copies were then normalized to themass 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, Invitro-gen™, ThermoFisher). Copy numbers of Chol-DsiSTAT3were quantitated by ddPCR using two different amounts oftemplate cDNA [tumor: 6.67, 33.5 ng; lungs: 6.67, 33.35 ng(Chol-DsiSTAT3 alone), 1.334, 13.4 ng (Chol-DsiSTAT3polyplexes); 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-loopquantitative Custom TaqMan Small RNA Assays (Invitrogen™,ThermoFisher) for the antisense strand of Chol-DsiSTAT3, andBioRad ddPCR reagents/consumables as directed.

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

Tumor growth delay of primary murine 4T1 breast tumors

All procedures were approved by the University of NebraskaMedical Center Institutional Animal Care and Use Committeeincluding guidelines for the humane treatment of animals.Primary tumors were prepared in female BALB/c mice asdescribed7 but hair was first removed from the region above thetumor cell inoculation site (mammary fat pad #4) by shaving/applying Nair cream (removed with warm water and gauze after30 s then rinsed with 70% ethanol) and 10-fold fewer 4T1 cells[5 × 105 4T1 cells total] were injected SQ to facilitate tumorvolume measurements by 3D surface scanning (TumorImager,Biopticon). Tumor volumes and body weights were measuredevery other day before treatment, then daily until 48 h after thelast treatment.

When tumor volumes reached 30 to 50 mm3 (day 0), vehiclealone [0.1 M HEPES, 0.15 M NaCl, pH 7.4, filter-sterilized] orvehicle containing the indicated Chol-DsiRNA polyplexes wasinjected (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 = 5mice) were compared at each time point by multiple t tests. Arate-based T/C ratio and associated P value12 were calculated asdirected using the author-provided excel spreadsheet. On day 8(48 h after final dose), tumors were isolated and stored at −80 °C,and Stat3 protein levels were determined by Western blot(Section 2.11).

Quantitation of Stat3 protein in primary 4T1 tumors by Westernblot

Total protein was extracted from thawed tumors using CellExtraction Buffer (Invitrogen) containing 1 mM PMSF, 1×protease inhibitor cocktail, and 1× EDTA (Thermo Scientific)and quantified using Pierce BCA protein assay (ThermoScientific) as directed. Protein lysates [40 μg] were resolved bySDS-PAGE (Mini-PROTEAN TGX Precast Protein Gels, Bio-

Rad) and transferred (Mini Trans-Blot® Cell, BioRad) to PVDFmembranes as directed. PVDF membranes were incubated withrocking in blocking buffer [5% (w/v) nonfat dry milk in TBSTbuffer] (r.t., 1 h), rinsed with TBST (r.t., 5 s), incubated withprimary antibodies (β-actin [sc-47778] and Stat3 [sc-8019],Santa Cruz Biotechnology) [1:200 in TBST] (4 °C, overnight),rinsed 3× with TBST (r.t., 5 min), and incubated with secondaryantibody (horseradish peroxidase-conjugated mouse IgGκ bind-ing protein; m-IgGκ BP-HRP [sc-516102], Santa Cruz Biotech-nology) [1:5000 in TBST] (r.t., 1 h), and rinsed 3× with TBST asabove. The blot was incubated with HRP substrate (LuminataClassico Western HRP, Sigma) as directed, visualized (MyECLimager, Thermo Scientific), and the average ratios of Stat3/β-Actin protein band intensities ±SD (n = 2 measurements) weredetermined by imaging densitometry (ImageJ software).

Pharmacokinetics of Chol-DsiSTAT3 after i.v. administration

All procedures were approved by the University of NebraskaMedical Center Institutional Animal Care and Use Committeeincluding guidelines for the humane treatment of animals. Chol-DsiSTAT3 alone and Chol-DsiSTAT3 polyplexes were preparedfor in vivo studies (Section 2.5) and injected (0.1 mL) into the tailvein of female BALB/c mice (n = 5 mice) at 2.5 mg Chol-DsiSTAT3/kg (Section 2.10). Blood (~0.1 mL) from treated anduntreated mice (plasma background) was collected into Li-heparinized tubes (0.3 mL Microvette Tubes, Sarstedt) from asubmandibular bleed (5 mm Goldenrod lancet, Braintree Scien-tific) and plasma supernatants (2500 RCF, 15 min) (~50 μL) werestored at −80 °C. A standard curve for Chol-DsiSTAT3 wasprepared by spiking Chol-DiSTAT3 or Chol-DsiSTAT3 poly-plexes [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 at3:1 plasma (v:v), vortexing, incubating (r.t., 5 min), addingwater-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) was pipetted into a 96-well black (clearbottom) plate. Fluorophore (Quant-iT™ RiboGreen® RNAReagent 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 (Molec-ular Devices SpectraMax ID3, top read). Samples and standardswere diluted as necessary to fall within the linear range offluorescence measurements. The average plasma concentrationof Chol-DsiSTAT3 at each time point ± propagated SD (n = 5mice) was then calculated as the difference between the plasmaconcentration 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.

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

All procedures were approved by the University of NebraskaMedical Center Institutional Animal Care and Use Committeeincluding guidelines for the humane treatment of animals. Chol-DsiSTAT3 alone and Chol-DsiSTAT3 polyplexes were preparedfor in vivo studies (Section 2.5) and injected (0.1 mL) intofemale 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, organsand tumors were isolated, weighed, suspended in RNAprotectTissue 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 andindependently homogenized (Precellys Evolution technology,Bertin Instruments) in QIAzol Lysis Reagent (Qiagen) [10 μLQIAzol Lysis Reagent:1 mg tissue] on “Hard” mode. A standardcurve for Chol-DsiSTAT3 and Chol-DsiSTAT3 polyplexes wasprepared for each organ or tumor by spiking in Chol-DsiSTAT3or 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 theinitial “Hard” mode homogenates of the respective organs ortumors isolated from untreated mice. SDS [5% (w/v) indeionized H2O] [20 μL:1 mg tissue] and water-soluble choles-terol (Sigma) [10 mg/mL in deionized H2O] [0.2 μL:1 mgtissue] were added to the initial “Hard” mode homogenates andfurther homogenized on “Soft” mode. A portion of the finalhomogenate (300 μL; normalizes to 10 mg of tissue sample forRNA extraction) was heated (92 °C, 5 min), cooled (r.t., 10 s),and vortexed (4 cycles of heating, cooling, vortexing).13 TotalRNA was extracted by column purification after preparing thefinal homogenate as directed (miRNeasy Mini Kit, Qiagen,Hilden, Germany) and quantitated by ABS260 (NanoDropspectrophotometer, ThermoFisher Scientific). The averagemass of Chol-DsiSTAT3 (ng)/mass of tissue (mg) ± propagatedSD (n = 2 tissue/tumor pieces from n = 5 mice) was quantitatedby RT-ddPCR (Section 2.9) and compared in each tissue bynonparametric two-tailed t tests with Holm–Sidak correction.

Dose escalation of Chol-DsiCTRL polyplexes

All procedures were approved by the University of NebraskaMedical Center Institutional Animal Care and Use Committeeincluding guidelines for the humane treatment of animals. Micewere acclimated to the animal room for approximately 7 days todetermine that they were healthy based on observed behavior andweight gain. Mice were randomized into treatment cohorts byweight and sex. Chol-DsiCTRL alone and Chol-DsiCTRLpolyplexes were prepared for in vivo studies as described(Section 2.5). On day 0, vehicle alone or vehicle containingChol-DsiCTRL or Chol-DsiCTRL polyplexes was injected

(0.2 mL) biweekly into the tail vein of male (n = 6 mice) andfemale BALB/c mice (n = 6 mice) at an initial dose of 3.12 mgChol-DsiCTRL/kg and bodyweight was measured daily until4 days after the final dose. The dose for each consecutiveinjection was increased following a modified Fibonacci scheme(100%, 65%, 52%, 40%, 29%, 33%, 33% increase over previousdose) to a maximum of 50 mg Chol-DsiCTRL/kg (8 doses over28 days). Mice were euthanized at the end of the study by anoverdose of Fatal-Plus® (Vortech Pharmaceuticals) (150 mg/kgbody weight). Average daily body weights ±SEM (n = 6 mice)within each sex were compared to vehicle by 2-way ANOVA

Chronic dosing of Chol-DsiCTRL polyplexes

All procedures were approved by the University of NebraskaMedical Center Institutional Animal Care and Use Committeeincluding guidelines for the humane treatment of animals. FemaleBALB/c mice, Chol-DsiCTRL alone, and Chol-DsiCTRL poly-plexes 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 tailvein of male (n = 6 mice) and female BALB/c mice (n = 10mice)at the indicated dose of Chol-DsiCTRL/kg. Bodyweight wasmeasured on each day of injection and 3 days after the final dose (9doses 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 thethymus, lungs, liver, spleen, kidneys, and bone marrow, and acomplete blood count (CBC) including platelet count, serum liverenzymes, creatinine, and BUN. Average values ±SEM (n = 10mice or less as indicated) of treatment groups were compared tovehicle by 2-way ANOVA where appropriate.

Results

Activity of Chol-DsiSTAT3 polyplexes in murine syngeneic 4T1breast cancer epithelial cells

Triple negative breast cancer (TNBC) is a subtype of breastcancer 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, andrecurrence, and the worst stage-dependent prognosis.14 Al-though TNBC represents only 15% of new breast cancerdiagnoses, it accounts for ~30% of breast cancer-related deaths(~150,000 deaths worldwide) and has fewer treatment optionsdue to the absence of ER, PR, and HER2.15

Several therapeutically relevant proteins have been identifiedin TNBC where suppression could potentially improve treatmentoutcomes.16 One such protein, signal transducer and activator oftranscription factor 3 (STAT3), is a receptor tyrosine kinase(RTK)-associated signaling protein that is activated andupregulated by cytokines and growth factors found in thetumor microenvironment.17 Receptor activation of STAT3results in STAT3 dimerization, nuclear translocation, bindingto STAT3-specific DNA binding elements, and subsequenttranscription of genes involved in the regulation of celldifferentiation, proliferation, apoptosis, angiogenesis, metastasis,

Figure 2. Suppression of murine STAT3 mRNA by DsiSTAT3 and Chol-DsiSTAT3 polyplexes in murine 4T1 breast cancer epithelial cells 24 h after treatment.4T1 cells were (A) electroporated alone or in the presence of inactive DsiCTRL (white bar) or active DsiSTAT3 (gray bar) [300 nM] then incubated at 37 °C for24 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] complexedwith 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 thendetermined 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.


and immune responses.18 Furthermore, STAT3 is one of themain transcription factors involved in the immunomodulation ofcancer19 and elevated expression of activated STAT3 isassociated with poor prognosis for breast cancer and manyother solid tumors.18,20 Thus, given the role of STAT3 in breastand other solid tumors and presence of similar STAT proteins(STAT 1, 2, 4, 5, and 6)21, there is great interest in specificallyinhibiting the expression of STAT3.

The syngeneic murine breast cancer epithelial cell line, 4T1, is agood model for TNBC because it lacks ER, PR, and HER2, sharessubstantialmolecular featureswith humanTNBC22, can be grown inimmune competent female BALB/c mice, is poorly immunogenic,has rates of growth and patterns of metastasis that resemble humanbreast cancer, and the extent of late stage disease is comparable tostage IV breast cancer.23,24 STAT3 is also important in the 4T1breast tumor model as inhibition of STAT3 by shRNA inhibitstumor formation and the frequency of spontaneous metastases.25

Delivery of siSTAT3 to 4T1 cells using membrane-penetratingpeptides also inhibits 4T1 invasion and migration in vitro26 andintratumoral administration of shSTAT3-expressing plasmidsinhibits primary tumor growth and spontaneous lung metastases.27

We previously found that Chol-DsiRNA polyplexes increasethe potency of Chol-DsiLUC against stably expressed luciferasein 4T1-Luc cells in vitro and as part of primary 4T1-Luc breasttumors after i.v. administration.11 Thus, we hypothesized thatChol-DsiRNA polyplexes can increase the potency of Chol-DsiRNA against therapeutically relevant genes expressed in 4T1cells such as STAT3.

To identify an active DsiSTAT3 sequence and an inactiveDsiCTRL sequence against murine STAT3 mRNA in murinesyngeneic breast cancer epithelial cells, we electroporated 4T1cells with inactive DsiCTRL or murine DsiSTAT3 based oncommercially available siSTAT3 sequences and compared

normalized murine STAT3 mRNA copy numbers vs. electropo-ration-only 4T1 cells 24 h after treatment by RT-ddPCR (Figure2, A). Electroporation with DsiSTAT3 decreased STAT3 mRNAcopy numbers 81% below electroporation-only 4T1 cells (Figure2, A, gray bar), whereas electroporation with DsiCTRL had noeffect (Figure 2, A, white bar). Thus, the DsiSTAT3 sequence issufficiently active and the DsiCTRL sequence is sufficientlyinactive against murine STAT3 mRNA expression in murinesyngeneic breast cancer epithelial cells.

To determine if Chol-DsiRNA polyplexes increase the potencyof Chol-DsiRNA against a therapeutically relevant target gene inmurine syngeneic breast cancer epithelial cells, we treated 4T1cells with Chol-DsiCTRL or Chol-DsiSTAT3 complexed withPLL[30]-PEG[5K] at N/P 1 (50 wt% Chol-DsiRNA) andcompared normalized murine STAT3 mRNA copy numbers tountreated 4T1 cells 24 h after treatment by RT-ddPCR (Figure 2,B). Transfection with Chol-DsiSTAT3 polyplexes decreasedSTAT3 mRNA copy numbers 64% below untreated 4T1 cells(Figure 2, B, black bar), whereas transfection with Chol-DsiCTRLpolyplexes decreased STAT3mRNAcopy numbers by 6% (Figure2, B, white bar). These results indicate that STAT3 suppression in4T1 cells is due primarily to the activity of complexed Chol-DsiSTAT3 and not potential non-specific effects of Chol-DsiRNApolyplexes. Thus, Chol-DsiRNA polyplexes increase the potencyof Chol-DsiRNA against a therapeutically relevant target gene inmurine syngeneic breast cancer epithelial cells.

Potency of Chol-DsiSTAT3 polyplex activity in primary murinesyngeneic 4T1 breast tumors after i.v. administration

We previously found that Chol-DsiLUC polyplexes formedwith PLL[30]-PEG[5K] at N/P 1 (50 wt% Chol-DsiLUC)suppress the luminescence of stably expressed luciferase in

Figure 3. Dose response and kinetics of STAT3 mRNA suppression in primary murine 4T1 breast tumors after i.v. administration of Chol-DsiSTAT3polyplexes. (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 ofinactive 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 femaleBALB/c mice bearing a single subcutaneous 4T1 tumor (~30 to 50 mm3) in the mammary fat pad. After 48 h, average ratios of murine STAT3 to murine HPRT1mRNA 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 ANOVAwith Dunn's post-test. A half maximal ED50 was calculated from a nonlinear fitof 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/Pratio 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 themammary fat pad. At the indicated time point after injection, average ratios of murine STAT3 mRNA copies to murine HPRT1 mRNA copies in primary 4T1breast tumors ± propagated SD (n = 5 mice) were determined by RT-ddPCR then normalized and compared to vehicle alone at the same time point bynonparametric Mann–Whitney two-tailed t test. Results are representative of at least two independent experiments.


early-stage primary murine syngeneic 4T1-Luc breast tumors by~78% after daily i.v. injections of 2.5 mg Chol-DsiLuc/kg overthree days, whereas uncomplexed Chol-DsiLuc under the samedosage regimen has no effect.11 Daily injections at a constantdose, however, precluded determining the actual potency ofChol-DsiRNA polyplexes in primary murine syngeneic breasttumors.

To determine the potency of Chol-DsiRNA polyplexesagainst a therapeutically relevant target gene in primary murinesyngeneic breast tumors after i.v. administration, we intrave-nously administered increasing doses of Chol-DsiSTAT3 (up to5 mg/kg) or a single maximum 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 STAT3mRNA copy numbers in early-stage primary 4T1 breast tumorsto treatment with vehicle alone by RT-ddPCR (Figure 3, A). Wechose a 48-h timepoint given that (i) Chol-DsiLUC polyplexesmaximally suppress the luminescence of stably expressedluciferase in primary murine 4T1 breast tumors 48 h after thefirst daily i.v. injection of a three-day regimen11 and (ii)luciferase activity closely matches luciferase mRNA and proteinlevels due the short intracellular half-life of luciferase protein(~2 h).28

Chol-DsiSTAT3 polyplexes (Figure 3, A, black circles)suppressed STAT3 mRNA copy numbers in primary 4T1 breasttumors to a maximum of 54% vs. vehicle alone at the highestdose (5 mg Chol-DsiSTAT3/kg) with a half-maximal ED50 of0.3 ± 0.1 mg/kg, whereas inactive Chol-DsiCTRL polyplexeshad no effect at the highest dose of Chol-DsiSTAT3 polyplexes(5 mg Chol-DsiCTRL/kg) (Figure 3, A, white circle). Theseresults indicate that the suppression of STAT3 mRNAexpression in primary 4T1 tumors is due to the activity ofcomplexed Chol-DsiSTAT3 and not potential non-specificeffects of the Chol-DsiRNA polyplexes. Thus, Chol-DsiRNApolyplexes greatly increase the potency of Chol-DsiRNA against

a therapeutically relevant target gene in primary murinesyngeneic breast tumors after i.v. administration.

Kinetics of Chol-DsiSTAT3 polyplex activity in primary murinesyngeneic breast tumors after i.v. administration

We previously found that Chol-DsiLUC polyplexes maxi-mally suppress the luminescence of stably expressed luciferasein primary murine syngeneic 4T1-Luc breast tumors 48 h afterthe first daily i.v. injection of a three-day regimen at 2.5 mgChol-DsiLuc/kg and maintain near-maximal suppression at least48 h after the last injection.11 Multiple daily i.v. injections andthe possibility of dose depot effects in the primary tumor bypotential 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 breasttumors.

To determine the kinetics of Chol-DsiSTAT3 Polyplexactivity in primary murine syngeneic breast tumors after i.v.administration, we intravenously injected Chol-DsiSTAT3complexed with PLL[30]-PEG[5K] at N/P 1 (50 wt% Chol-DsiSTAT3) and compared normalized STAT3 mRNA copynumbers in early-stage primary murine 4T1 breast tumors tovehicle alone every 24 h over 96 h (four days) by RT-ddPCR(Figure 3, B). We chose an i.v. dose of Chol-DsiSTAT3polyplexes that did not maximally suppress STAT3 mRNAexpression in primary 4T1 breast tumors (0.5 mg Chol-DsiSTAT3/kg) (Figure 3, A, black circles) to exclude possibletumor dose saturation effects on the kinetics of Chol-DsiSTAT3activity in the primary tumor.

Chol-DsiSTAT3 polyplexes (Figure 3, B, black circles)suppressed STAT3 mRNA copy numbers in primary 4T1 breasttumors to a maximum of 43% vs. vehicle alone after 48 h butsuppression decreased to 11% after 24 h (72 h post-injection).Thus, similar to Chol-DsiLuc Polyplex activity against the

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 withPLL[30]-PEG[5K] at N/P ratio 1 (50 wt% Chol-DsiRNA) was injected at 2.5 mg Chol-DsiRNA/kg 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 ateach 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 ofthe same Western blot were used to generate (B). Results are representative of at least two independent experiments.


luminescence of stably expressed luciferase in primary 4T1-Lucbreast tumors after the last daily i.v. injection of 2.5 mg Chol-DsiRNA/kg11, a single dose of Chol-DsiSTAT3 polyplexes thatis unlikely to saturate the primary tumor is maximally active inprimary murine breast tumors 48 h after i.v. administration butmaintains maximal mRNA suppression less than 24 h.

Activity of Chol-DsiSTAT3 polyplexes against the growth ofprimary murine syngeneic breast tumors after i.v. administra-tion

STAT3 is critical for the growth of 4T1 murine breasttumors.25,27 ,29 Thus, given that Chol-DsiSTAT3 polyplexessuppressed the expression of STAT3 mRNA in primary 4T1tumors after i.v. administration (Figure 3), we expected thatChol-DsiSTAT3 polyplexes would also inhibit 4T1 tumorgrowth.

To determine if Chol-DsiSTAT3 polyplexes are therapeuti-cally active against primary murine syngeneic breast tumors afteri.v. administration, we intravenously injected Chol-DsiSTAT3 orinactive Chol-DsiCTRL complexed with PLL[30]-PEG[5K] atN/P 1 (50 wt% Chol-DsiRNA) [2.5 mg Chol-DsiRNA/kg] everyother day to early-stage 4T1 breast tumor-bearing mice andcompared tumor volumes to vehicle alone over 8 days by 3Dsurface scanning (Figure 4, A, Figure S2). We administered2.5 mg Chol-DsiSTAT3/kg to ensure polyplexes maximalsuppression of STAT3 mRNA expression in primary 4T1 breasttumors (Figure 3, A, black circles).

Chol-DsiSTAT3 polyplexes (Figure 4, A, black circles)inhibited the growth of primary murine 4T1 breast tumors at arate-based T/C ratio of 8.6%12 vs. vehicle alone (Figure 4, A,white triangles), whereas inactive Chol-DsiCTRL polyplexes(Figure S1, white circles) had no effect on the growth of primarymurine 4T1 breast tumors vs. vehicle alone (Figure S1, whitetriangles) under the same dosage regimen. Consistent withSTAT3 mRNA suppression (Figure 3, A) and the inhibition ofprimary 4T1 breast tumor growth (Figure 4, A, black circles),

Chol-DsiSTAT3 polyplexes suppressed 50% of total murineStat3 protein in primary 4T1 breast tumors vs. vehicle alone 48 hafter the final i.v. dose (day 8) (Figure 4, B & C). Furthermore,average body weights of mice from all treatment groupsgradually increased over the course of the study (not shown),indicating that (i) tumor growth inhibition is due to the activity ofcomplexed Chol-DsiSTAT3 and not animal weight loss and (ii)Chol-DsiSTAT3 polyplexes are not toxic under the currentdosage regimen. Thus, given that the National Cancer Institute atNIH defines high anti-tumor activity as T/C ratios ≤10% at non-toxic doses12, Chol-DsiSTAT3 polyplexes have high anti-tumoractivity against primary murine syngeneic breast tumors after i.v.administration.

Pharmacokinetics of Chol-DsiSTAT3 and Chol-DsiSTAT3polyplexes in healthy female BALB/c mice after i.v. adminis-tration

DsiRNAmust enter the cytosol of target cells to be cleaved byDicer into siRNA and incorporated into RISC complexes beforesuppressing complementary mRNA.30 Thus, Chol-DsiRNApolyplexes are expected to increase the potency of Chol-DsiRNAin primary syngeneic breast tumors after i.v. administration, inpart, by affecting the pharmacokinetics and distribution of Chol-DsiRNA.

To first determine if Chol-DsiRNA polyplexes formed withPLL[30]-PEG[5K] affect the pharmacokinetics of Chol-Dsi-STAT3, we intravenously injected Chol-DsiSTAT3 alone orcomplexed with PLL[30]-PEG[5K] at N/P 1 (50 wt% Chol-DsiSTAT3) [2.5 mg Chol-DsiSTAT3/kg] into the tail veins ofhealthy female BALB/c mice. We then indirectly determinedplasma concentrations of Chol-DsiSTAT3 over time as anincrease in total RNA extracted from the plasma of treated vs.untreated mice (Figure 5) given that total RNA in plasma fromuntreated mice is relatively constant (not shown). The highvariability of plasma concentrations and limited number of timepoints (Figure 5, A & B) prevented complete and accurate

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 tailveins of healthy female BALB/c mice and average plasma concentrations of Chol-DsiSTAT3 ± SD (n = 5 mice) were determined indirectly at each time pointas 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 thecurrent scale.

Table 1Pharmacokinetic parameters of Chol-DsiSTAT3 and Chol-DsiSTAT3polyplexes 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 concentrations at time point = 0 (C0) and mean residencetime (MRT0-∞) of Chol-DsiSTAT3 were determined by non-compartmentalanalysis (Phoenix WinNonlin) and average area under the curve (AUC0-∞) ofChol-DsiSTAT3 (±SD from 5 mice) was determined by the linear logtrapezoidal rule of the respective PK profiles (Figure 5).


compartmental analysis of the PK profiles. Despite the limitedanalysis, 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 poly-plexes may increase the potency of Chol-DsiSTAT3 in primarysyngeneic breast tumors after i.v. administration, in part, byincreasing plasma exposure to Chol-DsiSTAT3.

Distribution of Chol-DsiSTAT3 and Chol-DsiSTAT3 polyplexesin 4T1 breast tumor-bearing female BALB/c mice

To next determine if Chol-DsiSTAT3 polyplexes affect thedistribution of Chol-DsiSTAT3 to primary murine syngeneicbreast tumors and normal tissues after i.v. administration, weintravenously injected Chol-DsiSTAT3 alone or complexed withPLL[30]-PEG[5K] at N/P 1 (50 wt% Chol-DsiSTAT3) [2.5 mgChol-DsiSTAT3/kg] into the tail vein of primary 4T1 breasttumor-bearing female BALB/c mice and compared the distribu-tion of Chol-DsiSTAT3 in perfused organs and tumors fifteenminutes post-injection by RT-ddPCR of the anti-sense strand ofChol-DsiSTAT3 (Figure 6). Chol-DiSTAT3 polyplexes (Figure6, 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 distri-bution, Chol-DsiRNA polyplexes (Figure 6, black bars) alsoincreased the distribution of Chol-DsiSTAT3 4-fold to thekidneys [4 ± 2 (SD) vs. 1.0 ± 0.4 ng Chol-DsiSTAT3/mgorgan], 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 (Figure6, white bars). Furthermore, although much lower overall levelswere observed than in other tissues, Chol-DsiRNA polyplexesincreased the distribution of Chol-DsiSTAT3 4.6-fold to thebrain [0.41 ± 0.05 ng (SD) vs. 0.088 ± 0.007 ng Chol-Dsi-STAT3/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.

Dose escalation of Chol-DsiCTRL polyplexes by i.v. adminis-tration

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 aftermultiple daily i.v. injections at 2.5 mg/kg without affecting bodyweight 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, weintravenously injected healthy male and female BALB/c micewith vehicle alone or vehicle containing increasing doses ofChol-DsiCTRL alone or complexed with PLL[30]-PEG[5K] atN/P 1 (50 wt% Chol-DsiCTRL) up to 50 mg Chol-DsiCTRL/kgtwo times a week over 28 days (8 injections total) and comparedbody weights (Figure 7, A, Table S1), terminal body and tissueweights (Table S2), the presence of liver cysts (Table S3), andhistopathology of the liver, spleen, kidneys, and femurs afterdecalcification (Table S4). We focused on inactive Chol-

Figure 6. Effect of Chol-DsiRNA polyplexes on the distribution of Chol-DsiSTAT3 in 4T1 breast tumor-bearing mice after i.v. administration. Chol-DsiSTAT3alone [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 (blackarrows) bearing single, orthotopic (mammary fat pad) 4T1 tumors (~30 to 50 mm3). After 15 min, the average ng of Chol-DsiSTAT3/mg tissue ± SD (n = 5mice) was determined by RT-ddPCR of the DsiSTAT3 antisense strand and compared in each tissue by nonparametric two-tailed t tests with Holm–Sidakcorrection.

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 bodyweights ±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 complexedwith PLL[30]-PEG[5K] at N/P 1 (50 wt% Chol-DsiCTRL) was injected into the tail veins of healthy female BALB/c mice biweekly at the indicated dose (blackarrows). 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).


DsiCTRL polyplexes (Table 2) to separate the possibility ofadditional toxicity by the suppression of STAT3 by Chol-DsiSTAT3 polyplexes.

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

Dose escalation with Chol-DsiCTRL increased relative liverweights by 10% in male mice and decreased total and relativeliver weights by ~8% in female mice compared to vehicle alonebut was not associated with histopathologic changes. Chol-DsiCTRL polyplexes also increased total and relative spleenweights by ~37% in male mice and decreased total and relativeliver weights by 5 to 7% compared to vehicle alone (Table S2).

The liver did not show any histopathologic changes and thespleens only showed differences in blood congestion but withoutabnormalities in the lymphoid structures.

Chol-DsiCTRL polyplexes had the highest number ofsurviving male and female mice with liver cysts (4 out of 6),but the number 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 occurred in BALB/c mice and were ofsimilar size and frequency between treatment groups. Further-more, most liver cysts were microscopic in size and differencesin numbers between treatment groups may have been due tosampling. As such, cystic changes in the liver are not consideredto be treatment related.

Liver, spleen, kidney, and femur histology was normal inmale and female mice from all treatment groups with occasionalmice having changes that are common in BALB/c mice butwithout differences between groups and thus are not consideredtreatment related (Table S4). Two deaths occurred with vehicle

Table 2Representative characteristics of Chol-DsiCTRL, PLL[30]-PEG[5K], and Chol-DsiCTRL polyplexes.

Sample Endotoxin EU/mg (±SD) LoadingChol-DsiCTRL wt%

Hydrodynamic Diameternm (±SD)

Zeta potentialmV (±SD)

Chol-DsiCTRL 0.104 (n.r.)a N/A N/A N/APLL[30]-PEG[5K] 0.0171 (0.0008)b N/A N/A N/AChol-DsiCTRL polyplexes (N/P 1) 0.023 (0.002)b 50 33 (2)c 5.2 (0.7)d

a Determined by manufacturer (IDT).b Determined by Endochrome-K™ kit.c Calculated from a nonlinear fit of the lognormal distribution of Chol-DsiCTRL polyplex diameters in 0.1 M HEPES, pH 7.4 (Figure S2).d Determined in 0.1 M HEPES, pH 7.4.


alone (Figure 7, A, day 18: one male and one female) and onedeath occurred with Chol-DsiCTRL (Figure 7, A, day 8, onemale), whereas no deaths occurred with Chol-DsiCTRLpolyplexes, suggesting that deaths were likely caused by thestress of multiple i.v. injections. Thus, i.v. administration ofChol-DsiCTRL alone or complexed with PLL[30]-PEG[5K] atN/P 1 appears to be well tolerated by healthy male and femalemice to at least 50 mg Chol-DsiCTRL/kg.

Chronic dosing with Chol-DsiCTRL polyplexes by i.v. admin-istration

To assess the toxicity of Chol-DsiRNA polyplexes in miceafter chronic dosing by i.v. administration, we intravenouslyinjected healthy female BALB/c mice with vehicle alone orvehicle containing uncomplexed Chol-DsiCTRL at 50 mg/kg orChol-DsiCTRL complexed with PLL[30]-PEG[5K] at N/P 1(50 wt% Chol-DsiCTRL) at 25 and 50 mg Chol-DsiCTRL/kgbiweekly over 28 days (9 injections total) and compared bodyweights (Figure 7, B, 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 7, B,white triangles), uncomplexed Chol-DsiCTRL (Figure 7, B,white circles), or Chol-DsiCTRL polyplexes at 25 mg Chol-DsiCTRL/kg (Figure 7, B, black/white squares) or 50 mg Chol-DsiCTRL/kg (Figure 7, B, black squares) continued to gainweight up to four days after the last dose despite deaths with50 mg/kg Chol-DsiCTRL alone (day 14) and 50 mg/kg Chol-DsiCTRL polyplexes (days 3, 14 [2 mice], 21). Mice from alltreatment groups also had similar rates of food and waterconsumption (Table S6) and terminal body weights (Table S7).

Chronic i.v. administration of Chol-DsiCTRL polyplexesincreased total and relative spleen weights by 22% at 25 mgChol-DsiCTRL/kg and 29% at 50 mg/Chol-DsiCTRL/kg andincreased total and relative liver weights by 10% at 50 mg Chol-DsiCTRL/kg compared to vehicle alone (Table S7) although nohistological abnormalities were observed in the lungs, liver,kidneys, spleen, brain, thymus, or femurs of any treatment groupcompared to vehicle alone. There were occasional animals ineach group with minimal changes commonly seen in BALB/cmice and were, consequently, not considered treatment related.Changes in the tails at the injection sites were similar betweengroups with changes ranging from mild chronic inflammation to

ulceration with acute inflammation, thus related to the repeatedinjections and not the administered material (Table S8).

WBC, hemoglobin, hematocrit, lymphocytes, and plateletsfrom all treatment groups were also within normal values inBALB/c mice (Charles River Laboratories) (Table S9) andtrauma observed around tail injection sites was consistent withdamage 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 belowmaximum FDA limits for i.v. administration to a 20 g mouse perhour (0.1 EU/mg drug)31, indicating that deaths were likelycaused by the stress of multiple i.v. injections and not theadministered 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 atleast 50 mg Chol-DsiCTRL/kg.

Discussion

Our study provides evidence that Chol-DsiRNA polyplexesformed with PLL[30]-PEG[5K] are highly potent in solid primarymurine syngeneic breast tumors and well tolerated after i.v.administration.We found that Chol-DsiRNApolyplexes (i) greatlyincrease the potency (half-maximal ED50 of 0.3 ± 0.1 mg Chol-DsiRNA/kg) (Figure 3, A) and therapeutic activity (rate-based T/Cratio of 8.6%) (Figure 4, A) of Chol-DsiRNA against anendogenous, therapeutically relevant target gene (STAT3) inearly-stage primary murine 4T1 breast tumors after i.v. adminis-tration 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 escalationover 28 days (8 injections total, 2 per week) (Figure 7, A, TablesS1-S4) and by healthy female BALB/c after chronic i.v.administration at 50 mg Chol-DsiCTRL/kg over 28 days (9injections total, 2 per week) (Figure 7, B, Tables S5-S9).

Our study also provides evidence that Chol-DsiRNApolyplexes formed with PLL[30]-PEG[5K] may increase thepotency of Chol-DsiRNA in primary murine syngeneic tumors,in part, by increasing plasma exposure and subsequentlocalization to the tumor. We found that Chol-DsiRNApolyplexes increase the AUC0-∞ of Chol-DsiSTAT3 in plasma13-fold (Figure 5, Table 1) and distribution to primary 4T1 breasttumors 2.7-fold (Figure 6) vs. uncomplexed Chol-DsiSTAT3after i.v. administration.


We previously found that Chol-DsiLUC polyplexes suppressluciferase activity (indirect measure of LUC mRNA) in primary4T1-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 maintainednear-maximal suppression at least 48 h after the final dose.11 Incontrast, Chol-DsiSTAT3 polyplexes suppressed STAT3 mRNAin primary 4T1 tumors a maximum of ~54% after a single dose of5 mg Chol-DsiSTAT3/kg and maintained maximal suppressionless than 24 h after a single dose of 0.5 mg Chol-DsiSTAT3/kg(Figure 3, B).

There are several possible reasons that may individually orcollectively explain why Chol-DsiRNA polyplexes suppressSTAT3 mRNA to a lesser extent and shorter duration than stablyexpressed LUCmRNA in 4T1 breast tumors. The first possibilityis that multiple dosing with Chol-DsiLUC polyplexes (a dailydose of Chol-DsiLUC polyplexes over three days vs. one dose ofChol-DsiSTAT3 polyplexes) at a higher dose (2.5 mg Chol-DsiLUC/kg vs. 0.5 mg Chol-DsiSTAT3/kg) may saturateprimary 4T1 tumors and, consequently, increase the extent andduration of LUC mRNA suppression vs. STAT3 mRNAsuppression. The second possibility is that 4T1 cells may bemore easily transfected by Chol-DsiRNA polyplexes than othercells within the primary 4T1 breast tumor given that LUCmRNAis only expressed in stably transfected 4T1-Luc cells within 4T1-Luc tumors, whereas STAT3 is expressed in 4T1 cells as well astumor-associated cells (e.g., mammary gland tissue)32 andtumor-infiltrating cells.19 Consistent with the second possibility,the third possibility is that steady-state levels of endogenousSTAT3 mRNA in 4T1 breast tumors are higher than stablyexpressed LUC mRNA in 4T1-Luc breast tumors, in part, due tohigher transcription rates of STAT3 mRNA caused by cytokineand growth factor signaling within the tumor microenvironment,a longer intracellular half-life33, and the additional expression ofSTAT3 mRNA by tumor-associated and tumor-infiltrating cells.Thus, initial steady-state levels of STAT3 mRNA may be moredifficult to overcome, less accessible to Chol-DsiRNA poly-plexes, and persist longer in primary 4T1 tumors compared tosteady-state levels of LUC mRNA in primary 4T1-Luc tumors.The fourth possibility is that using fewer 4T1 cells to formprimary 4T1 breast tumors (1 × 106 4T1-Luc cells in theprevious study11 vs. 1 × 105 4T1 cells in the current study tofacilitate 3D surface scanning) decreases tumor vascularizationand subsequent accessibility to cells within 4T1 tumors. Thisdifference in the number of 4T1 cells used for tumor inoculation,however, is unlikely to affect tumor vascularization given thatsimilar levels of tumor vascularization and necrosis are observedin primary 4T1 breast tumors after inoculation between 500 and1 × 106 4T1 cells.34 These possibilities can be best resolved inthe future by directly comparing the activities of Chol-DsiRNApolyplexes and Chol-siRNA polyplexes against the same mRNAtarget in the same tumor model under the same dosage regimen.

In summary, our results indicate that Chol-DsiRNA poly-plexes formed with PLL[30]-PEG[5K] greatly increase thepotency of Chol-DsiRNA molecules in primary murine synge-neic breast tumors and are well tolerated after i.v. administration.Thus, Chol-DsiRNA polyplexes may be a good candidate forPhase I clinical trials of RNAi-mediated therapeutic approachesin breast cancer and other solid tumors.

Acknowledgments

This work was supported by NIH/NCATS 1R41TR001902-01A1 (JAV), NIH/NCI R01CA228524 (RKS), NIH/NIDA5P01DA028555 (YA), Fred & Pamela Buffett Cancer CenterSupport Grant (P30CA036727) and Pediatric Cancer ResearchGroup Support, State of Nebraska, LB 417 (DC, ZY), Havlik-Wall Professorship (SMC), Nebraska Research Initiative (NRI)Collaborative award (SRD), and the University of NebraskaPresidential Graduate Fellowship (VVA). We thank UNMCComparative Medicine Research Technical Service for perform-ing SQ and tail vein injections and Karen L. Pennington (CohenLab) for technical support of the toxicity studies.

Credit Author Statement

Zhen Ye:Methodology, Investigation, Formal analysis, Datacuration Mai Mohamed Abdelmoaty.: Investigation, Formalanalysis, Data curation Vishakha V. Ambardekar: Methodol-ogy, Investigation Stephen M. Curran: Methodology, Investi-gation, Formal analysis, Writing – reviewing & editing,Validation, Supervision Shetty Ravi Dyavar: Methodology,Formal analysis, Supervision Lora L. Arnold: Methodology,Investigation, Formal analysis, Data curation Samuel M.Cohen: Methodology, Investigation, Formal analysis, Supervi-sion Devendra Kumar: Formal analysis Yazen Alnouti:Formal analysis, Writing – reviewing & editing, SupervisionDon W. Coulter: Conceptualization, Resources, Writing –reviewing & editing, Funding Acquisition Rakesh K. Singh:Conceptualization, Writing – reviewing & editing Joseph A.Vetro: Conceptualization, Methodology, Validation, Formalanalysis, Resources, Data curation, Writing – original draft,Writing – reviewing & editing, Supervision, Visualization,Project administration, Funding acquisition.

Appendix A. Supplementary data

Supplementary data to this article can be found online athttps://doi.org/10.1016/j.nano.2021.102363.

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