Journal of Controlled Release 198 (2015) 1–9

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Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel

Cancer-targeted MDR-1 siRNA delivery using self-cross-linked glycol chitosan nanoparticles to overcome drug resistance Ji Young Yhee a, Seungyong Song b, So Jin Lee a, Sung-Gurl Park c, Ki-Suk Kim c, Myung Goo Kim a,d, Sejin Son a, Heebeom Koo a, Ick Chan Kwon a,e, Ji Hoon Jeong d, Seo Young Jeong b, Sun Hwa Kim a,⁎, Kwangmeyung Kim a,⁎ a

Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Hwarangno 14-gil 6, Seongbuk-gu, Seoul 136-791, Republic of Korea Department of Life and Nanopharmaceutical Science, Kyung Hee University, 26 Kyunghee-daero, Dongdaemun-gu, Seoul 130-701, Republic of Korea Next-Generation Pharmaceutical Research Center, Korea Institute of Toxicology, Korea Research Institute of Chemical Technology, Daejeon 305-600, Republic of Korea d School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea e KU-KIST School, Korea University, 1 Anam-dong, Seongbuk-gu, Seoul 136-701, Republic of Korea b c

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Article history: Received 7 August 2014 Accepted 18 November 2014 Available online 4 December 2014 Keywords: Glycol chitosan Multi-drug resistance Nanoparticles Polymerized siRNA siRNA delivery

a b s t r a c t P-glycoprotein (Pgp) mediated multi-drug resistance (MDR) is a major cause of failure in chemotherapy. In this study, small interfering RNA (siRNA) for Pgp down-regulation was delivered to tumors to overcome MDR in cancer. To achieve an efficient siRNA delivery in vivo, self-polymerized 5′-end thiol-modified siRNA (poly-siRNA) was incorporated in tumor targeting glycol chitosan nanoparticles. Pgp-targeted poly-siRNA (psi-Pgp) and thiolated glycol chitosan polymers (tGC) formed stable nanoparticles (psi-Pgp-tGC NPs), and the resulting nanoparticles protected siRNA molecules from enzymatic degradation. The psi-Pgp-tGC NPs could release functional siRNA molecules after cellular delivery, and they were able to facilitate siRNA delivery to Adriamycin-resistant breast cancer cells (MCF-7/ADR). After intravenous administration, the psi-Pgp-tGC NPs accumulated in MCF7/ADR tumors and down-regulated P-gp expression to sensitize cancer cells. Consequently, chemo-siRNA combination therapy significantly inhibited tumor growth without systemic toxicity. These psi-Pgp-tGC NPs showed great potential as a supplementary therapeutic agent for drug-resistant cancer. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Multi-drug resistance (MDR) limits the therapeutic potential of anticancer drugs, resulting in failure of cancer treatment. In an attempt to explain MDR, a variety of factors and mechanisms were suggested, including 1) decreased cellular uptake or increased efflux of drugs, 2) increased drug detoxification, and 3) genetic alteration that could be influenced on drug-targeted enzyme levels or DNA repair capacity [1, 2]. In particular, P-glycoprotein (Pgp)-mediated drug efflux has been regarded as the most important mechanism of the MDR regulation. Pgp, also known as MDR protein 1 (MDR-1), is a typical ATP-binding cassette membrane transporter that effuses a broad range of drugs to decrease their intracellular accumulation [3]. Overexpression of Pgp and up-regulation of its functional activity in cancer cells can decrease their chemo-sensitivity against anti-cancer drugs. Over the past few decades, considerable efforts have been devoted to overcoming Pgp-mediated MDR of cancer. The early MDR reversal trials included small-molecule agents of Pgp inhibitors, such as verapamil and cyclosporine A [4,5]. These drugs could directly regulate the expression or activity of Pgp, but they often cause chronic toxicity and systemic side ⁎ Corresponding authors. E-mail addresses: [email protected] (S.H. Kim), [email protected] (K. Kim).

http://dx.doi.org/10.1016/j.jconrel.2014.11.019 0168-3659/© 2014 Elsevier B.V. All rights reserved.

effects such as bradycardia and immunosuppression [6,7]. More recently, sequence-specific gene silencing by small interfering RNA (siRNA) has been studied to block MDR-1 gene expression in drug-resistant cancer cells [8,9]. The sequence-specific gene silencing offers a relatively safe method for down-regulation of Pgp without side effects. As natural siRNA molecules show poor cellular uptake and low stability against the nuclease, efficient carrier systems for siRNA are indispensable prerequisites for successful siRNA delivery. Traditional siRNA carriers include cationic polymers and liposomes, which are generally highly toxic and low in target-site accumulation [10–12]. To allow a wider variety of choices of carrier for siRNA, the siRNA backbone has been recently structurally modified [13–16]. Prudent modifications of siRNA could improve its physiochemical properties for being incorporated in gene carriers with improved stability and higher binding affinity for carriers. In particular, self-polymerized 5′-end thiol-modified siRNA (poly-siRNA) showed increased binding affinity for carriers through higher molecular weight, anionic charges, and functional thiol groups [14,17]. Based on these, the poly-siRNA can be incorporated into carriers with lower cationic charge density, which have lower toxicity in vivo. In the current study, we designed and synthesized a poly-siRNA for Pgp suppression (psi-Pgp) to overcome MDR in cancer. To achieve safe and efficient delivery, the psi-Pgp was encapsulated in tumorhoming glycol chitosan nanoparticles which are biocompatible. Glycol

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chitosan polymers were chemically modified with sulfhydryl groups, and resulting thiolated glycol chitosan polymers (tGC) and psi-Pgp formed stable and condensed nanoparticles (psi-Pgp-tGC NPs). The efficacy of gene silencing and chemo-siRNA combination therapy was investigated in an Adriamycin-resistant variant human breast cancer cell line and the tumor mice model. The results provided clear evidences of in vivo Pgp down-regulation focused on the systemic siRNA delivery, compared to the previous studies [18,19]. Based on the advantages of poly-siRNA, this work highlights the potential of psi-Pgp-tGC NPs as a supplementary therapeutic agent to overcome the limitations in MDR cancer chemotherapy. 2. Materials and methods 2.1. Materials Glycol chitosan (GC, Mw = 250 kD; degree of deacetylation = 82.7%), dithiothreitol (DTT), and ethylenediaminetetraacetic acid (EDTA) were supplied by Sigma-Aldrich Corporation (St. Louis, MO). For fluorescence labeling, near-infrared fluorescence (NIRF) dye Flamma™ (FPR-675; Bioacts, Seoul, Korea), fluorescein isothiocyanate (FITC; Sigma, St. Louis, MO) isomer and Cyanine dye 3 (Cy3; Mirus bio, Madison, WI) were prepared. Sulfosuccinimidyl 6-[3′(2-pyridyldithio)-propionamido] hexanoate (Sulfo-LC-SPDP) were from Pierce (Rockford, IL). A human MDR-1 siRNA duplex that consisted of 5′-r(CGGAAGGCCUAAUG CCGA A) dTdT (sense) and 5′-r(UUCGGCAUUAGGCCUUCCG) dTdG (antisense) was synthesized and supplied by Bioneer (Daejeon, Korea). As a negative control, Pgp non-related sequence of poly-siRNA (psi-ns) was prepared to exclude the non-specific gene silencing effects. The psi-ns was synthesized with the sense strand 5′-r(UGUAGAUGGACUUGAACUC)dTdT, and antisense strand 5′-(GAGUUCAAG UCCAUCUACA) dTdT. For the polymerization of siRNA monomers, each sense and antisense strand of siRNA was modified with thiol group at the 5′-terminal end. All solutions were made up in RNase-free distilled water (Invitrogen, Carlsbad, CA) and autoclaved prior to use. All chemicals and reagents were of analytical grade and were used without further purification. 2.2. Synthesis of psi-Pgp encapsulated glycol chitosan nanoparticles (psi-Pgp-tGC NPs) To build up self-cross-linked psi-Pgp-tGC NPs, functional thiol groups were introduced to the GC polymers as described in the previous study [14]. Briefly, GC polymers (15 mg) were dissolved in phosphate buffer (pH 7.4, 5 mL), followed by the addition of sulfo-LC-SPDP (3 mg). The mixture was incubated overnight with gentle agitation, and the disulfide bridge generated by sulfo-LC-SPDP was reduced with 10 mM of DTT treatment for 3 h at pH 3.5. The solution was dialyzed for 96 h in distilled water using a dialysis tube (molecular weight cutoff = 12–14 kD, Spectrum Laboratories, Rancho Dominquez, CA) to be lyophilized. The degree of thiol modification in the GC polymers was determined using Ellman's assay. For the synthesis of poly-siRNA, a single siRNA molecule was designed to have free thiol groups at the 5′-ends of each strand [14,20]. The sulfhydryl group-exposed siRNA molecules were self-polymerized in a HEPES buffer (10 mM, pH 8.0). The N,N,N′,N′-tetramethylazodicarboxamide (1 mg, 5.8 μmol) was added to the HEPES buffer and gently vortexed to form poly-siRNA. After 24 h, poly-siRNA molecules were purified using a desalting column and then lyophilized. The resulting poly-siRNA (50 μg/100 μL HEPES buffer) was mixed with tGC (250 μg/100 μL HEPES buffer) at a weight ratio of 1, 10, and 20 and incubated at 37 °C for 1 h. 2.3. Characterization of psi-Pgp-tGC NPs Formation of the psi-Pgp-tGC NPs was confirmed using gel retardation assay and transmission electron microscopy (TEM, CM200 electron microscope, Philips). Reducibility of psi-tGC NPs were visualized in

polyacrylamide gels. The psi-tGC NPs were incubated with DTT (5 mM), and gel electrophoresis was performed to detect 21 bps sized monomeric siRNA molecules. The size and surface charge of psi-PgptGC NPs were measured by dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments Ltd., Worcestershire, UK) and electrophoretic light scattering spectrometer (Ostuka, Electronics Co., Ltd., Japan). The psi-Pgp-tGC samples (0.3 mg/100 μL HEPES buffer) for measuring size and surface charge were prepared in distilled water (1:20 diluted), and the final pH of sample was 8.1. Stability of the psi-Pgp-tGC NPs against serum degradation was visualized in polyacrylamide gels. Each psi-Pgp and psi-Pgp-tGC NPs was incubated in fresh rat serum at 37 °C for 0 to 24 h. After the incubation, disulfide linkages were cleaved by DTT treatment (0.1 M), and the remaining RNA molecules at 21 bp size were visualized by a gel document imaging system (MiniBIS Pro, DNR Bio-Imaging Systems, Jerusalem, Israel). 2.4. In vitro transfection and cytotoxicity of psi-Pgp-tGC NPs Adriamycin-resistant variant human breast cancer cell line MCF-7/ ADR was obtained from Dr. Dae-Duk Kim (College of Pharmacy, Seoul National University, Seoul, Korea). The cells were maintained in RPMI 1640 media (Gibco, Grand Island, NY) with 10% FBS (Gibco, Grand Island, NY) and 1% penicillin/streptomycin at 37 °C. To visualize the cellular uptake of psi-Pgp-tGC NPs, Cy3-labeled siRNA and FITC-labeled tGC were used for complexation. The MCF-7/ADR cells (5 × 104 cells/ 35 mm2 dish) were treated with dual-labeled psi-Pgp-tGC NPs (100 nM of siRNA concentration) for 4 h. The transfected cells were fixed with 4% paraformaldehyde to observe using an IX81-ZDC focus drift compensating microscope (Olympus, Tokyo, Japan). The cytotoxicity of psi-Pgp-tGC NPs was evaluated by MTT assay. The psi-Pgp formulated in commercial cationic liposomal transfection agent (Lipofectamine™ 2000; denoted as LF, Invitrogen, Carlsbad, CA) was used to compare the cytotoxicity. The cells in 96-well flatbottomed plates (5 × 103 cells/well) were exposed to different concentrations of siRNA formulations for 6 h. The cell viability was measured by observing optical absorbance differences at 570 nm after MTT solution treatment. The data were expressed as the relative cell viability (%) compared to the untreated control cells and were presented as the mean ± standard deviation (SD, n = 5). 2.5. In vitro gene silencing and changes in drug sensitivity Western blotting with MDR-1 (Pgp) antibody was performed to evaluate the in vitro gene-silencing efficiency of the psi-Pgp-tGC NPs. MCF-7/ADR cells (5 × 105 cells/100π dish) were exposed to the psiPgp-tGC NPs (100 nM, 5 mL) for 10 h. The equivalent dose of psi-Pgp formulated in LF was used as a positive control, and treatment without siRNA molecules were used as a negative control. After 48 h of further incubation in fresh RPMI media, total protein was extracted from the cells using a Mammalian Cell Lysis Kit (Sigma, St. Louis, MO). The protein concentrations were determined by BCA assay, and proteins in each sample (25 μg) were separated in 7.5% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes (GE healthcare, Buckinghamshire, UK). The memebrane was divided and incubated in antibody specific for MDR-1 (1:100 diluted in 5% skim milk, Santa Cruz Biotechnology, Santa Cruz, CA) and β-actin (abcam, MA, USA), respectively. Anti-mouse IgG-HRP (1:3,000 diluted) and chemiluminescence detection (Intron Biotechnology, Seoul, Korea) was used to detect protein expressions. The change in anti-cancer drug resistance of the psi-Pgp-tGC NPtreated MCF-7/ADR cells was also studied. The psi-Pgp (100 nM) was transfected in MCF-7/ADR cells (2 × 103 cells/96-well flat-bottomed plates well). After 48 h, the cells were treated with 0 to 10 μM of DOX for 48 h. The cell viability was measured by MTT assay as described

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above. In addition, intracellular distribution of DOX was observed under a fluorescence microscope.

Total blood was collected at 6 h post-injection to analyze the activities of ALT and AST.

2.6. Adriamycin-resistant mammary adenocarcinoma xenograft

2.10. Statistics

All the animal experiments were performed in compliance with the relevant laws and institutional guidelines of the Korea Institute of Science and Technology. To establish the MCF-7/ADR xenografts, female nude mice (4.5 weeks of age; Charles River, Washington, MA) were prepared. MCF-7/ADR cells (5 × 106 cells/100 μL) suspended in fresh RPMI media containing 50% Basement Membrane Matrix (growth-factor-reduced and phenol red-free, BD Bioscience, CA, USA) were subcutaneously injected into the left flank of the mice. When the volume of tumor was approximately 75 mm3 in size, the mice were randomly divided into control and experimental groups.

The differences in cell viability, Pgp expression, and tumor growth were analyzed using Student's t-test for pairs of control and psi-PgptGC NP-treated groups. p-Value less than 0.05 was considered as statistically significant. All statistical analyses were performed using the software SPSS (version 11.0, SPSS, Chicago, IL).

2.7. In vivo NIRF imaging Biodistribution of psi-Pgp-tGC NPs was monitored using timedependent in vivo NIRF imaging. The FPR-675-labeled psi-Pgp was used for in vivo NIRF imaging, and naked psi-Pgp (n = 3) and the psiPgp-tGC NPs (n = 3) were intravenously injected into MCF-7/ADR tumor-bearing mice (30 μg siRNA/head). NIRF signals were noninvasively monitored using the eXplore Optix system (ART Advanced Research Technology Inc., Montreal, Canada) for up to 48 h. After 48 h, tumors and major visceral organs were excised from the mice to compare the NIRF intensity at excised organs using a 12-bit CCD camera (Image Station 4000 MM; Kodak, New Haven, CT). 2.8. Tumor growth and target gene silencing in vivo MCF-7/ADR tumor-bearing mice were prepared as randomized into 3 different groups for treatments (PBS-control, DOX only, and psi-PgptGC + DOX-treated groups; n = 3 per each group), when the diameter of the tumors increased to 75 mm3. All treatment agents were intravenously administered via tail vein. The DOX (2.5 mg/kg/head) was injected into the mice once every 4 days for 3 weeks. The psi-Pgp-tGC NP (30 μg siRNA/head) injection was conducted 1 day prior to every DOX treatment. Tumor growth of the mice was recorded from the first day of treatment by measuring tumor volume (width2 × length / 2). All mice were sacrificed at 22 days post-treatment to obtain the tumor tissues for quantitative analysis of Pgp. Western blotting was performed to verify the gene silencing in the tumors as described above. In addition, a small fraction of tumor tissues was fixed in formalin and embedded in paraffin for immunohistochemistry. The tissue slides of each group were incubated with anti-MDR-1 (1:200 diluted) for 2 h at room temperature, and then HISTOSTAIN®-PLUS Kit (Invitrogen, Carlsbad, CA) was used as the secondary detection reagent. 2.9. Toxicological assay The signs and symptoms of toxicity were monitored from the initiation of treatment. Histologic examination of major visceral organs was performed after the scheduled treatment time. In addition, blood biochemistry was performed to evaluate systemic toxicity of psi-PgptGC + DOX (2.5 mg/kg). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST), the indicators of the hepatotoxicity, were measured to evaluate acute toxicity of the psi-Pgp-tGC + DOX (2.5 mg/kg). Since reference dose of DOX for MCF-7/ADR tumor treatment was 4–10 mg/kg [21,22], conventional strategy of high dose DOX (7.5 mg/kg) treated group was also prepared. Twenty of balb/c mice (5 weeks of age; Charles River, Washington, MA) were randomly divided into 4 different groups for treatments (control-PBS, psi-PgptGC (1.2 mg/kg, 30 μg siRNA contents included) only, high dosed DOX (7.5 mg/kg), and psi-Pgp-tGC (1.2 mg/kg, 30 μg siRNA contents included) + low dosed DOX (2.5 mg/kg) tread groups; n = 5 per each group).

3. Results 3.1. Preparation and characterization of psi-Pgp-tGC NPs Prior to the thiol-modification of GC, naive cationic GC and anionic poly-siRNA were incubated to confirm whether they spontaneously formed complexes by electrostatic interactions. TEM images and size distribution graph indicated that poly-siRNA and GC also could form nanoparticles, but loosely bound structures resulted in large sized aggregates which show around 700 nm of diameter with incomplete encapsulation of the poly-siRNA in GC carrier (Supplementary information, Fig. S1). Consequently, GC polymers were also thiolated to improve the binding affinity and enhance the intramolecular cross-linking for condensing nano-structures. Both GC polymer and siRNA molecules were thiol-modified (Scheme 1A), and the resulting poly-siRNA and tGC polymers were spontaneously assembled into nanoparticles through charge–charge interaction and chemical cross-linking (Scheme 1B). As many other chitosan based nanoparticles [23], the psi-Pgp-tGC NPs are expected to enter the cytoplasm at neutal to acidic pH (pKa of chitosan = 5–7) and to escape the lysosomes by changes of endosomal pH. Endogenic glutathione would induce the degradation of the psi-PgptGC NPs in cytosol, and released siRNA molecules could result in target-specific Pgp silencing (Scheme 1C). Under optimal conditions, 7% of amine residues on GC polymers were modified with thiol groups. The dithiol-modified sense and antisense strands of siRNA were annealed and self-polymerized to show a ladder-like migration pattern in polyacrylamide gel (Fig. 1A left). The molecular weight distribution of the poly-siRNA was between 21 bp and 350 bp, and 50% of siRNA molecules were above 100 bp on average. The complete retardation of poly-siRNA was achieved at a 1:10 weight ratio of psi-Pgp to tGC, indicating the complete encapsulation of siRNA molecules into the NPs (Fig. 1B). Importantly, the psi-Pgp-tGC NPs showed only 21 bp sized siRNA molecules after DTT treatment. It suggested that the psi-Pgp-tGC NPs released monomeric siRNA under a reductive environment (Fig. 1A right). The psi-Pgp-tGC NPs were observed as spherical particles with 200 to 300 nm of diameter in TEM images, and their average hydrodynamic diameter was ca. 269.8 ± 10.9 nm as measured by DLS (Fig. 1C). Although the psi-Pgp-tGC NPs were chemically condensed, they are structurally flexible that can pass through 0.2 μm cellulose acetate filter membrane (shown in Supplemental Fig. S2). The slightly positively charged surface (ξ potential = 7.7 ± 3.55 mV) suggested that the negatively charged psi-Pgp would be efficiently encapsulated in tGC polymers. The psi-Pgp-tGC NPs could protect siRNA molecules from enzymatic degradation (Fig. 1D). In fresh rat serum, poly-siRNA was degraded after 12 h, whereas the poly-siRNA in psi-Pgp-tGC NPs showed no significant siRNA degradation even after 24 h. 3.2. Cellular uptake and cytotoxicity of psi-Pgp-tGC NPs in vitro The cellular uptake of the psi-Pgp-tGC NPs was observed in MCF-7/ ADR cells using the dual fluorescence dye-labeled nanoparticles (Fig. 2A). Naked psi-Pgp-treated cells showed vacant fluorescence, indicating extremely low cellular uptake of psi-Pgp. In contrast, distinct red and green spots were observed in the psi-Pgp-tGC NP-treated cells. Red

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Scheme 1. Formation of psi-Pgp-tGC NPs and their intracellular delivery for target gene silencing.

and green spots of poly-siRNA and tGC were spread throughout the cytosol, and the merged images clearly showed the co-localized psi-Pgp and tGC in the cytoplasm. Compared to the psi-Pgp formulated in the commercialized transfection agent LF (psi-Pgp-LF), the psi-Pgp-tGC NPs showed lower cytotoxicity in vitro (Fig. 2B). At high particle concentrations N 59.4 μg/mL (400 nM of siRNA included), the psi-Pgp-tGC NP-treated cell showed above the 95% of viability in MTT assay. However, psi-Pgp-LF led to 50% of cell death at the identical dose for siRNA transfection. 3.3. Gene silencing and changes in drug sensitivity in psi-Pgp-tGC NP-treated cancer cells Pgp down-regulation in psi-Pgp-tGC NP treated cells was quantitatively analyzed using Western blotting assay (Fig. 2C). The psi-PgptGC NPs (100 nM of siRNA dose) induced significant Pgp suppression in MCF-7/ADR cells. On average, 62% of Pgp was decreased after psiPgp-tGC NPs treatment. In immunofluorescence, psi-Pgp-LF treated

and psi-Pgp-tGC treated cells showed low fluorescence of Pgp. However, psi-ns-in LF and psi-ns-tGC NP treated cells, indicating sequencespecific Pgp suppression of psi-Pgp-tGC NPs in vitro (See Supplementary, Fig. S3). The cellular uptake of DOX was also monitored after psi-Pgp transfection (Fig. 3A). Before the psi-Pgp transfection, cellular distribution of DOX was limited in cytosol. By contrast, both psi-Pgp-tGC NP and psi-Pgp-LF-treated cells showed fluorescence of DOX in nuclei. Compared to the MCF-7/ADR cells without psi-Pgp transfection, the psiPgp-tGC NP-treated cells showed enhanced nuclear accumulation of DOX at 5 μM of treatment. Nuclear delivery of DOX is essential for tumoricidal effect, because DOX presents cytotoxic effect after forming a complex with proteasomes to be transported to the nucleus [24]. Therefore, the changes in cellular distribution of DOX suggest that the psi-Pgp-tGC NPs can induce the changes in drug sensitivity. Changes in drug sensitivity to DOX were further studied in psi-Pgp transfected cells. The MCF-7/ADR cells with and without psi-Pgp-tGC NPs treatment were exposed to different concentrations of DOX to

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determine the changes in drug sensitivity. Compared to the control cells, the cell viability was significantly decreased in psi-Pgp-tGC NPtreated cells (Fig. 3B). IC50 of DOX was observed around 5 nM after the psi-Pgp transfection, whereas un-transfected MCF-7/ADR cells exhibited limited toxicity with 75% viability at the same dose. These results suggest that the psi-Pgp-tGC NPs increased DOX sensitivity in MDR cancer cells by a knockdown of the MDR-1 gene. 3.4. In vivo tumor-targeting properties of psi-Pgp-tGC NPs The particle structure was stable in serum positive condition for 24 h (Fig. S4), and the psi-Pgp-tGC NPs were systemically injected. Timedependent whole-body NIRF images showed the biodistribution of the psi-Pgp-tGC NPs in MCF-7/ADR tumor-bearing mice (Fig. 4A). After intravenous injection, no significant increase of tumor NIRF was observed in the psi-Pgp-injected mice. The NIRF signals were mainly detected in the kidney at the early time, indicating rapid renal clearance of psiPgp. By contrast, the psi-Pgp-tGC NPs started to accumulate at tumor site, and maximum NIRF intensity in the tumor was recorded at 24 h post-injection. The ex vivo NIRF imaging of the excised visceral organs and tumor also showed high accumulation of the psi-Pgp-tGC NPs at the tumor (Fig. 4B). The psi-Pgp-tGC NPs-injected mice exhibited 2.7fold higher NIRF intensity at the tumor compared to the psi-Pgpinjected group (Fig. 4C).

psi-Pgp treated group was excluded for the negligible gene silencing effects of poly-siRNA without carrier system [14,25]. DOX-only and psi-Pgp-tGC + DOX groups all showed reduced tumor volumes compared to the control group (Fig. 5A). In particular, the psi-Pgp-tGC NP co-treatment significantly enhanced anti-cancer activity of DOX. The mean tumor volume of psi-Pgp-tGC + DOX groups was 63.6% and 49.6% of the DOX-only and control group, respectively. The psi-nstGC + DOX did not induce the Pgp suppression or retardation of tumor growth, compared to the DOX only treated group (Fig. S5). It suggested that the MDR-overcoming effect of psi-Pgp-tGC NPs is due to the sequence-specific Pgp suppression. The Western blot analysis showed the successful Pgp silencing in the tumor tissues of the psi-Pgp-tGC NP-treated group, and the mean intensity of bands was decreased as 7.8% of the control tumor (Fig. 5B). The result of immunohistochemistry was closely correlated with the result of Western blot analysis (Fig. 5C). The psi-Pgp-tGC + DOX group showed a high degree of cell death with only a minimal Pgp expression, while the DOX only treated group exhibited distinct Pgp overexpression at the plasma membrane of tumor cells. These results all supported that the improved therapeutic effect of low dosed DOX (2.5 mg/kg) is mainly due to the increased drug sensitivity based on the Pgp suppression by cancer-targeted delivered psi-Pgp.

3.6. Systemic toxicity of psi-Pgp-tGC NPs 3.5. Pgp silencing and MDR-overcoming effects of the psi-Pgp-tGC NPs in vivo Tumor growth of 3 different groups was recorded for 3 weeks to investigate MDR-overcoming effects of psi-Pgp-tGC NPs. The naked

All mice (n = 9) had survived after 22 days, and any behavioral abnormalities and significant weight loss were not noticed in the period of experiment. In the histologic examination, discernible hemorrhage, infarction, or wide range of apoptosis was not observed in any of the

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Fig. 2. In vitro cellular uptake, cytotoxicity, and gene silencing of psi-Pgp-tGC NPs. (A) Cellular uptake of naked psi-Pgp and psi-Pgp-tGC NPs in cultured MCF-7/ADR cells. (B) Cytotoxicity of psi-Pgp-tGC NPs and psi-Pgp-LF (C) Pgp down-regulation in psi-Pgp-tGC NP-treated cells using Western blotting. Lipofectamine 2000 (LF) was used as a positive control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

increased in of psi-Pgp-tGC + DOX (2.5 mg/kg) and high dosed DOX (7.5 mg/kg) treated group, but psi-Pgp-tGC + DOX treated group demonstrated significantly lower ALT and AST than high dosed DOX treated group (Fig. 5D and E). In particular, the ALT level of psi-Pgp-tGC + DOX

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visceral organs including kidney, liver, spleen, and lung of psi-Pgp-tGC NP + DOX (2.5 mg/kg)-treated mice (See Supplementary, Fig. S6). In the blood biochemistry, control and psi-tGC treated groups showed ALT and AST in normal range [26]. ALT and AST were both

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Lung Heart Liver SpleenKidneyTumor

Fig. 4. In vivo NIRF imaging of NIRF dye labeled psi-Pgp-tGC-injected MCF-7/ADR tumor-bearing mice. (A) Real-time whole-body NIRF imaging of the tumor-bearing mice after intravenous injection of naked psi-Pgp (upper) and psi-Pgp-tGC NPs (lower). Circle = site of tumor. (B) Images of excised tumor and visceral organs at 48 h post-injection. (C) NIRF intensity at excised tumor and vital organs of psi-Pgp and psi-Pgp-tGC-injected mice (n = 3/group).

treated mice was in the normal range. The results of blood chemistry indicated that the psi-Pgp-tGC NPs did not cause acute toxicity. Moreover, the psi-Pgp-tGC NP + DOX (2.5 mg/kg) protocol could reduce the toxicity of conventional therapy of high dose of DOX (7.5 mg/kg) for MDR-related cancer treatment. 4. Discussion Therapeutic efficacy of chemotherapy is limited by MDR, and the nanoparticle platform has been extensively investigated in the field of MDR cancer therapy. As drug-loaded nanoparticles partially bypass the drug-resistance mechanism [27,28], nanoparticle formulation itself improves the therapeutic effect by selective accumulation of anticancer drugs in cancer cells. Indeed, nanoparticles for delivery of the MDR-1 gene targeting siRNA demonstrated considerable progress in overcoming MDR resulting from specific down-regulation of Pgp [8–12]. Several reports suggested that the siRNA delivery for Pgp down-regulation had great potential for MDR reversal [29,30]. However, traditional methods for siRNA delivery via intratumoral administration are not appropriate to treat a wide range of cancers, such as lifethreatening metastatic cancer. To achieve cancer-targeted delivery of MDR-1 gene targeting siRNA through systemic administration, the siRNA molecules were selfpolymerized to be incorporated into the tumor-homing glycol chitosan nanoparticles. In our previous study, intravenously injectable psi-tGC NPs showed great potential as an efficient and safe in vivo siRNA delivery system for cancer treatment [14]. The psi-tGC NPs induced efficient target gene silencing in tumors without unintended immune stimulation and side effects. The psi-Pgp, synthesized in this study, was successfully

incorporated into the tGC NPs in the same manner. The molecular weight distribution of psi-Pgp showed slightly shorter chains of siRNA polymers than the poly-siRNA targeting different mRNA sequences [14,17], and it would be attributed to the sequential differences in siRNA, which can influence its physical properties [31]. Despite the sequential differences in siRNA, the resulting psi-Pgp was successfully encapsulated in the tGC NPs and showed great potential as a siRNA delivery system. The psiPgp-tGC NPs could protect siRNA molecules from enzymatic degradation, and they were completely degraded to release monomeric siRNA under the reductive conditions. Intravenously injected psi-Pgp-tGC NPs exhibited improved tumor accumulation in MCF-7/ADR tumor-bearing mice, and they successfully down-regulate Pgp expression. The Pgp suppression in tumors was closely related to the drug sensitivity to DOX. The dose of DOX for MCF-7/ADR tumor treatment is generally 4–10 mg/kg [21,22], and a sub-therapeutic dose of DOX (4 mg/kg, once per week) induced only marginal inhibition of tumor growth [32]. At the therapeutic dose (10 mg/kg per week), DOX showed severe systemic toxicity such that the survival rate of the mice was 60% after 30 days [22]. In this study, we also confirmed that liver enzymes of ALT and AST were significantly increased for acute toxicity of DOX at 7.5 mg/kg. However, after the treatment of psi-Pgp-tGC NPs, we inhibited tumor growth using a sub-therapeutic dose of DOX (2.5 mg/kg) with reduced systemic toxicity. The psi-Pgp-tGC NPs potentiated DOX-mediated inhibition of tumor growth, and the psi-Pgp-tGC NP-treated group showed significantly lower tumor volume compared to the control and DOX-only treated groups. Changes in subcellular distribution of DOX also supported that Pgp silencing increased the drug sensitivity in psi-Pgp-tGC NP-treated cells. Altered subcellular DOX distribution may indicate the changes in drug resistance in drug-resistant

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Fig. 5. In vivo gene silencing in tumor and supplementary therapeutic effects of psi-Pgp-tGC in MCF-7/ADR tumor-bearing mice. (A) Tumor growth curve of the control, DOX-only, and psiPgp-tGC + DOX-treated groups for 22 days. (B) Pgp expression in MCF-7/ADR tumors after 22 days. (C) Histology (Top, H&E stain) and Pgp expression in tumor tissues (Bottom, immunohistochemistry: brown). (D) Alanine aminotransferase (ALT) and (E) aspartate aminotransferase (AST) enzyme level in the blood of mice.

variants [9,33–35]. The Pgp silencing in MCF-7/ADR cells enhanced cytoplasmic/nuclear DOX accumulation, leading to a higher therapeutic effect of DOX. In conclusion, the treatment of psi-Pgp-tGC NPs enables effective MDR-related cancer therapy with a lower dose of DOX. In addition, the use of low-dose DOX consequentially reduced side effects of systemic toxicity. To summarize, this study describes a systemic delivery of siRNA for overcoming MDR and combinational therapy with DOX. The Pgptargeting siRNA was self-polymerized, and the resulting psi-Pgp was successfully loaded in the nanoparticles. The psi-tGC NPs showed high levels of accumulation in tumor tissues based on EPR effect, and they successfully down-regulated Pgp of the MDR-related tumor. As the tumor growth was effectively inhibited with a sub-therapeutical dose of DOX, no considerable symptoms or histopathological changes for systemic toxicity were observed. Notably, the intravenous route of administration would allow the treatment of a wide range of cancers, particularly for metastatic

cancer, with improved patient compliance. These findings suggest that psi-Pgp-tGC NPs have great potential as a supplementary therapeutic agent for the treatment of MDR-related tumor. Acknowledgments This study was supported by the National Research Foundation of Korea (NRF-2013K1A1A2A02050115 and 2010-0027955) and Development and commercialization of molecular diagnostic technologies for lung cancer through clinical validation (1004393) by the MOTIE, Korea. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jconrel.2014.11.019.

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