Simultaneous Delivery of Electrostatically Complexed Multiple Gene

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Simultaneous Delivery of Electrostatically Complexed Multiple Gene-Targeting siRNAs and an Anticancer Drug for Synergistically Enhanced Treatment of Prostate Cancer Eunshil Choi, Wonjae Yoo, Jae Hyung Park, and Sehoon Kim Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00227 • Publication Date (Web): 20 Jul 2018 Downloaded from http://pubs.acs.org on July 22, 2018

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Molecular Pharmaceutics

Simultaneous Delivery of Electrostatically Complexed Multiple Gene-Targeting siRNAs and an Anticancer Drug for Synergistically Enhanced Treatment of Prostate Cancer

Eunshil Choi1,†, Wonjae Yoo1,2,†, Jae Hyung Park2 and Sehoon Kim1,3,*

1

Center for Theragnosis, Korea Institute of Science and Technology (KIST), Seoul 136-791, Korea;

2

School of Chemical Engineering, College of Engineering, Sungkyunkwan Univeristy, Suwon 440-

746; 3Division of Bio-Medical Science & Technology, KIST School, Korea University of Science and Technology (UST), Seoul 136-791, Korea †

These authors equally contributed to this work.

KEYWORDS: gene silencing, Bcl-2, survivin, androgen receptor, prostate cancer, drug delivery

ABSTRACT Simultaneous silencing of multiple apoptosis-related genes is an attractive approach to treat cancer. In this paper, we present a multiple gene-targeting siRNA/drug delivery system for prostate cancer treatment with high efficiency. Bcl-2, survivin and androgen receptor genes involved in the cell apoptosis pathways were chosen as silencing targets with three different siRNAs. The colloidal nanocomplex delivery system (< 10 nm in size) was formulated electrostatically between anionic siRNAs and a cationic drug (BZT), followed by encapsulation with Pluronic F-68 polymer. The

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formulated nanocomplex system exhibited sufficient stability against nuclease-induced degradation, leading to successful intracellular delivery for the desired therapeutic performance. Silencing of targeted genes and apoptosis induction were evaluated in vitro on human prostate LNCaP-LN3 cancer cells by using various biological analysis tools (e.g., real-time PCR, MTT cell viability test, and flow cytometry). It was demonstrated that when the total loaded siRNA amounts were kept the same in the nanocomplexes, the simultaneous silencing of triple genes with co-loaded siRNAs (i.e., Bcl-2, survivin, and AR-targeting siRNAs) enhanced BZT-induced apoptosis of cancer cells more efficiently than the silencing of each single gene alone, offering a novel way of improving the efficacy of gene therapeutics including anticancer drug.

INTRODUCTION Among malignant tumors, prostate cancer is one of the most common kind of cancer in men with hardly noticeable early symptoms and rapid development of metastasis.1,2 Androgens, which are essential for prostate gland development, are internalized into prostate cells by the androgen receptor (AR) and help prostate cancer cells to survive.3–5 Prostate cancer is androgen-dependent in the early stage and can be cured by surgery or hormone regulation, but turns progressive and androgen-independent in the later stage, making it hard to eradicate.4,6,7 It has also been known that development of prostate cancer is related to the expression of apoptosis-related proteins that exist in many cancer cells such as Bcl-28 and survivin9,10. Upon appropriate death-inducing stimuli, cells inactivate anti-apoptoic proteins and undergo caspase-related apoptosis process.11,12 However, because of the delicate inter-connection of many anti/pro-apoptotic genes and their complementary effects, it has been noted that single gene inhibition might result in limited apoptotic responses.13–17 Therefore, if it is possible to concurrently control the expression of multiple key proteins in prostate


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cancer cells, for example, Bcl-2, survivin, and AR, it may create a more efficient treatment to the prostate cancer. Among many cancer treatment strategies, siRNA/drug combination therapy has emerged as a new potential therapeutic strategy because siRNA with a target-specific gene knockdown capability18–22 can lead to the more efficient treatment of cancer than a single drug-based chemotherapy alone.23–31 To obtain a desired therapeutic result, it is also a pre-requisite to develop a bio-safe and effective delivery carrier because free siRNA has poor cell-membrane permeability and undergoes easy degradation in serum.32,33 Much effort has been committed to create an adequate non-viral gene carrier system by utilizing a traditional electrostatic polycomplexation method between polyanionic siRNA and cationic polymers (or liposome).34–37 However, such designed systems possess several clinically-unfavorable features including cytotoxicity of the cationic agents (mostly amine-derivatized)38–40 and chemical instability of liposomes41. We previously presented a more clinically-relevant siRNA/drug delivery formulation by which the Bcl2 gene-targeting siRNA (Bcl-2-siRNA) can synergistically induce cell apoptosis in cooperation with co-delivered anticancer drug.42 In the study, the hydrophobically associated multiple monocomplex (HMplex) system with a small colloidal size (< 10 nm) was formulated between Bcl2-siRNA and a cationic anticancer drug benzethonium chloride (BZT)43 in the presence of a biocompatible Pluronic F-68 polymer44. The HMplex was utilized for the dual therapy of breast cancer by stably delivering both siRNA and BZT into tumor cells. Depending on the target gene or the type of cancer, the HMplex-based siRNA carrier platform can extensively be utilized to deliver a wide range of siRNAs against various anti-apoptotic proteins. In this paper, we report a multiple gene-targeting siRNA/drug nanocomplex (m-NNC) for prostate cancer treatment with high efficiency. Triple siRNAs targeting cell death-related genes,



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Bcl-2, survivin, and AR were directly complexed with BZT via electrostatic interaction, and encapsulated by Pluronic F-68 polymer. To better understand the effect of the simultaneous intracellular delivery of multiple siRNAs on cell survival, we also prepared the NNC systems containing non-targeting siRNA (n-NNC) or single-targeting siRNAs (i.e., one of Bcl-2, survivin, and AR-targeting siRNA) (s-NNC). Cell uptake and the corresponding combinatorial therapy of mNNC were investigated in vitro by using human prostate LNCaP-LN3 cancer cells relative to those control NNCs. We demonstrate that when the total loaded siRNA quantities are kept the same, the m-NNC containing all of Bcl-2, survivin, and AR-targeting siRNA exhibits the highest efficiency to induce cell apoptosis among the tested NNC compositions. The presented results suggest that the simultaneous delivery of triple siRNAs and anticancer drug offer a promising strategy to improve the efficacy of gene/drug therapeutics.

EXPERIMENTAL SECITON

Materials. Custom siRNAs, scrambled siRNA (scRNA), Cy5.5-labeled siRNAs, and agarose gel loading buffer were purchased from Bioneer (Daejeon, Korea). Lipofectamine2000 was purchased from Invitrogen (USA). Custom PCR primers for each gene were purchased from Macrogen (Seoul, Korea). All siRNA and primer sequences are provided in Table 1. SYBR® Gold and Trizol were purchased from Invitrogen (Carlsbad, CA, USA). Heparin sodium salt, chloroform, isopropanol,

3-(4,

5-Dimethyl-2-thiazolyl)-2,

5-diphenyl-2H-tetrazolium

bromide

(MTT),

chlorpromazine, methyl-β-cyclodextrin, and amiloride were purchased from Sigma-Aldrich (St. Louis, MO, USA). The first antibodies for Western blotting were purchased from Santa Cruz Biotechnology (CA, USA) for Bcl-2, androgen receptor, and β-actin and R&D systems (MN, USA)


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for survivin, and the second antibody from Bethyl Laboratories (TX, USA). Annexin V-FITC/PI apoptosis detection kit was purchased from BioVision, Inc (Milpitas, CA, USA). Preparation and Characterization of NNCs. Therapeutic NNCs were prepared by stirring 4.65 µg of siRNA (or scRNA) and varying quantities of BZT (26.4−66 µg) for 1 h at room temperature in 250 µL of neutral PBS containing 1.25 mg of Pluronic F-68 polymer. The hydrodynamic size by dynamic light scattering (DLS) and zeta potential of the prepared NNCs were measured by using a zeta-sizer (Nano-ZS, Malvern, UK) as suspended in PBS at room temperature. To observe NNCs by transmission electron microscopy (TEM, FEI Tecnai G2 F20 at 200 kV), a drop of the as-made NNC solution was dried on a 200 mesh copper grid coated with carbon and negatively stained with treated with 2wt% uranyl acetate solution. For optimization of the N/P ratio, aliquots of the NNC solutions prepared at a varying charge ratio were loaded onto 2% agarose gel for electrophoresis in TBE buffer solution at 100 V. After electrophoresis, the SYBR Gold-stained siRNA bands were detected by gel documentation system (MiniBis Pro, DNR Bio-Imaging Systems, Israel). Cell Culture. Human prostate LNCaP-LN3 cancer cells were obtained from Korean Cell Line Bank (KCLB, Korea). Cell culture media, antibiotics, fetal bovine serum (FBS) were purchased from Welgene Inc. (Korea). LNCaP-LN3 cells were cultured in Minimum Essential Medium (MEM) with 10% FBS and antibiotics in a humidified 5% CO2 incubator at 37℃. Other cancer cells, PC-3 (androgen-independent prostate cancer cell line) and MDA-MB-231 (breast cancer cell line) were also purchased from Korean Cell Line Bank (KCLB, Korea) and cultivated similarly to LNCaP-LN3 cells, in Dulbecco’s modified Eagle’s medium (DMEM) and RPMI 1640 with 10% FBS and antibiotics, respectively. Gel Retardation Assay. The structural stability of NNCs was evaluated by using FBS and heparin. NNCs (or free siRNA) were incubated with 50% FBS/neutral PBS at 37℃ for various time periods



(0−16 h), immediately after which aliquots of each sample were treated with heparin to release the intact siRNA from NNCs and analyzed by gel electrophoresis. For a heparin competition assay at various heparin amounts, NNCs containing 1µg of siRNA were incubated with 0−500 µg of heparin for 30 min at room temperature, and analyzed by gel retardation assay. In addition, the siRNA release from NNCs was detected by photoluminescence (PL) spectroscopy using QuantaMaster 40 (QM 40) UV/VIS steady state spectrofluorometer (Photon Technology International, USA). Cell Uptake. LNCaP-LN3 cells were seeded onto 35 mm coverglass bottom dishes (2 ×105 cells/well) and incubated for 2 days in 10% FBS-containing MEM. At a confluence of 70−80%, cells were incubated with free Cy5.5-labeled siRNA or Cy5.5-labeled siRNA-containing NNCs for 2 h in plain MEM, washed with DPBS, and fixed with 4% paraformaldehyde. Optical fluorescence images of cells were taken using a LEICA DMI3000B equipped with a Nuance FX multispectral imaging system (CRI, USA). To quantitatively determine cellular uptake efficiency, LNCaP-LN3 cells were seeded onto 12-well culture plates (2 × 105 cells/well) and incubated to reach 70−80% confluence. Next, cells were incubated with Cy5.5-tagged free siRNA or Cy5.5-labeled siRNAcontaining NNCs for 2 h in serum-free media, washed with DPBS, trypsinized, collected by centrifugation, and analyzed by flow cytometry (Guava easyCyteTM Flow Cytometers, EMD Millipore, USA). To study possible cell uptake mechanisms, cells were pre-treated with cell uptake inhibitors, chlorpromazine (10 µg/mL), methyl-β-cyclodextrin (10 mM), and amiloride (50 µM) at 37 oC for 1 h, respectively, or incubated at 4 oC for 1 h in serum-free media. Cells were then washed with DPBS and treated with Cy5.5-labeled siRNA-containing NNCs under the same condition as described above. Gene Silencing and Western Blotting. LNCaP-LN3 cells were seeded onto 12-well culture plates (2 × 105 cells/well) and incubated until cells reached 70-80% confluence. Cells were incubated for


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2 h in serum-free MEM with various NNCs at the constant charge ratio of 8 and total RNA concentration of 17 nM. Next, cells were washed once with DPBS and kept incubated for another 46 h in serum-rich MEM. To evaluate the gene silencing effect of the delivered siRNAs, the above treated cells were trypsinized and harvested by centrifugation. After a total RNA was isolated from the cells using Trizol (Invitrogen, USA), its complementary DNA was synthesized by using High Capacity RNA-to-cDNA kit (Applied Biosystems, USA). Next, the produced DNA was mixed with SYBR Green master Mix (Applied Biosystems, USA) and a gene-specific customized primer (or βactin primer), and then processed for real time quantitative reverse transcription polymerase chain reaction (RT-qPCR) by using a StepOnePlus real-time PCR system (Applied Biosystems, USA). The results were given as the mean ± standard deviation (STD) (n = 3) after normalized against those for the internal standard, β-actin. The threshold cycle (Ct) values in RT-qPCR were provided in Supporting Information (Table S1). For Western blotting, proteins of each cell sample were collected by lysis buffer, separated by gel electrophoresis, and transferred to blotting membranes. The membranes were subsequently treated with antibodies and ECL reagents (Bio-Rad, USA) according to the manufacturers’ protocols, and then visualized with ATTO image analyzer (ATTO, Japan). Cytotoxicity and Apoptosis Assay. For cytotoxicity assay of various treatments, cells were seeded onto 96-well culture plates (1 × 104 cells/well) and incubated to reach 70−80% confluence. Cells were then incubated with the NNCs (N/P = 8; total RNA concentration= 17 nM) for 2 h in plain MEM, washed once with DPBS, and kept incubated in fresh 10% FBS-MEM for another 46 h. The cells were again washed with DPBS and examined with the colorimetric MTT test with a microplate reader (λ= 540 nm) (Spectra Max 340, Molecular Devices, Sunnyvale, CA, USA). For cell apoptosis assay, the cells were also treated in the same procedure as for the gene silencing



experiment except for the annexin V-FITC/propidium iodide (PI) staining step 48 h post-treatment (BioVision, Inc, USA). The apoptotic cell levels were analyzed by flow cytometry (Guava easyCyteTM Flow Cytometers, EMD Millipore, USA). Statistical Analysis. All experiments were carried out at least three times, and the data were represented as the mean ± STD. Statistical significance was analyzed by Student’s two-tailed t-test.

Table 1. Sequences of oligonucleotides

RESULTS AND DISCUSSION The m-NNC was prepared (Figure 1A) according to modification of our previously reported protocol42. The mixture of multiple siRNAs was composed of the same portion of Bcl-2, survivin, and AR-siRNA. BZT was chosen because of its cationic character as well as the anticancer effect43 so that it could be electrostatically complexed with polyanionic siRNA in aqueous solution and allowed for the simultaneous cell uptake with co-loaded siRNAs. A polymeric surfactant, Pluronic


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F-6844 was used to offer high water-solubility and colloidal stability to the resulting system and to efficiently deliver the loaded drugs into cells. siRNA/BZT complexation was achieved by simply mixing the two ingredients in PBS for 1 h at room temperature in the presence of Pluornic F-68 polymer. In the absence of the surfactant, hydrophilic siRNA and BZT were rapidly coagulated and formed larger water-insoluble nanoclusters (131.8 ± 75.7 nm, Table 2). On the other hand, the predissolved surfactant readily incorporates the siRNA/BZT aggregates into the hydrophobic core of its micellar structure44, drastically reducing the resulting particle sizes (ca. < 10 nm in diameter) as measured by DLS (Figure 1B and Table 2) and TEM (Figure 1D; tiny white dots pointed by arrows). The employed concentration of F-68 polymer (6.0 × 10-4 M) was higher than its critical micelle concentration (4.8 × 10-4 M)44, thus favorably forming a micellar structure to encapsulate siRNA/BZT drug molecules. To obtain a tightly-bound stable NNC structure, the charge ratio between BZT ammonium and siRNA phosphate (N/P) was optimized by varying the amount of BZT. As shown in the gel electrophoresis assay (Figure 1B), the band indicating free siRNA gradually diminished as the N/P ratio increased and completely disappeared at the charge ratio of 8 or above, indicating completion of siRNA/BZT complexation. The zeta potential of the resulting complex structure changed accordingly and turned closer to zero (-1.7 ± 0.2 mV when N/P = 8 or higher) from a more negative value (-8.57 ± 2.92 mV when N/P = 4) (Figure 1B).



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Figure 1. (A) Schematic depiction of the therapeutic NNC composition, (B) agarose gel retardation assay of the NNCs at various N/P ratios, (C) hydrodynamic size (black square) and zeta potential (white circle) of the NNCs measured by DLS, and (D) TEM image of the NNCs.


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Table 2. Hydrodynamic diameter and zeta potential of the NNCs with various siRNA/drug combinations.

a

data represented as the mean ± STD (n = 5)



In order to comparatively study a therapeutic effect of the m-NNC, non-targeting or single-targeting NNCs (i.e., n-NNC or s-NNCs) were also prepared with scrambled siRNA and one of Bcl-2, survivin, and AR-targeting siRNAs, respectively (Figure 1A), by following the same procedure as described above. As shown in Figure 2A, no sign of uncomplexed siRNA was visible at the charge ratio of 8 for all of the NNCs with different siRNA compositions. The hydrodynamic size and the surface charge of the complexes were reproduced in consistence with the above-optimized values (Table 2). The stability of the resulting complexes was tested by incubating them in 50% serumcontaining PBS at 37°C for 16 h and subsequently exposing the serum-treated samples to polyanionic heparin to release the intact siRNA. As presented in Figure 2B, unprotected free siRNA completely disappeared after 4 h of serum treatment. On the other hand, the siRNA complexes were distinctively more stable, showing the release of unbroken siRNA after 14 h of serum incubation (Figure 2C). The heparin-induced siRNA release behavior of the complex was further investigated by varying the concentration of heparin and by using Cy5.5-tagged siRNA. As displayed in Figure 2D, the free siRNA-indicating gel band appeared brighter with the increase of heparin concentration, proving the promoted siRNA release from the complex. Also, fluorescence intensities of the NNCbound Cy5.5-siRNA were measured by using PL spectroscopy, showing the fluorescence recovery depending on the heparin concentration (Figure 2E). The complex initially showed a very weak fluorescence signal in the absence of heparin because of the selfquenched aggregation of Cy5.5 in the tightly-bound siRNA/BZT complex structure42, but

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displayed immediate recovery of fluorescence with the addition of heparin, indicating disappearance of the self-quenching effect by decomplexation.

Figure 2. Agarose gel assay showing (A) NNC formation with various siRNAs at the charge ratio of 8, (B) nuclease-induced degradation of free siRNA, (C) response of the m-NNC to the nuclease (top) and subsequent heparin treatment (bottom), and (D) siRNA release from the m-NNC depending on the heparin amount. (E) PL spectra of Cy5.5tagged NNC in PBS with and without heparin treatment. (F: free siRNA, B: Bcl-2, S: survivin, A: AR)

Cellular uptake of the complexes was evaluated with LNCaP-LN3 cells by using optical fluorescence microscopy and flow cytometry. For fluorescence imaging, the cells were incubated with uncomplexed free Cy5.5-siRNA or Cy5.5-siRNA-containing NNCs (N/P = 8 at the constant total siRNA concentration of 17 nM) for 2 h at 37 oC and washed with DPBS. As seen in Figure 3A, the cells treated with s-NNC or m-NNC displayed bright Cy5.5 fluorescence signals in the cytoplasm, which indicated that the NNCs underwent 13



decomplexation upon cellular uptake, resulting in release of the stably protected Cy5.5siRNA and recovery of the initially quenched fluorescence emission. On the other hand, free siRNA-treated cells emitted very weak fluorescence because the unprotected free siRNA molecules were rarely taken up by cells and quickly degraded in the nuclease-rich cellular environment33, matching with the above-shown serum/heparin treatment results (Figure 2B−C). A greater degree of cell internalization of the complexed siRNA was also confirmed by flow cytometry-based cell sorter analysis. The cell population emitting Cy5.5-originated fluorescence largely increased for the NNC-treated cells, compared to the free siRNA-treated case (~54−55% for both s-NNC and m-NNC with respect to the untreated cell control (Figure 3B (i-iv)). It is noteworthy that the NNCs exhibited the approximately same cell uptake efficiencies regardless of their siRNA sequences (i.e., targets), proving that the NNC system is simple yet versatile enough to co-load a variety of siRNAs on demand and to efficiently deliver those siRNAs into cells at the same time. It is the most likely that cellular uptake of the formulated NNCs was mediated through an energy-dependent endocytosis process. As shown in Figure 3C, the cell uptake of NNCs was drastically reduced after the pretreatment of cells at 4 oC for 1 h (27.6 ± 0.8%, compared to the cell uptake efficiency with no such pretreatment). At a constant temperature of 37 oC, the cell uptake of NNCs was reduced by pretreatment with chlorpromazine and amiloride, which are inhibitors of clathrin-mediated endocytosis and micropinocytosis, respectively,45–48 to a similar extent (83.2 ± 7.7% and 85.5 ± 7.2% , respectively). Methyl-β-cyclodextrin, an inhibitor of caveolae-involved endocytosis45–48 did not influence on the cell internalization of NNCs under the same condition (99.5 ± 10.5%). 14


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Figure 3. (A) Fluorescence images and (B) flow cytometry analysis showing the intracellular delivery of NNCs into LNCaP-LN3 cells after 2 h incubation (scale bar, 1 µm; i, s-NNC with Bcl-2-siRNA; ii, s-NNC with survivin-siRNA; iii, s-NNC with ARsiRNA; iv, m-NNC). (C) Relative cell uptake efficiencies under 1 h pre-treatment of cells with various inhibitors or low temperature (4 oC) before incubation with Cy5.5-labeled siRNA-containing m-NNC. *p < 0.05 and ***p < 0.001 compared with no pretreatment.

In order to evaluate the gene silencing effect of the cell-internalized siRNAs, cells were treated in the same procedure used for the cell uptake experiments and then additionally incubated for another 46 h in serum-rich media (total siRNA concentration = 17 nM). 15



The level of each gene expression was measured by using real time RT-qPCR. The result (Figure 4A) showed that free siRNAs and the scRNA-containing n-NNC only caused negligible down-regulation of genes (< 5% with respect to the NNC-untreated control level). In contrast, the mRNA expression for Bcl-2, survivin, and AR gene was distinctively reduced in the s-NNC-treated cells, reaching 62.1 ± 2.5, 62.8 ± 2.9, and 65.1 ± 4.3%, respectively. When cells were treated with the m-NNC cells, the level of each gene expression was 73.4 ± 6.1 (Bcl-2), 76.0 ± 2.0% (survivin), and 73.8 ± 5.4 (AR), respectively, which was comparable to the result obtained by using a reference siRNA delivery carrier, lipofectamine 2000. This result again demonstrates that the NNC structure is stable enough to protect and deliver the complexed siRNA into cells, successfully inducing a targeted gene silencing event even at a small amount of the individual siRNAs (i.e., ~5.7 nM).

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Figure 4. Down-regulation of (A) mRNA expression of each gene and (B) expression of each protein in LNCaP-LN3 cells after treatment with various therapeutic NNCs. **p < 0.01 compared with n-NNC.

On the basis of the above-discussed intracellular siRNA delivery and the subsequent gene silencing effect, it is expected that cells are subjected to apoptotic events in the cell death pathways related to each protein. That is, siRNAs targeting Bcl-2 and survivin lead to activation of the caspase-involved apoptosis process.8–10,49,50 The AR is also involved in the cell apoptosis mechanism associated with Bcl-2 and survivin in a complex protein network.51–53 Indeed, protein expression of the NNC-treated cells proves that the loaded siRNAs specifically induced down-regulation of each targeted protein (Figure 4B; indicated by red dotted lines). Comparatively, non-targeting n-NNC caused no substantial impact on the expression level of proteins (Supporting Information Figure S1). Considering the apoptosis-inducing anticancer effect of BZT43, it is further anticipated that the therapeutic efficacy toward LNCaP-LN3 cells can be synergistically enhanced by combining BZT and the siRNA-induced down-regulation of the target proteins. 17



Cell survival and apoptosis levels after various treatments were analyzed by MTT cytotoxicity assay and by annexin V-FITC/PI staining. The MTT cytotoxicity test (Figure 5A) revealed that free siRNAs against all of the three silencing targets were not toxic to cells. Also, there was no significant difference between the untreated cell control and the cells incubated with the siRNA/BZT-unloaded empty polymeric carrier (i.e., Pluronic F68 surfactant). When cells were treated with the scRNA-containing n-NNC, the cell survival decreased to 71.5 ± 3.9%, which is primarily attributed to the BZT toxicity. Treating cells with the Bcl-2, survivin or AR-targeting s-NNC reduced the viabilities to 63.1 ± 4.8, 63.0 ± 5.1, 63.0 ± 3.9%, respectively (about 8.5% lower than n-NNC), thus demonstrating that the gene silencing in each case enhanced the BZT-induced chemotherapy. More significantly, the triple-targeting m-NNC containing all of the three siRNAs caused greater cell death than s-NNCs, decreasing the cell survival down to 54.6 ± 4.6% (about 16.9% lower than n-NNC and additionally ~8.4% lower than s-NNCs). These results demonstrate that simultaneous shutdown of the multiple apoptosisassociated genes had a greater impact on the cell viability than a single gene silencing. We could not observe a notable toxicity of m-NNC against normal cells (e.g., NIH-3T3), which further confirms the target-specific functioning of the NNC system (Supplementary Information, Figure S2).

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Figure 5. (A) MTT cytotoxicity assay and (B) flow cytometry-analyzed apoptosis assay of LNCaP-LN3 cells treated with various NNCs. The number in the right bottom of the quadrant graph indicates the percentage of the early apoptotic cells. *p < 0.05, **p < 0.01, ***p < 0.001 compared with n-NNC or free BZT; #p < 0.05; n.s. compared with untreated control cells.

In response to the promising result of the cytotoxicity of the NNCs with various siRNA combinations, we continued to figure out the apoptotic cell populations for each treatment by using flow cytometry analysis (Figure 5B). The percentage of the apoptotic cells was estimated by analyzing the resulting quadrant graph showing annexin V-FITCpositive cells. No PI-positive cells appeared for all treatments, indicating the absence of cell necrosis event. For comparison, 6.9% of the apoptotic cells were detected for the nNNC-treated cells, reflecting the co-delivered BZT toxicity. The m-NNC-incubated cells resulted in 14.0% of the apoptotic cells, while s-NNCs resulted in only ~10%. To our 19



interest, all of the tested s-NNCs resulted in similar apoptotic cell populations with ignorable differences. This tendency again rationalizes our choice of siRNAs in the NNCs, indicating that all of Bcl-2, survivin, and AR gene play a key role in the survival of LNCaP-LN3 cells, in good agreement with the previous literatures3–5,51,53. For all of the treated cells, it appeared that the resulting cells were only in the early apoptosis stage, which is probably because of the relatively low siRNA and BZT quantity. Dual genetargeting NNCs that were composed of two of Bcl-2, survivin, and AR-siRNAs, also showed enhanced therapeutic effect on LNCaP-LN3 cells under the same condition, resulting in the cell apoptotic levels between the single-targeting and the triple-targeting NNCs (Supplementary Information, Figure S3). When a higher concentration of siRNA (35 or 70 nM) was tested at a constant N/P charge ratio, a similar multi-targeting effect was observed with overall decrease in the cell survival (Supplementary Information, Figure S4A). When the NNC-treated cells were incubated for different periods of time (i.e., 24 h or 72 h) at a fixed siRNA dose (17 nM), no significant difference was shown in the cell viability compared to the current condition (i.e., 48 h incubation) (Supplementary Information, Figure S4B). The current system is versatilely applicable to various types of human cancer cells such as breast cancer cell line (e.g., MDA-MB-231) as well as a different cell line of prostate cancer (e.g., PC-3) (Figure 6). In the case of PC-3 cells, AR-targeting siRNA did not have a meaningful impact on the cell survival because of the androgen-independence of PC-3 cells54, further validating a gene-specific therapeutic performance of the cell-internalized NNC system. Taken together, the presented results demonstrate that that our multiple gene-targeting drug delivery system is capable of significantly promoting apoptosis, 20


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suggesting that more siRNA combinations can be incorporated into the current nanocomplex platform for further developing multi-targeting siRNA therapeutics with higher efficiency.

Figure 6. (A) MTT cytotoxicity test of PC-3 prostate cancer cells with various NNCs (total siRNA concentration = 35 nM; N/P = 8). (B) MTT cytotoxicity assay and (C) Bcl2 gene silencing of MDA-MB-231 breast cancer cells treated with n-NNC or Bcl-2 targeting s-NNC (total siRNA concentration = 35 or 70 nM; N/P ratio = 8).

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CONCLUSIONS We have demonstrated that the concurrent intracellular delivery of three different siRNAs along with an anticancer drug BZT could be utilized for improving treatment of prostate cancer by silencing multiple apoptosis-related genes. In order to make the current system more clinically applicable, further research would involve incorporation of cell-specific targeting surface-functional groups and stability of the tiny Pluronicsurfaced system during systemic circulation, not to mention various apoptosis-related proteins and the related types of cancer. With such considerations, the presented report would offer a facile multiple-targeted strategy to eradicate prostate cancer with even higher efficiency.

ASSOCIATED CONTENT Supporting Information. Supplemental information (i.e., cytotoxicity of the NNCtreated normal cells, cell studies for dual-targeting NNCs, cytotoxicity of the cells treated with m-NNC with a higher siRNA concentration or under a different incubation time, and Ct values) is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Tel: +82-2-958-5924 Fax: +82-2-958-5909

Funding Sources

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This work was supported by grants from the Korea Health Industry Development Institute (HI15C1540), the Development of Platform Technology for Innovative Medical Measurements Program from Korea Research Institute of Standards and Science (KRISS–2017-GP2017-0020), and the Intramural Research Program of KIST. ACKNOWLEDGEMENT The authors thank Dr. Yeong-Su Jang and Dr. Mihue Jang for helpful discussion during the gene silencing experiment, and specially thank Dr. Seo-Young Kwak for her kind help during Western blotting.

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Simultaneous Delivery of Electrostatically Complexed Multiple Gene

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