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Development of Bioactive PEGylated Nanostructured Platforms for Sequential Delivery of Doxorubicin and Imatinib to Overcome Drug Resistance in Metastatic Tumors Biki Gupta, Thiruganesh Ramasamy, Bijay Kumar Poudel, Shiva Pathak, Shobha Regmi, Ju Yeon Choi, Youlim Son, Raj Kumar Thapa, Jee-Heon Jeong, Jae-Ryong Kim, Han-Gon Choi, Chul Soon Yong, and Jong Oh Kim ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b09163 • Publication Date (Web): 27 Feb 2017 Downloaded from http://pubs.acs.org on February 28, 2017

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ACS Applied Materials & Interfaces

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Development of Bioactive PEGylated Nanostructured Platforms for Sequential Delivery of Doxorubicin and Imatinib to Overcome Drug Resistance in Metastatic Tumors

Biki Gupta1, Thiruganesh Ramasamy1, Bijay Kumar Poudel1, Shiva Pathak1, Shobha Regmi1, Ju Yeon Choi1, Youlim Son2, Raj Kumar Thapa1, Jee-Heon Jeong1, Jae Ryong Kim2, Han-Gon Choi3, Chul Soon Yong1*, Jong Oh Kim1*

1

College of Pharmacy, Yeungnam University, 214-1, Dae-Dong, Gyeongsan 712-749, South

Korea 2

Department of Biochemistry and Molecular Biology, College of Medicine, Yeungnam

University, Daegu, 705-717, Republic of Korea 3

College of Pharmacy, Institute of Pharmaceutical Science and Technology, Hanyang

University, 55, Hanyangdaehak-ro, Sangnok-gu, Ansan 426-791, South Korea

*Corresponding author: Prof. Jong Oh Kim, Ph.D. Tel: +82-53-810-2813, Fax: +82-53-810-4654, E-mail: [email protected] *Co-corresponding author: Prof. Chul Soon Yong, Ph.D. Tel: +82-53-810-2812, Fax: +82-53-810-4654, E-mail: [email protected]

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Abstract Metastasis of cancers accounts for almost the entirety of cancer-related deaths. In this study, we report a PEGylated-nanostructured platform for co-administration of doxorubicin (DOX) and imatinib (IMT) intended to effectively inhibit metastatic tumors. The DOX- and IMT-co-loaded nanostructured system (DOX/IMT-N) is characterized by an excellent encapsulation potential for both drugs and shows sequential and sustained drug release in vitro. DOX/IMT-N significantly inhibited the in vitro proliferation of MDA-MB-231 and SK-MEL-28 cells. The inhibitory effect on in vitro proliferation of the cells was significantly greater than the effect of free DOX, DOX/IMT cocktail, or the nanostructured system housing DOX only (DOX-N). DOX/IMT-N remarkably enhanced cellular drug uptake, resulting in enhanced apoptosis, caused by significant increases in the expression levels of apoptotic marker proteins. Intravenous administration of DOX/IMT-N to MBA-MB-231 xenograft tumor-bearing mice resulted in significantly improved inhibition of tumor progression compared to DOX, DOX/IMT or DOX-N. Therefore, the nanostructured DOX/IMT-N system could potentially aid in overcoming drug resistance in metastatic tumors and improve the effectiveness of metastatic tumor therapeutics.

Keywords: metastasis, doxorubicin, imatinib, nanostructured platform, drug delivery, chemoresistance.


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INTRODUCTION Cancer metastasis is responsible for nearly 90 percent of all cancer-related deaths.1 Breast cancer metastases are the most common of metastases, with an exceptionally high proportion of metastasizing primary breast tumors.2 Breast cancer is the leading cause of cancer-related deaths of women worldwide.3 Metastases of breast cancer at distant sites, rather than the primary tumors are primarily responsible for deaths.4–6 The general treatment modality for breast cancer is surgery in conjunction with adjuvant chemotherapy, with a view to eradicate the micrometastases believed to be present even at the earliest stages.7 Anthracyclines, taxanes, and other cytotoxic drugs are frequently used, as single agents or in combination, for adjuvant chemotherapy in breast cancer.8 The response rates, however, have been typically low and recurrence of cancer metastases is common.9,10 Resistance to chemotherapeutic agents is viewed as a major impediment to successful treatment of metastatic breast cancer. A number of mechanisms are responsible for chemotherapy resistance of breast cancers, most importantly overexpression of P-glycoprotein (P-gp) and other ATP-binding cassette (ABC) transport proteins.11 Doxorubicin (DOX), an anthracycline chemotherapeutic drug, has been extensively used and widely researched as adjuvant chemotherapy in breast cancer, as a single agent and in combination with taxanes and other chemotherapeutics.12–14 Despite promising results in the initial phases of treatment with DOX, recurrence and/or metastasis of breast cancer are common in the long run, primarily as a result of development of multidrug resistance (MDR). Impaired nuclear translocation of DOX in the tumor cells, resulting from increased nuclear and cytoplasmic P-gp expression levels, has been cited as the major mechanism leading to MDR in breast cancer.15,16 In addition to acquired chemoresistance in tumors such as breast cancer, DOX



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is also associated with intrinsic chemoresistance, especially in melanomas. Melanoma cells highly express a group of ABC transporters reported to mediate DOX resistance.17 Combination of DOX and imatinib (IMT) has been reported to effectively reverse intrinsic and acquired resistance associated with DOX by inhibiting ABC-transporter function.18 Additionally, reversal of chemoresistance to a number of agents has been widely reported using nanomedicine-based drug delivery systems, which offer critical advantages over administration of free therapeutics.19 Lipid-based nanocarriers have demonstrated excellent outcomes by overcoming P-gp mediated efflux, sequestering drugs at tumor sites via enhanced permeability and retention (EPR), and escaping endosomal clearance once internalized.20–22 In this study, we prepared PEGylated nanostructured platforms comprising of a hybrid lipid core co-loaded with DOX and IMT (DOX/IMT-N). We performed physical characterization of the system, and evaluated in vitro cytotoxicity in MDA-MB-231 cells and in vivo anticancer effect on MDA-MB-231 xenograft model, comparing DOX/IMT-N to DOX monotherapy, DOX and IMT combination (DOX/IMT), and DOX-loaded nanostructured platforms (DOX-N). In addition, in vitro cytotoxicity was evaluated in SK-MEL-28 cells, malignant melanoma cells exhibiting intrinsic resistance to DOX, in order to investigate whether the system was capable of overcoming intrinsic DOX resistance, in addition to acquired drug resistance characteristic of MDA-MB-231 cells.

EXPERIMENTAL METHODS Materials


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Doxorubicin hydrochloride (DOX · HCl) was received from Dong-A Pharmaceutical Company (Yongin, South Korea), while IMT was obtained from LC Laboratories (Woburn, Massachusetts, USA). Glyceryl monostearate (GMS) was purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan), Labrafil M 1944 CS (LbM, oleoyl polyoxy-6-glycerides) from Gattefosse (St Priest, France), soya-lecithin (SL) from Junsei Co. Ltd. (Tokyo, Japan), poloxamer 188 (Px188) from BASF (Ludwigshafen, Germany), and polyoxyethylene (40) stearate (POS; PEG-stearate), dextran sulfate sodium (DS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and coumarin-6 from Sigma-Aldrich Co. (St Louis, MO, USA). Primary cleaved caspase3, cleaved caspase-9, and cleaved poly(ADP-ribose) polymerase (PARP) antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Secondary anti-GAPDH antibody was obtained from Abcam plc (Cambridge, UK), while secondary β-actin antibody was obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). All antibodies were stored at -20 °C and were diluted before use as recommended. All other chemicals used during the study were of reagent grade and were used with no further purification.

Cancer cell lines Human breast adenocarcinoma (MCF-7; MDA-MB-231) and human skin malignant melanoma (SK-MEL-28) cell lines were initially obtained from the Korean Cell Line Bank (Seoul, South Korea). MCF-7 and MDA-MB-231 cell lines were cultured in HyClone Dulbecco’s high glucose modified Eagles medium (DMEM; GE Healthcare Life Sciences, Utah, USA) supplemented with 10 % fetal bovine serum (FBS), 100 U/mL penicillin G sodium and 100 µg/mL streptomycin sulfate, and incubated at 37 °C in 5 % CO2 atmosphere. SK-MEL-28 cells were cultured in Hyclone minimum essential medium with Earle’s balanced salt solution (MEM/EBSS; GE



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Healthcare Life Science, Utah, USA) containing 2.0 mM L-glutamine and 10 mM HEPES (4-(2hydroxyethyl)-1-piperazineethanesulfonic acid) buffer. The medium was supplemented with 10 % FBS, 100 U/mL penicillin G sodium and 100 µg/mL streptomycin sulfate, and the cells were incubated at 37 °C in 5 % CO2 atmosphere.

Development of DOX-resistant MDA-MB-231 cells DOX-resistant MDA-MB-231 cells were developed based on an approach previously reported by Bao et al.15 The resistant variants were generated by repeatedly culturing MDA-MB-231 cells in the presence of 0.2 µmol/L DOX. The cells that proliferated well in the presence of doxorubicin after several passages qualified as DOX-resistant MDA-MB-231 cells, and were named MDAMB-231(R) cells. The DOX-resistant variants of MDA-MB-231 cells, i.e. MDA-MB-231(R), were characterized against non-DOX treated MDA-MB-231 cells (Figure S1).

Preparation of PEGylated DOX/IMT-N PEGylated DOX/IMT-N was prepared by combining emulsification and ultrasonication. To elaborate, GMS and POS were melted at 70 °C, mixed together with LbM and SL, dissolved in DMSO and allowed to cool at 25°C. DOX and IMT were dissolved in DMSO and mixed with the lipids to constitute the organic phase. The organic phase was slowly injected into the aqueous phase, comprising of an aqueous solution of Px188, and vigorously stirred for approximately 10 minutes to allow for emulsification. The emulsion was subjected to ultrasonication using a probe sonicator (Vibra-Cell, SONICS) and cooled in an ice bath, to yield PEGylated DOX/IMT-N. The surfactant and DMSO were washed away by diffusion across a dialysis membrane (molecular weight cut-off 3.5 kDa). DOX/IMT-N were freeze-dried and stored at 4 °C. Thereafter, the


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nanoparticles were adequately re-dispersed in purified water as and when required. For preparation of DOX-N and blank nanoparticles, the above procedures were reproduced precluding the addition of IMT, and DOX and IMT, respectively.

Characterization of DOX/IMT-N

Particle size and ζ-potential Dynamic light scattering (DLS) was used to record the hydrodynamic particle size and ζpotential of DOX/IMT-N using a Nano-S90 ZetaSizer (Malvern Instruments, UK). All the experiments were performed in triplicate, at a scattering angle of 90° and a temperature of 25 °C. Adequate dilutions of the samples in purified water were obtained prior to each measurement.

Morphological characterization The morphology of DOX/IMT-N was studied by transmission electron microscopy (TEM). DOX/IMT-N was pre-stained with 2 % w/v phosphotungstic acid, deposited on a carbon filmcoated copper grid, and dried by exposure to mild to moderate infrared radiation. TEM images were observed and recorded under an H7600 transmission electron microscope (Hitachi, Japan).

Drug loading and encapsulation The loading capacity (LC) and encapsulation efficiency (EE) of the nanoparticles were determined by dissolving a known quantity of freeze-dried DOX/IMT-N in methylene chloride and quantifying DOX and IMT. DOX content was determined by UV/vis-spectrophotometry (PerkinElmer U-2800, Hitachi, Tokyo, Japan) at 458 nm. IMT content was ascertained by high-



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performance liquid chromatography (HPLC; Hitachi, Tokyo, Japan) using a previously reported method.23 LC and EE were calculated as percentage of drug content with respect to the total amount of nanoparticles and percentage of drug content with respect to the amount of the added drug, respectively.

Differential scanning calorimetry Differential scanning calorimetry (DSC) thermograms of freeze-dried DOX/IMT-N, freeze-dried blank nanoparticles, DOX, and IMT were obtained using a DSC-Q200 differential scanning calorimeter (TA Instruments, USA). The DSC scans were performed by heating the samples from 20 to 240 °C at a heating rate of 10 °C/min in a dynamic nitrogen atmosphere.

X-ray diffraction X-ray diffraction (XRD) patterns of freeze-dried DOX/IMT-N, freeze-dried blank nanoparticles, DOX, and IMT were recorded using a vertical goniometer and X-ray diffractometer (X’pert PRO MPD diffractometer, Almelo, The Netherlands). The diffractometer measured Ni-filtered CuKαradiation (voltage 40 kV; current 30 mA) scattered in the crystalline regions of the sample. The XRD scans were performed over a diffraction angle (2 θ) ranging from 10° to 60° at a scanning rate of 5 °/min.

In vitro drug release study In vitro release of DOX and IMT from DOX/IMT-N, and DOX from DOX-N, was evaluated at pH 7.4 and pH 5.0 by dialysis.24 A sample of system under study (1 mL) was placed in a dialysis bag (molecular weight cut-off 3.5 kDa), which was pre-hydrated overnight in phosphate-buffered


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saline (PBS), at pH 7.4 or acetate buffer solution (ABS), at pH 5.0. The dialysis bag was clipped at both ends, dipped in a 50 mL tube containing 40 mL of PBS (pH 7.4) or ABS (pH 5.0), and placed in a water bath shaker (HST-205 SW; Hanbaek ST Co., South Korea) synchronized at 100 strokes per minute at a temperature of 37 °C. Aliquots (0.5 mL) of the release media were sampled at predetermined time intervals and replaced with equal volumes of fresh media. DOX and IMT content in the samples was determined as described above.

In vitro cytotoxicity study Optimization of the drug combination Effects of varying the molar ratio of DOX and IMT in DOX/IMT-N on the proliferation of MDA-MB-231(R) cells were studied to determine the optimal combination ratio for the drugs. For this purpose, DOX/IMT-N containing DOX and IMT in molar ratios of 1:1, 1:2, 1:5, 1:10, and 1:20 were prepared along with DOX-N and IMT-N. MDA-MB-231(R) cells were seeded in 96-well plates (1×104 cells per well) in 10 % FBS-supplemented high glucose DMEM and incubated overnight at 37 °C. The medium was removed from each well, and above formulations were added in appropriate dilutions so that the final concentration of DOX/IMT-N as a total drug concentration in each well was uniform at 0.5 µg/mL. The viable cells in each well after 48-hour incubation were determined as per protocol described for the MTT assay below. DOX/IMT-N loaded with DOX and IMT in the so-determined optimal molar combination were utilized for further studies.

MTT assay



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Effects of treatment with free DOX, free DOX/IMT combination (molar ratio 1:5), DOX-N, and DOX/IMT-N on the proliferation of MCF-7, MDA-MB-231(R), and SK-MEL-28 cells were evaluated using the MTT assay. For this purpose, 1×104 cells per well, in 10 % FBSsupplemented high glucose DMEM or in 10 % FBS-supplemented MEM/EBSS containing 2 mM L-glutamine and 10 mM HEPES (depending on the cell line), were seeded in 96-well plates and incubated overnight at 37 °C. Thereafter, the culture medium was removed from each well, followed by the addition of different dilutions of DOX, DOX/IMT, DOX-N, and DOX/IMT-N (in serum-free DMEM or MEM/EBSS). After 48-hour incubation at 37 °C, the medium was removed, the wells were washed twice with PBS, 100 µL MTT (1.25 mg/mL in serum-free DMEM or MEM/EBSS) was added to each well, and the plates were incubated at 37 °C for 3 h, following which 100 µL DMSO was added to each well. The number of viable cells was determined by measuring the absorbance at 570 nm using a microplate reader (Multiskan EX, Thermo Scientific, Waltham, MA, USA). Cell viability was calculated using the formula: cell viability = (Asample / Acontrol) × 100 %, where A stands for absorbance at 570 nm. MTT assay was also performed using blank nanoparticles to determine the compatibility of the system with the cells.

Cellular uptake study FACS analysis MCF-7, MDA-MB-231(R) and SK-MEL-28 cells (2×105 cells per well) were seeded in 6-well plates, incubated overnight at 37 °C, and treated with DOX/IMT-N, with replicates constructed by varying drug concentration and incubation time in order to determine concentration- and time-dependence of the cellular uptake. After incubation, the cells were washed twice with cold


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PBS, detached from the well surfaces, and re-dispersed in 1 mL of cold PBS. Cellular uptake of DOX in various groups was determined by fluorescence-activated cell sorting (FACS) analysis using FACS Verse (BD Biosciences, San Jose, CA, USA). Untreated cells were used as internal control. At least 10 000 events were acquired and analyzed per sample. Cellular uptake of DOX/IMT-N was compared with that of free DOX. For this purpose, the cells were incubated for 2 h with DOX and DOX/IMT-N at a concentration of 5 µg/ml.

Fluorescence microscopy The cells (2×105 cells per well) were seeded in 6-well plates, incubated overnight at 37 °C, and treated with DOX/IMT- and coumarin-co-loaded nanostructured platforms (DOX/IMT/Co-N) followed by incubation at 37 °C for 2 h. The cells were washed twice with cold PBS and observed under an inverted fluorescence microscope system (Nikon Eclipse Ti, Nikon, Tokyo, Japan). The images were captured and analyzed by NIS-Elements BR 4.20.00 microscope imaging software (Nikon, Tokyo, Japan).

Apoptosis assay PE-annexin V/7-amino actinomycin D (7-AAD) apoptosis kit was used for the apoptosis assay of MCF-7, MDA-MB-231(R) and SK-MEL-28 cell-lines. Briefly, 3×105 cells per well were seeded in a 6-well plate and incubated overnight at 37 °C. The cells were treated with DOX, IMT, DOX/IMT, DOX-N, and DOX/IMT-N (in equivalent drug concentration of 2 µg/mL). After 12-h incubation at 37 °C, the cells were washed twice with cold PBS, detached from the well surfaces, and dispersed in 90 µL of 1X annexin V binding buffer. PE-annexin and 7-AAD (5 µL each) were added, and the cells were incubated at room temperature in the dark for 15 min. Finally,



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900 µL of 1X annexin V binding buffer was added and the cell samples were analyzed on a BD FACS Verse Flow Cytometer (BD Biosciences, San Jose, CA, USA). Untreated cells were used as internal control. At least 10 000 events were acquired and analyzed per sample.

Western blot analysis Western blot analysis was performed to detect relevant protein markers to aid evaluation of the apoptotic activity of the cancer cells after drug treatment. 5×105 cells per well were seeded in a 6-well plate, incubated overnight at 37 °C, and treated with DOX, IMT, DOX/IMT, DOX-N, and DOX/IMT-N (in equivalent drug concentration of 2 µg/mL) for 12 h. The cells were washed twice with cold PBS, detached from the wells, and lysed by adding M-PER protein extraction reagent (ThermoFisher Scientific Inc., Rockford, IL, USA) with a protease inhibitor. The extracted proteins were separated using SDS-PAGE gel and transferred to a PVDF (polyvinylidene difluoride) membrane (Millipore, Billerica, MA, USA) by electrophoresis. The membrane, after blocking with 5 % skim milk in Tris-buffered saline with 1 % Tween 20 (TBST), was incubated with specific antibodies (diluted as per recommendations in 5 % bovine serum albumin) against studied proteins or with anti-GAPDH or anti-β-actin antibodies (diluted as per recommendation in 5 % bovine serum albumin) as a loading control. The membrane was washed three times with TBST, incubated with the prescribed secondary antibody (diluted as per recommendation in 5 % skim milk), washed with TBST again, and soaked in SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA). The specific proteins were detected by exposing the membrane in an automated image acquisition system (LAS-400 mini, luminescent image analyzer, Fujifilm, Tokyo, Japan).


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In vivo antitumor study Development of MDA-MB-231(R) xenograft models Six-week-old female BALB/c nude mice were subcutaneously injected in the right flank region with 1×107 MDA-MB-231(R) cells dispersed in 100 µL of serum-free DMEM mixed with equal volume of Corning Matrigel Matrix Phenol Red-Free. When the tumor sizes reached ~ 100 mm3, the mice were divided randomly into 6 groups of six mice each (n = 6). The mice were kept at 20 ± 2 °C and 50-60 % relative humidity throughout the study period. All animal-handling procedures were in accordance with the protocols approved by the Institutional Animal Ethical Committee, Yeungnam University, South Korea.

Drug administration and data collection Five of the above six groups of xenograft mice were subjected to intravenous (i.v.) administration of DOX, IMT, DOX/IMT, DOX-N, and DOX/IMT-N in the tail vein, and the sixth group was utilized as untreated control. Each formulation, in the dose equivalent to 5 mg drug/kg mouse body weight, was administered on days 0, 3, 7, and 10. Tumor dimensions and body weights of the mice were evaluated at pre-determined times. Respective tumor volumes were calculated based on the formula: volume= ½ × (longest dimension) × (shortest dimension) 2

.

Tumor histopathological study Upon completion of the in vivo antitumor study period, representative tumor masses from each group were removed from euthanized mice, fixed in formalin, and sent for histopathological study. The histopathological and histomorphometric changes of the tumor masses were observed



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by general hematoxylin and eosin staining. In addition, tumor cell apoptosis (caspase-3 and cleaved-PARP), angiogenesis (CD31/platelet endothelial cell adhesion molecule 1 (PECAM-1)) and proliferation (Ki-67) were detected by immunohistochemistry staining using primary antisera and avidin-biotin-peroxidase complex (ABC) methods. The tumor cell volumes (%/mm2 of tumor mass), mean caspase-3, PARP-, CD31- and Ki-67-immunolabeled cell percentages (%/mm2 of tumor mass) were calculated to give a clearer picture of the changes with various treatments.

Statistical analysis Statistical differences between the DOX/IMT-N-treated group and other treatment groups during MTT assay, western blot analysis, and in vivo antitumor study were determined by one-way ANOVA combined with Dunnett’s test at the significance level, p < 0.05. All observations were expressed as mean ± SD (n = 3).

RESULTS AND DISCUSSION Preparation and characterization of PEGylated DOX/IMT-N PEGylated DOX/IMT-N was prepared by combining emulsification and ultrasonication. The scheme of the PEGylated DOX/IMT-N system is shown in Figure 1A. To ensure high payload, DOX was subjected to ion-pairing with DS prior to encapsulation into the nanocarrier system. Cationic DOX upon complexation with anionic DS encapsulates readily into the nanocarrier system compared to free DOX, which achieves only a modest encapsulation owing to its poor lipophilicity.25,26 For optimization of the DS/DOX ratio, aqueous solutions of DS and DOX in


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different molar ratios (1:1, 1:10, 1:20, 1:30, and 1:40) were allowed to react, and the percentages of DOX undergoing complexation to form the DS-DOX polyelectrolyte complex were determined. As shown in Figure S2, percentage of DOX that undergoes ion-pairing with DS is maximum (~ 100 %) up to a DS/DOX ratio of 1:20. Further increase of the proportion of DOX decreases the efficiency of ion-pairing. Therefore, 1:20 was selected as the optimum DS/DOX ratio for forming the insoluble polyelectrolyte complexes.



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Figure 1. (A) Scheme of PEGylated doxorubicin and imatinib-co-loaded nanostructured platforms (DOX/IMT-N). (B) Transmission electron microscopy (TEM) images. (C) Differential scanning calorimetry (DSC) thermograms. (D) X-ray diffraction (XRD) diffractograms. (E) In vitro drug release profiles of DOX and IMT from DOX/IMT-N.


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Preliminary experiments were conducted to identify and optimize various factors that affect nanoparticle size and drug encapsulation. The ratio of the lipids to the PEG-derivative can play an important role in determining the particle size and drug encapsulation capability of nanocarriers.27 Consequently, the effects of GMS-to-POS ratio on particle size and DOXencapsulation efficiency of the PEGylated DOX/IMT-N were studied, and are reported in Figure S3. At GMS-to-POS-ratio of 2:1, DOX/IMT-N exhibited the smallest particle size and greatest DOX encapsulation efficiency. Hence, a ratio of 2:1 was used for preparation of the PEGylated DOX/IMT-N. Characterization of the final DOX/IMT-N revealed a particle size ~ 120 nm, PDI < 0.200, loading capacity ~ 15 % w/w, encapsulation efficiency ~ 100 % w/w, and ζ-potential < –30.0 mV. Nanocarriers with particle sizes < 200 nm ideally exhibit preferential accumulation at tumor sites due to leaky tumoral vasculature, which, along with prolonged circulation of the nanoparticle system, forms the core of passive tumor targeting by EPR.28,29 PEGylation of the current nanostructured platforms afforded the required prolonged circulation by avoiding reticuloendothelial clearance. Furthermore, capability of the nanocarriers to accommodate a high payload ensured delivery of ample amounts of the drug to the tumor site. The moderate surface charge exhibited by the nanocarriers conferred suitable physical and physiological stability of the system in circulation. Freeze-dried nanocarriers were observed to be excellently stable physically upon storage at 4 oC and fairly stable upon storage at 25 oC, as reflected by the respective changes in particle size, PDI and encapsulation efficiency of the reconstituted nanoparticles recorded over a period of 45 days (Table S1). TEM images revealed that the nanoparticles were spherical with a dense core and sizes ~ 120 nm (Figure 1B). In the DSC thermograms, the sharp endothermic peaks corresponding to the



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melting points of the crystalline drugs were absent in case of DOX/IMT-N (Figure 1C). Similarly, XRD diffractograms showed that the characteristic peaks of DOX and IMT were absent in DOX/IMT-N (Figure 1D). There results confirmed that the drugs were well encapsulated within the carrier system in an amorphous or molecularly dispersed state. Drug release studies from DOX/IMT-N, along with those from DOX-N, were performed at pH 7.4 (PBS) and pH 5.0 (ABS) by diffusion. As shown in Figure 1E, a sequential release of DOX and IMT was achieved from DOX/IMT-N. DOX- and IMT-release achieved in 24 h was ~ 39 and 29 %, respectively, at pH 7.4, and remarkably higher at ~ 55 and 71 %, respectively, at pH 5.0. A similar release trend was observed for the single drug-loaded system (Figure S4). Drug release from all formulations followed similar trends, characterized by a pattern of sustainedrelease with evident lack of initial burst release. Mathematical modeling of the release data for DOX/IMT-N revealed a good fit to the Higuchi and Korsmeyer-Peppas models (Table S2). In accordance with the Higuchi model, the proposed release mechanism could be the extraction of the drug by simple diffusion or leaching of the drug by release fluids.30 The release exponent values derived from fitting the release data to the Korsmeyer-Peppas model were all greater than 0.5, indicating that the drug release followed anomalous or non-Fickian diffusion involving drug diffusion from the lipid core as well as the erosion of the lipid core.31 Referencing the Higuchi model, whereby the amount of drug released is a function of the solubility of the drug in the release medium, along with the fact that both DOX and IMT exhibit greater solubility at lower pH, explains the accelerated release rates of the drugs at lower pH.30,32,33 The accelerated release rates at lower pH mirror the capability of DOX/IMT-N to remain relatively stable in circulation while undergoing physicochemical changes inside the tumor tissues to permit localized drug release inside the tumor microenvironment characterized by acidic pH.34,35


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In vitro inhibition of cell proliferation Optimization of DOX/IMT molar ratio in DOX/IMT-N was performed to determine the drug combination accomplishing the maximum inhibition of the cancer cell proliferation. Maximum inhibition of proliferation of MDA-MB-231(R) cells was achieved at DOX/IMT molar ratio of 1:5 (Figure S5). Hence, molar ratio of 1:5 for DOX/IMT was selected as the optimal ratio for effective treatment and was used for further studies.

Figure 2. In vitro cytotoxic effects of different treatments on (A) MCF-7 cells, (B) MDA-MB-231(R) cells, (C) SK-MEL-28 cells, and of (D) blank NPs on different cell-lines.



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MTT assay was performed to compare the cytotoxicity profiles of DOX/IMT-N, DOX, DOX/IMT, and DOX-N on DOX-sensitive MCF-7 cells and DOX-resistant MDA-MB-231(R) and SK-MEL-28 cell lines. Figure 2 shows the cytotoxicity profiles of the four treatment groups and blank nanoparticles on MCF-7, MDA-MB-231(R), and SK-MEL-28 cells. All four treatment groups triggered dose-dependent cytotoxicity in MCF-7, MDA-MB-231(R), and SK-MEL-28 cells. DOX/IMT-N induced significantly higher cytotoxicity compared to other treatment groups. Standard curves were obtained by fitting the cytotoxicity profiles to a four-parameter logistics model to determine the individual IC50 values (Table S3). DOX/IMT-N caused explicit reduction of the IC50 values in sensitive and resistant cell lines, compared to DOX. IC50 reduction was more pronounced in resistant cells. Higher IC50 values of DOX in both resistant cell lines illustrate the DOX chemoresistance of these cells. The main mechanism associated with observed chemoresistance is ABC transporter-mediated efflux, responsible for crossresistance to a number of anticancer agents (multidrug resistance) including DOX.36 Lipid nanoparticles incorporating DOX overcome multidrug resistance by enhancing cellular uptake and retention of the drug and bypassing ABC transporter-mediated efflux.37,38 In addition, DOX, in combination with IMT, reverses acquired and intrinsic DOX resistance by a number of mechanisms, including the inhibition of ABCB1 transporter.18 These reports offer an explanation for the enhanced cytotoxic effects of DOX/IMT-N on MDA-MB-231(R) and SK-MEL-28 cells. The blank nanoparticles exhibited no apparent cytotoxicity even at an extremely high dose of 1000 µg/mL in either of the cell-lines. These results indicate that the nanostructured platforms are appreciably compatible with the cells.

Cellular uptake and apoptosis


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Cellular uptake of DOX/IMT-N by MCF-7, MDA-MB-231(R), and SK-MEL-28 cells was studied by FACS analysis and fluorescence microscopy. Through FACS analysis, concentrationand time-dependent uptake patterns of DOX/IMT-N were observed in all three cell-lines (Figure S6). Additionally, uptake of DOX/IMT-N and DOX was compared (Figure 3).



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Figure 3. FACS analyses showing cellular uptake of DOX and DOX/IMT-N by (A) MCF-7, (B) MDAMB-231(R), and (C) SK-MEL-28 cells at 5 µg/mL drug concentration and 2 h incubation time.

No significant differences were observed between the uptake of DOX/IMT-N and DOX by MCF-7 cells. However, DOX/IMT-N uptake by resistant cell lines (MDA-MB-231(R) and SKMEL-28) was clearly greater compared to DOX. This finding illustrates the capability of DOX/IMT-N to enhance DOX uptake in MDA-MB-231(R) and SK-MEL-28 cells that normally exhibit resistance to uptake and cytosolic accumulation of DOX. Fluorescence microscopy further illustrated the uptake of the nanoparticle system by MCF-7, MDA-MB-231(R), and SKMEL-28 cells. Green fluorescence originating from coumarin loaded in the nanoparticles revealed the capability of the system to undergo endocytosis by not only the MCF-7 cells, but also the MDA-MB-231(R) and SK-MEL-28 cell lines, resulting in cytosolic accumulation (Figure 4). Intense red fluorescence of DOX in the nuclear region of all three cell-lines is in favor of the hypothesized translocation of DOX into the nucleus, after it is released from the nanoparticles in the cytosol.


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Figure 4. Fluorescence microscopic images of DOX/IMT- and coumarin-co-loaded nanoparticles in (A) MCF-7, (B) MDA-MB-231(R), and (C) SK-MEL-28 cells.

Flow cytometric analysis of apoptosis was performed to ascertain the percentage of cells in early and late apoptotic phases upon treatment with DOX, IMT, DOX/IMT, DOX-N, and DOX/IMTN, and compare and contrast the DOX/IMT-N-treated group with the other treatment groups in this context. The results of FACS analysis of apoptosis in MCF-7, MDA-MB-231(R), and SKMEL-28 cells upon treatment with the specified agents are shown in Figure 5. Free DOX induced a remarkable degree of apoptosis in MCF-7 cells as evident from significant proportion of cells in early and late apoptosis. DOX/IMT-N treatment slightly accelerated MCF-7 apoptosis. In both resistant cell-lines, DOX-induced apoptosis was not remarkable. However, treatment with DOX/IMT-N induced significant apoptosis in both MDA-MB-231(R) and SK-MEL-28



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cells. In both cell lines, levels of apoptosis induced by DOX/IMT-N were greater compared to DOX, DOX/IMT, and DOX-N.


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Figure 5. FACS analyses illustrating early and late apoptosis in (A) MCF-7, (B) MDA-MB-231(R), and (C) SK-MEL-28 cells. (a) Untreated control, (b) DOX, (c) IMT, (d) DOX/IMT, (e) DOX-N, and (f) DOX/IMT-N.



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Western blotting was performed to quantify the expression levels of various apoptotic marker proteins in MDA-MB-231(R) and SK-MEL-28 cells. Expression levels of cleaved-caspase-3, cleaved-caspase-9, and cleaved-PARP in MDA-MB-231(R) cells, and cleaved-caspase-3 and cleaved-caspase-9 in SK-MEL-28 cells upon treatment with DOX, IMT, DOX/IMT, DOX-N, and DOX/IMT-N compared to untreated control are shown in Figure 6. In the lower panels of Figure 6 the respective expression levels relative to control have been quantified (n = 3). Significant increments in the expression levels of the apoptotic marker proteins in both cell-lines in DOX/IMT-N-treated group compared to DOX-, DOX/IMT-, and DOX-N-treated groups offer additional proof of DOX/IMT-N inducing significant apoptosis in both resistant cell-lines. In addition, expression of the aforementioned marker proteins provides information regarding the plausible pathways that lead to apoptosis in these resistant cancer cells.


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Figure 6. Western blotting for (A) cleaved-caspase-3, cleaved-caspase-9, and cleaved-PARP expression levels in MDA-MB-231(R) cells and for (B) cleaved-caspase-3 and cleaved-caspase-9 expression levels in SK-MEL-28 cells in different treatment groups. ** p < 0.001, *** p < 0.0001.

Figure 7. In vivo antitumor study: (A) Tumor growth and (B) weight gain/loss profiles in MDA-MB231(R) xenograft mice upon treatment with DOX, IMT, DOX/IMT, DOX-N, and DOX/IMT-N compared to untreated control. The arrows indicate the four treatment points (5 mg/kg each), at 0, 3, 7, and 10 days. ** p < 0.001, *** p < 0.0001.

In vivo tumor regression in mice bearing MDA-MB-231(R) xenograft tumors



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The potential of DOX/IMT-N to suppress metastatic breast cancer was evaluated in mice bearing MDA-MB-231(R) xenograft tumors. DOX/IMT-N significantly reduced tumor size compared to tumor-bearing control group and DOX-, DOX/IMT-, and DOX-N-treated groups (Figure 7A and S7). In addition, DOX, as monotherapy and in combination with IMT, caused severe reduction in mice body weight. DOX/IMT-N and DOX-N, however, caused no significant reduction in body weight (p < 0.05) compared to control (Figure 7B). Body weight is considered an important indicator of general systemic toxicity.39 Therefore, we can deduce that DOX/IMT-N, and DOXN, may reduce the systemic cytotoxicity associated with DOX. These results indicate that DOX/IMT-N noticeably enhances the anticancer potential of free DOX, DOX in combination with IMT, and DOX monotherapy nanoparticles.

Figure 8. Immunohistochemical evaluation of MDA-MB-231(R) xenograft tumors in untreated control (a), DOX-treated (b), IMT-treated (c), DOX/IMT-treated (d), DOX-N-treated (e), and DOX/IMT-Ntreated (f) mice: (A) Tumor mass histopathological images, (B) Images of caspase-3 and PARPimmunolabeled cells, (C) Images of CD31 and Ki-67 immunoreactive cells. Each scale bar represents 120 µm.


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Figure 9. Proposed general mechanism of anticancer activity of DOX/IMT-N.

Histopathological and histomorphometric changes in MDA-MB-231(R) xenograft tumor masses in control (untreated), DOX-, IMT-, DOX/IMT-, DOX-N-, and DOX/IMT-N-treated mice are presented in Figure 8. Significant reduction in tumor-mass volume, significant increases in apoptotic markers caspase-3 and PARP-immunoreactive cells, and significant decreases in angiogenesis and cell proliferation markers CD31- and Ki-67-positive cells, were observed, in the order of DOX/IMT-N > DOX-N > DOX/IMT > DOX > IMT, compared to tumor-bearing untreated control. These results indicate that DOX/IMT-N exhibit improved



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antitumor activity compared to DOX, DOX/IMT, and DOX-N, through increase in tumor cell apoptosis mediated by inhibition of angiogenesis and tumor cell proliferation. The overall mechanisms believed to be responsible for the excellent activity of DOX/IMT-N are summarized in Figure 9. IMT in conjunction with DOX has been reported to interfere with the activation of STAT3-dependent survival pathways.18 Suppression of STAT3 activation is known to trigger tumor-cell apoptosis and cause inhibition of metastasis,40 leading to potentiation of DOX-induced apoptosis. In addition, IMT has been shown to inhibit ABCB1 function, which, along with the curbing of P-gp-mediated drug efflux by lipid-based nanocarriers, results in increased intracellular retention of DOX and sensitization of DOXresistant cells.18,21

CONCLUSIONS PEGylated nanostructured platforms co-loaded with DOX and IMT exhibited excellent in vitro and in vivo inhibition of metastatic breast cancer. DOX/IMT-N induced significantly greater inhibition of the in vitro proliferation of MDA-MB-231(R) cells compared to free DOX, combination of DOX and IMT, and DOX monotherapy-loaded nanoparticles. DOX/IMT-N overcame P-gp-mediated efflux of DOX, and enabled accumulation of DOX in the nuclear region of the cell. The capability of the nanocarrier to overcome MDR was augmented by IMT. As a result, the nanoparticle system triggered significantly higher levels of apoptosis of MDAMB-231(R) cells compared to DOX, DOX/IMT, and DOX-N. The in vitro findings were in agreement with in vivo antitumor studies in MDA-MB-231(R) xenograft mice, where


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DOX/IMT-N showed greater suppression of xenograft tumors compared to DOX, DOX/IMT, and DOX-N. Besides encouraging results in reversing acquired DOX resistance in MDA-MB-231(R) cells, DOX/IMT-N reversed intrinsic resistance to DOX in SK-MEL-28 melanoma cells significantly more than DOX/IMT or DOX-N. The findings of this study, combined with the potential of industrial application of lipid-based nanoparticles as drug delivery tools, signify the value of DOX/IMT-N for clinical applications.

ASSOCIATED CONTENT Supporting Information Optimization of the molar ratio of dextran sulfate to doxorubicin for loading into the nanocarrier system; effects on particle size and size, and doxorubicin-encapsulation efficiency of the nanostructured platforms due to changes in glyceryl monostearate to polyoxyethylene stearate ratio; in vitro release profile of DOX from DOX-N; optimization of the molar ratio of DOX and IMT combination; FACS analyses showing concentration- and time-dependent cellular uptake of DOX/IMT-N by MCF-7, MDA-MB-231(R), and SK-MEL-28 cells; representative photographs of tumors from each treatment group after incision; correlation coefficients and rate constants of fitted release models for DOX/IMT-N; calculated IC50 values of different treatment agents on different cancer cell lines.

AUTHOR INFORMATION Corresponding Authors



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*E-mail: [email protected] *E-mail: [email protected]

Notes The authors declare no conflict of interest.

ACKNOWLEDGEMENTS This research was supported by the National Research Foundation of Korea (NRF) grant funded

by

the

Korea

government

(MSIP)

(No.

2015R1A2A2A01004118,

2015R1A2A2A04004806) and by the Medical Research Center Program (2015R1A5A2009124) through the NRF funded by MSIP.

ABBREVIATIONS GMS, glyceryl monostearate LbM, labrafil M 1944 CS SL, soya-lecithin POS, polyoxyethylene (40) stearate DMSO, dimethyl sulfoxide DLS, dynamic light scattering TEM, transmission electron microscopy LC, loading capacity EE, encapsulation efficiency DSC, differential scanning calorimetry


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XRD, X-ray diffraction FACS, fluorescence-activated cell sorting

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