Journal of Controlled Release 192 (2014) 114–121

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Triple negative breast cancer therapy with CDK1 siRNA delivered by cationic lipid assisted PEG-PLA nanoparticles Yang Liu a, Yan-Hua Zhu a, Cheng-Qiong Mao a, Shuang Dou a,b, Song Shen a, Zi-Bin Tan a, Jun Wang a,b,c,⁎ a b c

CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Life Sciences and Medical Center, University of Science & Technology of China, Hefei, Anhui 230027, China Hefei National Laboratory for Physical Sciences at Microscale, Hefei, Anhui 230027, China High Magnetic Field Laboratory of CAS, University of Science and Technology of China, Hefei, Anhui 230026, China

a r t i c l e

i n f o

Article history: Received 27 November 2013 Accepted 2 July 2014 Available online 10 July 2014 Keywords: Triple-negative breast cancer Synthetic lethality siRNA delivery Cationic lipid assisted nanoparticle Cyclin-dependent kinase 1

a b s t r a c t There is no effective clinical therapy yet for triple-negative breast cancer (TNBC) without particular human epidermal growth factor receptor-2, estrogen and progesterone receptor expression. In this study, we report a molecularly targeted and synthetic lethality-based siRNA therapy for TNBC treatment, using cationic lipid assisted poly(ethylene glycol)-b-poly(D,L-lactide) (PEG-PLA) nanoparticles as the siRNA carrier. It is demonstrated that only in c-Myc overexpressed TNBC cells, while not in normal mammary epithelial cells, delivery of siRNA targeting cyclin-dependent kinase 1 (CDK1) with the nanoparticle carrier (NPsiCDK1) induces cell viability decreasing and cell apoptosis through RNAi-mediated CDK1 expression inhibition, indicating the synthetic lethality between c-Myc with CDK1 in TNBC cells. Moreover, systemic delivery of NPsiCDK1 is able to suppress tumor growth in mice bearing SUM149 and BT549 xenograft and cause no systemic toxicity or activate the innate immune response, suggesting the therapeutic promise with such nanoparticles carrying siCDK1 for c-Myc overexpressed triple negative breast cancer. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death in females worldwide [1]. Breast cancer is a heterogeneous disease with different morphologies, molecular profiles, clinical behaviors and responses to therapy [2]. The triple-negative breast cancer (TNBC) comprises about 15% of breast cancer cases and is immunohistochemically defined by a lack of expression of human epidermal growth factor receptor 2 (HER2), estrogen receptor (ER) and progesterone receptor (PR) [3]. TNBC is characterized by its biological aggressiveness, poor prognosis and lack of a therapeutic target in contrast to hormonal receptor positive and HER2-positive breast cancers [4]. Clinically relevant biomarkers have been used to guide the application of targeted therapeutics for receptor positive breast cancers. For example, in regard to ER-positive breast cancer, tamoxifen is an effective targeted therapy for hormone [5], while Herceptin is a targeted therapy for HER2-positve breast cancer [6]. Unfortunately, up to now, there is no targeted therapy for TNBC. The only modality of systemic therapy available for TNBC is chemotherapy with platinum [7] or epirubicin and paclitaxel alone or together [8], providing limited choices with unignorable side effects.

⁎ Corresponding author at: School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, China. Tel.: +86 551 63600335; fax: +86 551 63600402. E-mail address: [email protected] (J. Wang).

http://dx.doi.org/10.1016/j.jconrel.2014.07.001 0168-3659/© 2014 Elsevier B.V. All rights reserved.

Much attention has been paid to the investigation of biology insights and targeted therapy of TNBC. For example, E2F-regulated gene Chk1 is found to be highly expressed [9] and the DNA damage signaling kinase ATM is aberrantly reduced [10] in TNBC. β-catenin pathway activation, 53BP1 loss and defective repair of oxidative DNA damage are also reported in TNBC [11–13], providing further understanding of signal pathways that distinguish TNBC from other breast cancer subtypes. It has also been reported that a successful phase II study with carboplatin and iniparib shows efficacy and safety in patients with metastatic TNBC [14], owing to the discoveries in signal pathway studies. Nevertheless, targeted therapy for TNBC is still far from clinical application. Recent study by Goga et al. revealed that c-Myc, as an important oncoprotein, is overexpressed in TNBC [15,16]. The elevated c-Myc expression makes TNBC more proliferative and syntheticallly lethal with cyclin-dependent kinase 1 (CDK1) inhibition [15,16]. The concept of synthetic lethality refers to that mutation of one of two genes is compatible with viability but mutation of both leads to death [17]. Thus, targeting a gene that is synthetically lethal to a cancer-relevant mutation should kill only cancer cells and spare normal cells, and synthetic lethality therefore provides a conceptual framework for the development of cancer-specific cytotoxic agents [18–26]. It must be known that c-Myc inhibition is not technically available now because as a transcriptional factor the special structure of c-Myc augments the difficulty of its inhibitor design [16], and moreover, c-Myc inhibition in normal growing tissue such as skin, testis and GI tract may cause severe cytotoxicity [27]. On the other hand, CDK inhibitors have been developed

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over 20 years but none of them has been approved for clinical application [28]. Therefore, a bypass strategy is necessary for CDK1 inhibition in TNBC treatment. Small interfering RNA (siRNA) has been rapidly developed as a promising candidate for treatment of numerous diseases (e.g. neurodegenerative disorders, cancer and infectious diseases) [29–34], particularly with the assistance of a delivery system, which can be a lipidbased or polymer-based system, a peptide conjugate, single-chain fragment variable antibody fusion protein system and so on [35–39]. We have previously reported micelleplex and anti-Her2 single-chain antibody mediated siRNA delivery for cancer therapy [40,41]. In this study, as shown in Fig. 1, by using a cationic lipid assisted PEG-PLA nanoparticle system previously developed by us [42], we validated that systemic delivery of CDK1 siRNA (siCDK1) can be a molecularly targeted therapy of TNBC, which takes advantage of the synthetic lethal interaction between c-Myc and CDK1 in TNBC cells but not in normal mammary epithelial cells.

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2.3. siRNA All siRNA duplexes with 2′-OMe modification targeting CDK1 (siCDK1), c-Myc (siMyc) and negative control siRNA (siNC) were obtained from GenePharma Co. (Shanghai, China); their oligonucleotide sequences were shown below. Fluorescently labeled FAM-siRNA and Cy5-siRNA from GenePharma Co. were synthesized by modification of the 3′-end of the sense strand with fluorescein. siCDK1: sense strand, 5′-GGCACUGAAUCAUCCAUAUTT-3′; antisense strand, 5′-AUAUGGAUGAUUCAGUGCCTT-3′; siMyc: sense strand, 5′-CACCUAUGAACUUGUUUCATT-3′; anti-sense strand, 5′-UGAAACAAGUUCAUAGGUGTT-3′; siNC: sense strand, 5′-UUCUCCgAACgUgUCACgUdTdT-3′; anti-sense strand, 5′-ACgUgACACgUUCggAgAAdTdT-3′. 2.4. Preparation and characterization of nanoparticles with siRNA encapsulation

2. Materials and methods 2.1. Materials Poly(ethylene glycol)-block-poly( D , L -lactide) (PEG 5K -PLA 11K ) and the cationic lipid N,N-bis(2-hydroxyethyl)-N-methyl-N-(2cholesteryoxycarbonyl-aminoethyl) ammonium bromide (BHEMChol) were synthesized and purified according to previously reported procedure [42]. Insulin, hydrocortisone, cholera toxin, dimethyl sulfoxide (DMSO) and methyl thiazol tetrazolium (MTT) were purchased from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum (FBS) was purchased from Excell Bio (Shanghai, China). LipofectamineTM RNAi MAX, DMEM/F-12 medium, RMPI 1640 medium, horse serum and recombinant human epidermal growth factor (EGF) were obtained from Invitrogen (Carlsbad, CA).

2.2. Cell culture Human triple negative breast cancer cell line SUM149 from Asterand (Detroit, MI) was maintained in DMEM/F-12 medium with 5% FBS, 5 μg/mL insulin and 1 μg/mL hydrocortisone. Another human triple negative breast cancer cell line BT549 from the American Type Culture Collection (ATCC) (Manassas, VA) was grown in RMPI 1640 medium with 10% FBS and 5 μg/mL insulin. Human mammary epithelial cells MCF-10A from ATCC was cultured in DMEM/F-12 medium supplemented with 5% horse serum, 20 ng/mL EGF, 5 μg/mL insulin, 0.5 μg/mL hydrocortisone, 100 ng/mL cholera toxin. All the cell lines were cultured in a humidified atmosphere containing 5% CO2 at 37 °C.

Nanoparticles loaded with siRNA, denoted as NPsiRNA, were prepared by a double emulsion–solvent evaporation technique as previously described. Briefly, an aqueous solution of siRNA (0.2 mg) in 25 μL of RNase free water was emulsified by sonication for 60 s over an ice bath in 0.5 ml of chloroform containing 1.0 mg of BHEM-Chol, 25 mg of PEG 5k-PLA11k. This primary emulsion was further emulsified in 5 mL of RNase free water by sonication (80 W for 1 min) over an ice bath to form a water-in-oil-in-water emulsion. The mixture was then added to 50-mL round-bottom flask, and the solvent (chloroform and RNase free water) was concentrated under reduced pressure by a rotary evaporator to a volume of 1 mL. siRNA encapsulation efficiency, zeta potential and particle size measurements were conducted according to a high-performance liquid chromatography analysis procedure or using a Malvern Zetasizer Nano ZS90, as previously reported [43]. To confirm the encapsulation of siRNA, we performed the RNase protection assay. 20 pmol free siRNA or siRNA encapsulated by nanoparticles (NPsiRNA) were incubated with an equal volume of 50 μg/mL RNase A (Sangon Biotech, Shanghai, China) for 4 h at 37 °C. After enzyme treatment, an equal amount of NPsiRNA was incubated with heparin sodium solution (5 mg/mL, Sangon Biotech, Shanghai, China) for 10 min to exchange the siRNA from nanoparticles. The free siRNA, NPsiRNA and NPsiRNA treated with heparin were electrophoresed on a 20% polyacrylamide gel electrophoresis (PAGE) at a constant voltage of 50 V for 4 h in Tris/borate/EDTA buffer (TBE buffer; 89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.3). The siRNA bands were visualized with GelRed (Biotium, Hayward, CA) staining under a UV transilluminator at a wavelength of 365 nm.

2.5. Cellular uptake

Fig. 1. Schematic illustration of cancer therapy of triple negative breast cancer (TNBC) over-expressed c-Myc with CDK1 siRNA delivered by cationic lipid (BHEM-Chol) assisted poly(ethylene glycol)-b-poly(D,L-lactide) (PEG5K-PLA11K) nanoparticles.

SUM149, BT549 or MCF-10A cells (5 × 104) were seeded into 24well plates and cultured for 24 h to reach 50% confluence. The cells were then incubated at 37 °C with NPFAM-siRNA suspended in complete culture medium at a polymer concentration varied from 0.09 mg/mL to 0.55 mg/mL, and the corresponding concentration of FAM-siRNA were from 50 nM to 200 nM. FAM-siRNA (50 nM) transfected by Lipofectamine RNAi MAX and free FAM-siRNA (200 nM) were used as controls. After 2 h, the cells were trypsinized by 0.25% Trypsin-EDTA solution (Invitrogen, Carlsbad, CA) and washed twice by ice-cold phosphate buffered saline (PBS, 0.01 M, pH 7.4). The non-specific extracellular fluorescence was quenched by suspending the cells in 0.2% Trypan Blue for 2 min, then the cells were analyzed by flow cytometer using a FACS Calibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ) and analyzed by FlowJo Software (TreeStar, San Carlos, CA).

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2.6. In vitro gene silencing SUM149, BT549 or MCF-10A cells (10 × 104) were seeded into 6-well plates and cultured for 24 h. The cells were then incubated at 37 °C with NPsiCDK1 suspended in complete culture medium at concentrations of siCDK1 from 50 nM to 200 nM for 72 h. The total RNA of cells was then collected using RNAiso Plus (TaKaRa, Dalian, China). One microgram of total RNA was transcribed into cDNA using PrimeScriptTM RT reagent Kit (TaKaRa). To assess levels of CDK1 mRNA, real-time quantitative PCR (qPCR) was performed using FastStart Universal Probe Master (Roche Applied Science, Indianapolis) with forward 5′-AAGCTGGCTCTT GGAAATTGA-3′ and reverse 5′-ATGGCTACCACTTGACCTGTAGTT-3′ CDK1 primers. CDK1 mRNA levels were normalized against the housekeeping gene GAPDH using forward 5′-ATCAAGAAGGTGGTGAAGCAGG CA-3′ and reverse 5′-TGGAAGAGTGGGAGTTGCTGTTGA-3′ primers. PCR parameters consisted of 60 s of Taq activation at 95 °C, followed by 40 cycles of PCR at 95 °C × 20 s, and 1 cycle of 95 °C × 15 s, 57 °C × 60 s and 95 °C × 15 s. CDK1 mRNA levels were finally normalized to those of cells cultured in culture medium with PBS treatment. For Western blot analysis, cells transfected for 72 h as above were washed twice with cold PBS, and lysized in 50 μL of lysis buffer (50 mM NaCl, 50 mM EDTA, 1% Triton X-100) containing a protease inhibitor cocktail (Roche, Indianapolis, IN). The cell lysates with total protein (80 μg) was electrophoresed and further analyzed according to a standard process described previously [44]. Anti-CDK1 (1:1000) (Abcam, Austin, TX), anti-cleaved PARP (1:1000) (Cell Signaling Technology, Beverly, MA), anti-c-Myc (1:1000) (Cell Signaling Technology, Beverly, MA) or anti-β-Actin (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA) was used in the analyses. Goat anti-mouse or goat anti-rabbit HRP-conjugated antibodies (1:10,000) (Santa Cruz Biotechnology, Santa Cruz, CA) were used as the secondary antibody. The membranes were visualized using the ECL system (Pierce, Rockford, IL) and the expression levels of protein were normalized to actin protein expression levels. 2.7. CDK inhibitors Purvalanol A (Sigma-Aldrich, St. Louis, MO) was constituted in 100% DMSO and used at a concentration of 10 μM. Dinaciclib (Sigma-Aldrich, St. Louis, MO) was reconstituted either in 100% DMSO for cell culture use (10 nM) or in 20% HPBCD (hydroxypropyl cyclodextrin, Aladdin, Shanghai, China) at a dose of 50 mg/kg for mouse studies. 2.8. Proliferation assays The reduction in cell proliferation associated with decreasing CDK1 expression was assessed by MTT viability assay and colony formation assay in SUM149, BT549 and MCF-10A cells. For MTT viability assay, cells were seeded in 96-well plates at 5000 cells per well in 100 μL of complete culture medium for 24 h. Then various concentrations of NPsiCDK1 or CDK inhibitors were added and cells were incubated for an additional 72 h. The MTT stock solution was then added to each well to achieve a final concentration of 1 mg/mL, with the exception of the wells used as a blank, to which the same volume of PBS (0.01 M, pH 7.4) was added. After incubation for an additional 2 h, 125 μL of extraction buffer (20% SDS in 50% DMF, pH 4.7) was added to the wells and incubated 6 h at 37 °C. The absorbance was measured at 570 nm using a Bio-Rad 680 microplate reader. Cell viability was normalized to that of cells cultured in the culture medium with PBS treatment. For the colony formation assay, cells were treated for 72 h with various concentrations of NPsiCDK1 or CDK inhibitors followed by trypsinization and suspension in culture medium. One thousand cells of each group were plated in each well of 6-well plates. Cells were then incubated at 37 °C with 5% CO2 for 10 to 14 days. After removing the medium, cells were washed twice with PBS followed by staining with 0.5% crystal violet (Sangon Biotech, Shanghai, China) in ethanol

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for 5 min. Then, the cells were washed twice with distilled water and visualized. 2.9. Cell cycle analysis and cell apoptosis assay Cells were treated by various concentrations of NPsiCDK1 or CDK inhibitors for 72 h. After treatment, for cell cycle analysis, cells were collected and handled with DNA Reagent kit (BD PharMingen, San Diego, CA) following the manufacturer's protocol. Samples were acquired by flow cytometer using a FACS Calibur flow cytometer and gated to exclude debris and doublets. Cell cycle distribution was analyzed by FlowJo Software. For cell apoptosis assay, cells were collected and treated by Annexin-V-FITC FLUOS Staining Kit (Roche Applied Science, Indianapolis, IN). Cells were acquired by flow cytometer using a FACS Calibur flow cytometer and analyzed by FlowJo Software that Annexin-V positive population was considered as apoptotic cells. 2.10. Biodistribution of siRNA encapsulated nanoparticles in vivo For in vivo fluorescence imaging, female BALB/c nude mice bearing SUM149 tumors received an i.v. injection of either PBS, Cy5-siRNA encapsulated nanoparticles, or equivalent free Cy5-siRNA (10 μg). The animals were placed onto the warmed stage inside the IVIS Lumina light-tight chamber and anesthesia was maintained with 2.5% isoflurane. Image acquisition was performed at different time intervals on a Xenogen IVIS® Lumina system (Caliper Life Sciences, MA, USA). The animals were sacrificed 72 h after the injection of Cy5-siRNA encapsulated nanoparticles or free Cy5-siRNA and the organs were also imaged. The results were analyzed using Living Image® 3.1 software (Caliper Life Sciences, MA, USA). The distributions of Cy5-siRNA in tumor cells were analyzed using confocal laser scanning microscopy. Twelve hours after the injection of either PBS, Cy5-siRNA encapsulated nanoparticles, or equivalent free Cy5-siRNA (20 μg), the mice were sacrificed and the tumors were fixed in 4% paraformaldehyde overnight at 4 °C, then immersed overnight in 30% sucrose solution. Tumor tissues were sectioned (6 μm thick), permeated with 0.1% Triton X-100 and counterstained with Alexa Fluor® 488 (Invitrogen, Carlsbad, CA) and DAPI (Sigma-Aldrich, St. Louis, MO), then imaged using a Zeiss LSM 710 confocal microscope using a 40× objective. 2.11. Quantification of siRNA in tumor after the systemic administration To determination the siRNA in tumor, we performed the stem-loop RT-PCR for siRNA as Susan Magdaleno's group previously reported [45]. Three mice with SUM149 tumor were injected with free siCDK1 and three mice were injected with NPsiCDK1 with an equal amount of siCDK1 (20 μg). After 12 h, the mice were sacrificed and the tumors were harvested and immediately frozen on dry ice. The tumors were then placed directly into the lysis buffer and total RNAs were isolated using the mirVanaTM miRNA Isolation Kit (Life Technologies, Carlsbad, CA) according to the manufacturer's protocol. The assay for quantification of siRNAs consisted of two steps: reverse transcription (RT) and real-time PCR. The RT primer for siCDK1's sense strand features the similar stem-loop design which was provided by the Small RNA Assay Kit (Applied Biosystems, Foster City, CA). Typically, 10 ng of total RNA was used for RT reaction (GoscriptTM Reverse Transcription System, Promega, Madison, WI) following the manufacturer's protocol. Realtime PCR was performed using a standard TaqMan® PCR protocol on Agilent Mx3000P qPCR System. The 20 μL PCR reaction mixture included 2 μL RT products, 1 × TaqMan® Universal PCR Master Mix, 0.2 μM TaqMan® probe. The reaction was incubated at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. In order to determine the number of molecules that was delivered to the tumors, experiments were performed, by adding known amounts of siRNA (250, 125, 50, 10, 5, 1, 0.5, 0.2 and 0.1 pmols) to disrupted tumor lysate

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after homogenization. This allowed determining the amount of siRNA delivered based on comparison of Ct values between injected and standard samples. 2.12. Human triple-negative breast xenograft tumor model and tumor suppression study Female BALB/c nude mice (6 weeks old) were purchased from Vital River Laboratories (Beijing, China) and all animals received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals. The procedures were approved by the University of Science and Technology of China Animal Care and Use Committee. The xenograft tumor models were generated by subcutaneous injection of 100 μL SUM149 cells (5 × 106 per mouse) or BT549 cells (5 × 106 per mouse) mixed in 50% Matrigel (Becton Dickinson, Bedford, MA) into the second right mammary fat pad of nude mice. When the tumor volume was about 50 mm3, the mice were randomly divided, and treated by i.v. injection of PBS, free siCDK1, NPsiNC, NPsiCDK1, or i.p. injection of dinaciclib (50 mg/kg, twice per week). Tumor growth was monitored by measuring the perpendicular diameter of the tumor using calipers. The estimated volume was calculated based on the following equation: tumor volume = 1/2 × width2 × length. 2.13. In vivo gene silencing One day after the last treatment, the animals were sacrificed and the tumors were excised for real time PCR and Western blot. CDK1 antibody (Biolegend, San Diego, CA) was used for the detection of CDK1 expression in vivo. 2.14. Mouse cytokine and interferon response analyses After the last day of treatment, serum was collected and assayed for mouse IFN-γ, TNF-α, IL-6, AST, ALT and LDH using quantitative, enzyme-linked immunosorbent assay kit, following validation of each ELISA according to the manufacturer's instructions. Absorbance was read using a Bio-Rad microplate reader (Hercules, CA, USA) at 450 nm. 2.15. Statistical analysis

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significant gene silencing effect [46]. We have previously demonstrated that cationic lipid assisted polymer nanoparticles can mediate siRNA entrance to cancer cells and siRNA molecules can at least partially escape from the lysosomes [42]. To demonstrate that the nanoparticles can transport siRNA molecules into TNBC cells and normal mammary epithelial cells, fluorescence-labeled FAM-siRNA was encapsulated into nanoparticles and the resulting NPFAM-siRNA was incubated with SUM149, BT549 and MCF-10A cells at 37 °C for 2 h. As shown in Fig. 2A, FACS analyses indicated a dose-dependent intracellular FAM fluorescence of cells with the delivery of nanoparticles, while incubation of cells with free FAM-siRNA resulted in weak fluorescence in cells at background level, illustrating that nanoparticles can promote the transportation of FAM-siRNA into both TNBC and mammary epithelial cells. It may be noticed that Lipofectamine RNAi MAX mediated the most effective siRNA transportation to cells at a relatively lower dose; however, as a transfection agent designed for in vitro experiments, it could not be effectively used for further in vivo applications, particularly for systemic siRNA delivery. The biological activity of siRNA delivered by nanoparticles was further investigated with siCDK1. As shown in Fig. 2B, CDK1 mRNA and protein levels analyzed by qPCR and Western blot 72 h post transfection showed that free siCDK1, empty nanoparticles (NP) and NPsiNC failed to inhibit CDK1 expression in SUM149, BT549 and MCF-10A cells. However, NPsiCDK1 significantly down-regulated CDK1 mRNA and protein expression in all types of cells, which was again in a siCDK1 dosedependent manner from 50 nM to 200 nM. siCDK1 delivery by NPsiCDK1 at a concentration of 200 nM significantly knocked down CDK1 mRNA level in SUM149 cells, BT549 cells and MCF-10A cells to a level of 28.0 ± 1.4%, 37.6 ± 0.3% and 32.2 ± 1.7%, respectively, which were comparable with CDK1 gene silencing levels induced by siCDK1 (50 nM) carried by Lipofectamine RNAi MAX. 3.3. Synthetic lethality to TNBC cells by NPsiCDK1 It was found that c-Myc, a typical oncoprotein, was overexpressed in triple negative breast cancer and the overexpression of c-Myc made TNBC more proliferative but synthetically lethal with CDK1 inhibition [15]. We confirmed that as TNBC cells, SUM149 and BT549 cells overexpressed c-Myc oncoprotein when compared with mammary

The statistical significance of treatment outcomes was assessed using Student's t-test (two-tailed); p b 0.05 was considered statistically significant in all analyses (95% confidence level). 3. Results and discussion 3.1. NPsiRNA preparation By using a biocompatible and biodegradable PEG5K-PLA11K copolymer, with the assistance of BHEM-Chol, we prepared the siRNAencapsulated nanoparticles (NPsiRNA) and blank nanoparticles (NP) with an average diameter of 146.4 ± 5.9 nm and 128.8 ± 1.6 nm, respectively. Such siRNA-encapsulated nanoparticles exhibited high encapsulation efficiency of siRNAs (90.9%) and a sufficient loading content of siRNAs (0.69%, w/w) that were precisely determined by HPLC. The encapsulation of siRNA was also confirmed by the RNase protection assay. As shown in Figure S1, siRNAs were encapsulated by the nanoparticles with high encapsulation rate as there was little free siRNA detected after electrophoresis, while with the treatment of RNase A, the nanoparticle can protect the siRNA from enzymatic degradation, which showed the advantage of using the siRNA delivery system. 3.2. NPsiCDK1 mediated CDK1 expression down-regulation in vitro The siRNA molecules must be transported into cells before they can function in RNAi, and more siRNA in the cytosol would induce more

Fig. 2. Flow cytometric analyses of cells treated with FAM-siRNA-encapsulated nanoparticles at various concentrations for 2 h in 37 °C (A). NPsiCDK1-mediated gene silencing in SUM149, BT549 and MCF-10A cells determined by real time PCR and Western blot (B). LiposiCDK1 represents the transfection with Lipofectamine RNAi MAX at a siCDK1 dose of 50 nM. The dose of NPsiNC was 200 nM. NP represents the empty nanoparticles without any siRNA encapsulation.

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epithelial MCF-10A cells by Western blot analyses (Figure S2A). It was further demonstrated that c-Myc could not be a proper gene for molecularly targeted therapy of TNBC. As shown in Figure S2B, transfection of SUM149 and BT549 cells with siMyc carried by Lipofectamine RNAi MAX significantly down-regulated c-Myc gene expression, but it only induced very slight effect on cell death and cell apoptosis. Some cytotoxity was also observed in MCF-10A cells, indicating that downregulation of c-Myc would damage normal cells (Figure S2C and S2D). Although Myc is a key regulator of cell growth, proliferation, and survival which is deregulated in various cancer cells, there exist the following obstacles to develop Myc as a good therapeutic target [27,47]. First, Myc exerts its biological influence through protein–protein and protein–DNA interactions that have proven difficult to disrupt with small molecules [48–50]. Second, aberrant Myc expression is not due to mutation in the Myc gene itself but a consequence of its induction by ‘upstream’ oncogenic signals. Finally, Myc is essential for proliferation and stem cell compartment maintenance of regenerative adult tissues such as the gastrointestinal tract, skin and bone marrow [51, 52]. Together with the results as shown in Figure S2, these suggested that c-Myc might not be a potent target gene for TNBC therapy. Although c-Myc may not be a proper target for TNBC treatment, down-regulation of CDK1 can lead to synthetic lethality in TNBC cells with elevated c-Myc expression. To investigate whether TNBC cells are sensitive to NPsiCDK1 mediated CDK1 expression inhibition, we measured the cell viability following siCDK1 delivery to the cells, which was compared with treatment of purvalanol A, a small molecular inhibitor of CDK1 [53]. As shown in Fig. 3A, inhibition of CDK1 expression with NPsiCDK1 reduced the viability of SUM149 and BT549 cells. However, without elevated c-Myc expression, MCF-10A did not respond to CDK1 expression silencing by NPsiCDK1. The low viability of SUM149 and BT549 cells at 49.2% and 56.8% was observed when the cells were treated with NPsiCDK1 at a siCDK1 concentration of 200 nM, while with the same treatment, the viability of MCF-10A cells remained above 95%. Cell treatment with LiposiCDK1 led to more significant cell death at lower siCDK1 dose but it induced non-specific cytotoxicity effect even to MCF-10A cells. It must be mentioned that the CDK1 inhibitor purvalanol A induced severe cytotoxicity to both TNBC cells and normal mammary epithelia MCF-10A cells, which indeed cause other non-CDK kinase inhibition [15]. On the other hand, nanoparticles alone without

siCDK1 encapsulation did not show any cytotoxic effect on all the cells. Similar phenomenon was observed in the colony formation assay, which showed decreased clonogenicity in SUM149 and BT549 cells following treatment with NPsiCDK1 while sparing the normal cell line MCF-10A (Fig. 3B). These results suggested that the inhibition of cell proliferation is the result of a synthetic lethal relationship between c-Myc overexpression and CDK1 inhibition. Knockdown of CDK1 also induced apoptosis in TNBC cells. As shown in Fig. 4A, NPsiCDK1 induced cell apoptosis in a dose-dependent manner in c-Myc overexpressed SUM149 and BT549 but had little effect on normal breast mammary epithelia MCF-10A cells. NPsiCDK1 at a siCDK1 concentration of 200 nM caused 32.2% cell apoptosis in SUM149 cells and 29.5% cell apoptosis in BT549 cells, which was comparable with LiposiCDK1 induced cell apoptosis. Purvalanol A caused cell apoptosis in a large number of TNBC cells unsurprisingly but it also induced apparent cell apoptosis in normal breast cells. Similar to the cell proliferation assay described above, neither blank nanoparticles nor free siCDK1 induced significant cell apoptosis. Poly(ADP-ribose) polymerase (PARP) is a family of proteins involved in a number of cellular processes mainly including DNA repair and programmed cell death. Cleavage of PARP produced 89 kD protein fragment (c-PARP) which was a special marker of apoptosis [54]. The c-PARP protein levels in SUM149, BT549 and MCF-10A 72 h after different treatments were analyzed. As Fig. 4B shows, NPsiCDK1 led to a siCDK1 dose-dependent accumulation of c-PARP in SUM149 and BT549, but had little effect on MCF-10A cells. Purvalanol A induced c-PARP accumulation not only in Myc-overexpressed TNBC cells but also in normal human breast cells. This non-specific effect eventually limits its application in vivo. Thus, therapy strategy based on this synthetic lethal interaction with NPsiCDK1 was not only effective for TNBC cells with elevated c-Myc expression, but also did little harm to normal cells such as human mammary epithelial cells. Cyclin-dependent kinases are a conserved family of protein kinases that regulate orderly progression through the different phases of the cell cycle [55]. In mammalian cell division, there is clear evidence that CDK1 is essential and activated CDK1 at the G2-M transition is required for early mitosis [56]. Thus, there might be a concern in that CDK1 down-regulation mediated by NPsiCDK1 would cause cell cycle arrest, which further induces cell apoptosis. Indeed, in our experiments, it was observed that in SUM149, BT549 and normal MCF-10A cells,

Fig. 3. Cell viability (A) and colony formation (B) of SUM149, BT549 and MCF-10A cells with different treatments.

Fig. 4. Cell apoptosis induced by different treatments (A) and c-PARP expression change in different cells after treatment (B).

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delivery of siCDK1 induced dose-dependent accumulation of G2-M arrested cells (Figure S3), consistent with the essential role of CDK1 in cell cycle regulation. However, in c-Myc overexpressed TNBC cells, due to the role of c-Myc in promoting cell proliferation, the cell cycle arrest caused much more inhibition of cell viability and cell apoptosis than in normal breast cell line [15,57]. 3.4. Biodistribution of siRNA encapsulated nanoparticles in vivo The nanoparticle possesses a PEG protection corona, thus is expected to be beneficial for its accumulation in the tumor site through the “enhanced permeation and retention” (EPR) effect, which is also called passive targeting of nanoparticulate delivery system [58]. To demonstrate this, we administrated Cy5-siRNA encapsulated nanoparticles and free Cy5-siRNA by i.v. injection to SUM149 tumorbearing mice and monitored the fluorescence distributions in situ by fluorescence imaging. As shown in Fig. 5A, Cy5-siRNA was rapidly eliminated from the animal's body following the injection when it was not delivered by nanoparticles. The fluorescence was merely observed in the animal at 12 h after receiving an injection of free Cy5-siRNA. On the other hand, the Cy5-siRNA could be detected as long as 72 h post injection at the tumor site when encapsulated by the nanoparticles. Moreover, the fluorescence image of major organs at 72 h after the administration also showed the accumulation of Cy5-siRNA when carried by the delivery system (Fig. 5B). This demonstrated that the nanoparticles could indeed enhance siRNA accumulation in tumor tissues. To further examine whether nanoparticles can

Fig. 5. (A) Fluorescence images of SUM149 xenograft-bearing mice after intravenous (i.v.) injection of PBS (left), NPCy5-siRNA (middle) or free Cy5-siRNA (right) at different time. The tumor is at the second right mammary fat pad of mice. (B) Fluorescence images of major organs after i.v. injection of PBS, NPCy5-siRNA or free Cy5-siRNA at 72 h. (C) CLSM images show the distribution of siRNA in tumor following i.v. injection of PBS, NPCy5-siRNA or free Cy5-siRNA. siRNAs were labeled with red fluorescence. DAPI (blue) and Alexa Fluor 488 phalloidin (green) were used to stain cell nucleus and cytoskeleton, respectively.

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deliver siRNAs into tumor cells following its accumulation in tumor tissue, we sectioned the tumor tissues 12 h post injection. Confocal images exhibited strong Cy5-siRNA fluorescence when injected with NPCy5-siRNA in the tissue section (Fig. 5C); while little fluorescence was observed in the tumor injected with free Cy5-siRNA. Finally, we determined the siRNA content in the tumor issues treated with different formulations by stem-loop RT-PCR. The NPsiCDK1 exhibited a dominant accumulation in the tumor issues compared with free siCDK1 as shown in Figure S4. The nano-sized delivery system did help siRNAs accumulate in tumor tissue after systemic administration as proved above.

3.5. Tumor growth inhibition mediated by systemic delivery of NPsiCDK1 The tumor growth curve in Fig. 6A showed that systemic administration of NPsiCDK1 at a dose of 2 mg/kg siCDK1 per injection every other day significantly inhibited SUM149 tumor growth, with comparable effect to dinaciclib (CDK1 inhibitor widely used in vivo, whose cytotoxity is shown in Figure S5) injection, whereas neither NPsiNC at the same siRNA dose nor blank NP at the same concentration affected the tumor growth, indicating that NPsiCDK1 displayed a sequence-specific antitumor activity in vivo. Similar inhibition to BT549 tumor growth was observed (Fig. 6B). To explore how siRNA dose and administration frequency affected the outcome of tumor inhibition, we performed three doses of NPsiCDK1 at the same frequency with injection of 2 mg/kg, 1 mg/kg or 0.5 mg/kg on every second day, and three frequencies of administration at the same NPsiCDK1 dose of 2 mg/kg on every other day,

Fig. 6. Tumor growth curves of SUM149 (A&C) and (B&D) xenograft tumor model after treating with NPsiCDK1 at different doses and Dinaciclib at 50 mg/kg. Images of SUM149 (E) and BT549 (F) xenograft tumors at the final time point of treatment. NPsiCDK1-x represent different doses and administration frequencies of NPsiCDK1 which are 0.5 mg/kg every other day (NPsiCDK1-1), 1.0 mg/kg every other day (NPsiCDK1-2), 2 mg/kg every other day (NPsiCDK1-3), 2 mg/kg twice per week (NPsiCDK1-4) and 2 mg/kg once per week (NPsiCDK1-5), respectively. The data are shown as mean ± SD (n = 5). (A&B) *p b 0.01, compared with PBS. (C&D) *p b 0.01, compared with NPsiCDK1-4, #p b 0.01, compared with NPsiCDK1-5.

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Fig. 7. Body weight change of mice bearing SUM149 (A) and BT549 (B) xenograft tumor during treatment. Examination of mouse IFN-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-6 levels in the serum of SUM149 (C) and BT549 (D) xenograft-bearing mice after the last day of treatment. The data are shown as mean ± SD (n = 5). *p b 0.05; #p b 0.001, compared with PBS.

twice per week or once per week. As indicated in Fig. 6C and 6D, administration of NPsiCDK1 on every other day with a dose of 2 mg/kg and 1 mg/kg effectively reduced tumor growth, but injection with a dose of 0.5 mg/kg had much less effect on tumor inhibition. On the other hand, decreasing the administration frequency to twice per week or once per week at a dose of 2 mg/kg halted the tumor inhibition effect obviously in both tumor models. Fig. 6E&F and Figure S6 show the volume and tumor weight of SUM149 xenograft tumors at the final treatment time point. It should be mentioned that when dinaciclib inhibited the tumor growth, the body weight of mice with SUM149 and BT549 xenograft significantly decreased (Fig. 7A&B). Moreover, the AST, ALT and LDH level of the serum after dinaciclib treatment obviously increased (Figure S7), implying that the small molecular inhibitor might induce significant side effects during treatments. On the contrary, systemic administration of NPsiCDK1 showed little effect on the mice's body weight and liver damage-related cytokines in both tumor models (Fig. 7A&B, Figure S7, and Figure S8A&B). On the other hand, since doublestranded siRNA administration may activate the innate immune response, leading to non-specific anti-tumor effects [59], we analyzed for levels of mouse TNF-α, IFN-γ and IL-6 in the serum, taken on the last day of treatment. These results showed in Fig. 7C&D and Figure S8C&D that none of the cytokine or interferon responses significantly changed versus respective untreated controls. Collectively, these results establish that the anti-tumor effects of NPsiCDK1 here are not due to non-specific siRNA-mediated activation of the innate immune response. To further evaluate whether the inhibition of tumor growth observed upon treatment with NPsiCDK1 was related to CDK1 gene silencing in tumor cells, we examined CDK1 mRNA and protein expression levels in the tumors following treatment. The tumor masses from each group of mice were excised 24 h after the last injection and extracted RNA was analyzed using real-time PCR for CDK1 and GAPDH mRNA expression. As shown in Fig. 8, SUM149 tumors from mice treated with NPsiCDK1 exhibited significantly lower levels of CDK1 mRNA dependent on the dose and frequency of systemic administration. Western blot analysis of total protein from each group of SUM149 tumor mass using anti-CDK1 monoclonal antibodies showed a dose and frequency dependent knockdown of CDK1 protein expression levels in tumors from mice treated with NPsiCDK1. Such CDK1 protein expression knockdown was not seen in tumors from mice receiving control treatments. BT549 tumor mass from each mouse was also excised and analyzed for CDK1 mRNA or protein expression as well (Fig. 8, lower).

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Fig. 8. Expression levels of CDK1 mRNA (upper) and protein (lower) in SUM149 (A) and BT549 (B) tumor tissue at the end time point of treatment. Three samples of each group were randomly chosen for detection.

4. Conclusions We have demonstrated that triple negative breast cancer with c-Myc overexpression can be molecularly targeted with CDK1 siRNA delivered by cationic lipid assisted PEG-PLA nanoparticles through a synthetic lethal interaction between c-Myc and CDK1. NPsiCDK1 is able to significantly reduce the expression of CDK1 gene in cell culture, and with CDK1 knockdown by NPsiCDK1, it causes a significant increase in cell apoptosis in TNBC with c-Myc overexpression while not in normal human mammary epithelial cells. Systemic delivery of NPsiCDK1 significantly inhibits tumor growth in mice bearing SUM149 and BT549 xenograft, due to the reduced CDK1 expression. Importantly, the delivery of siCDK1 by this nanoparticle system does not cause in vivo systemic toxicity or activate the innate immune response, providing a novel approach for triple-negative breast cancer therapy. Acknowledgments This work was supported by the National Basic Research Program of China (973 Programs, 2010CB934001, 2013CB933900), the National High Technology Research and Development Program of China (863 Program, 2014AA020708), the National Natural Science Foundation of China (51125012, 51390482, 81302724) and the SRFDP (20133402110019). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jconrel.2014.07.001. References [1] A. Jemal, F. Bray, M.M. Center, J. Ferlay, E. Ward, D. Forman, Global cancer statistics, CA Cancer J. Clin. 61 (2011) 69–90. [2] A. Bosch, P. Eroles, R. Zaragoza, J.R. Vina, A. Lluch, Triple-negative breast cancer: molecular features, pathogenesis, treatment and current lines of research, Cancer Treat. Rev. 36 (2010) 206–215. [3] D.S. Tan, C. Marchio, R.L. Jones, K. Savage, I.E. Smith, M. Dowsett, J.S. Reis-Filho, Triple negative breast cancer: molecular profiling and prognostic impact in adjuvant anthracycline-treated patients, Breast Cancer Res. Treat. 111 (2008) 27–44. [4] J.S. Reis-Filho, A.N. Tutt, Triple negative tumours: a critical review, Histopathology 52 (2008) 108–118. [5] M. Clarke, R. Collins, C. Davies, J. Godwin, R. Gray, R. Peto, Early breast cancer trialists collaborative, Tamoxifen for early breast cancer: an overview of the randomised trials, Lancet 351 (1998) 1451–1467. [6] N.E. Hynes, H.A. Lane, ERBB receptors and cancer: the complexity of targeted inhibitors, Nat. Rev. Cancer 5 (2005) 341–354. [7] J.E. Uhm, Y.H. Park, S.Y. Yi, E.Y. Cho, Y. La Choi, S.J. Lee, M.J. Park, S.H. Lee, H.J. Jun, J.S. Ahn, W.K. Kang, K. Park, Y.H. Im, Treatment outcomes and clinicopathologic

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Triple negative breast cancer therapy with CDK1 siRNA delivered by cationic lipid assisted PEG-PLA nanoparticles.

There is no effective clinical therapy yet for triple-negative breast cancer (TNBC) without particular human epidermal growth factor receptor-2, estro...
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