Engineering of caveolae-specific self-micellizing anticancer lipid nanoparticles to enhance the chemotherapeutic efficacy of oxaliplatin in colorectal cancer cells

Accepted Manuscript Engineering of Caveolae-specific Self-micellizing Anticancer Lipid Nanoparticles to Enhance the Chemotherapeutic Efficacy of Oxaliplatin in Colorectal Cancer Cells Pasupathi Sundaramoorthy, Thiruganesh Ramasamy, Siddhartha Kumar Mishra, Keun-Yeong Jeong, Chul Soon Yong, Jong Oh Kim, Hwan Mook Kim PII: DOI: Reference:

S1742-7061(16)30322-1 http://dx.doi.org/10.1016/j.actbio.2016.07.006 ACTBIO 4317

To appear in:

Acta Biomaterialia

Received Date: Revised Date: Accepted Date:

21 March 2016 7 June 2016 5 July 2016

Please cite this article as: Sundaramoorthy, P., Ramasamy, T., Mishra, S.K., Jeong, K-Y., Yong, C.S., Kim, J.O., Kim, H.M., Engineering of Caveolae-specific Self-micellizing Anticancer Lipid Nanoparticles to Enhance the Chemotherapeutic Efficacy of Oxaliplatin in Colorectal Cancer Cells, Acta Biomaterialia (2016), doi: http:// dx.doi.org/10.1016/j.actbio.2016.07.006

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Engineering of Caveolae-specific Self-micellizing Anticancer Lipid Nanoparticles to Enhance the Chemotherapeutic Efficacy of Oxaliplatin in Colorectal Cancer Cells

Pasupathi Sundaramoorthy1,‡, Thiruganesh Ramasamy2,‡, Siddhartha Kumar Mishra3, KeunYeong Jeong1, Chul Soon Yong2, Jong Oh Kim2, Hwan Mook Kim1,*

1

Gachon Institute of Pharmaceutical Sciences, Gachon University, Incheon 406-840, Republic of

Korea 2

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

Korea 3

Department of Zoology, School of Biological Sciences, Dr. Harisingh Gour Central University,

Sagar 470003, India

‡

Both authors contributed equally.

Correspondence author: Hwan Mook Kim, Ph.D Gachon Institute of Pharmaceutical Sciences, Gachon University Incheon, 406-840, Republic of Korea Tel: +82-32-899-6446; Fax: +82-32-820-4829; [email protected]

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ABSTRACT Novel nanomaterials for the intracellular transport of therapeutic cargos have been actively sought to effectively breach cell-membrane barriers. In this study we developed novel selfmicellizing anticancer lipid (SMAL)-based pro-apoptotic nanoparticles (NPs) that enhance the accumulation and chemotherapeutic efficacy of oxaliplatin (OL) in colorectal cancer cells (CRCs). We demonstrated that NPs with special affinity to caveolae could be designed and based on this specificity, NPs effectively differentiated between endothelial cells (tumor cells) and epithelial cells, without the need for a cell-specific targeting moiety. We demonstrated a remarkable uptake of OL-loaded SMAL NPs (SMAL-OL) in HCT116 and HT-29 cells via the caveolae-mediated endocytosis (CvME) pathway. The higher accumulation of SMAL-OL in the intracellular environment resulted in a significantly elevated anticancer effect compared to that of free OL. Cell cycle analysis proved G2/M phase arrest, along with substantial presence of cells in the sub-G1 phase. An immunoblot analysis indicated an upregulation of pro-apoptotic markers (Bax; caspase-3; caspase-9; and PARP1) and downregulation of Bcl-xl and the PI3K/AKT/mTOR complex, indicating a possible intrinsic apoptotic signaling pathway. Overall, the ability of SMAL NPs to confer preferential specificity towards the cell surface domain could offer an exciting means of targeted delivery without the need for receptor-ligand-type strategies. KEYWORDS Caveolae-mediated endocytosis (CvME), self-micellizing anticancer lipid (SMAL), oxaliplatin, colorectal cancers, nanoparticles

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1. INTRODUCTION Colorectal cancer (CRC) is the third leading cause of cancer-related deaths, with approximately 50,000 deaths in the United States alone and around 650,000 deaths worldwide per year [1]. Gastrointestinal (GI) tract cancer is the second leading cause of death among male and female patients. Overall, the lifetime risk of developing CRC is about 1 in 20 (5.1%) [2]. Surgery is commonly employed as a curative treatment in various stages of early, as well as advanced, CRC. However, surgery can not eliminate all malignant cells, therefore warranting additional drug therapies such as chemotherapy [3]. The Food and Drug Administration approved oxaliplatin (OL), a third-generation platinum compound, for the treatment of stage III, advanced, or metastatic CRC [4]. OL acts by binding to DNA or RNA, and forms intrastrand adducts (platinum-DNA adducts), which disrupt DNA replication and transcription processes. This results in cell apoptosis, and eventually cell death [5]. However, the use of OL is still associated with a range of side effects, including myelotoxicity, acute and chronic peripheral neuropathy, cardiotoxicity, and severe GI tract disorders [6]. Moreover, conventional chemotherapeutic drugs often suffer from poor solubility, a narrow therapeutic window, and poor systemic distribution patterns, which may result in treatment failure in cancers. In order to improve the therapeutic efficacy and reduce the toxicities of platinum drugs, various delivery systems (liposomes, polymeric nanoparticles, polymeric capsules and inorganic nanoparticles) have been studied, which can direct the anticancer drugs to solid tumors by virtue of the enhanced permeation and retention (EPR) effect [7, 8]. Nevertheless, a lack of adequate cellular uptake, carrier-mediated toxicity and poor dispersion stability of nanocarriers has hampered the clinical translation of OL in CRC [9].

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Nanoparticulate drug delivery systems (Nano-DDS) have emerged as promising strategy for the specific delivery of anticancer drugs [10, 11]. After circulating in the bloodstream, nanoparticles (NPs) could passively accumulate in the tumor tissue [12]. In this context, any nanomaterial that can enhance the chemotherapeutic response of a drug moiety, while being non-toxic to normal cells, is highly desired (a pro-apoptotic agent) [13, 14]. Bioactive lipids such as sphingolipids play an important role in signal transduction pathways, especially growth arrest, cell proliferation and pro-apoptotic effects in cancer cells [22]. They control various membrane-associated signaling pathways, which are actively involved in growth and apoptosis of cancer cells. Moreover, sphingolipids have been reported to induce apoptosis by generating reactive oxygen species (ROS), caspase expression and Bax translocation. Recently, it has been established that sphingolipid-based novel self-micellizing anticancer lipid (SMAL) could selectively induce cell death via the activation of apoptosis and autophagy in CRC cell lines. [15, 16]. Through understanding this clinical outcome, we sought to develop a SMAL-based Nano-DDS that serves the dual function of a drug delivery agent with antitumor activity. Recently, great efforts have been made to identify effective and new pharmacological targets that can increase cancer-targeting efficiency [17]. Despite the great advancement in cancer biology, a generalized approach of targeting the therapeutic agent to malignant cells is a big challenge due to the variability in the expression of targets in different patients and at different stages of cancers [18]. Therefore, a highly generalized approach, which can differentiate between various cell types found within the tumor microenvironment, without the need for ligand/receptor-based active targeting, could be of great importance. In this context, lipid raft-based cellular uptake (through caveolae) is one of the processes involved in cell uptake of extracellular particles. Caveolin-1(CAV1) is a major structural protein in the caveolae (flask-shaped invaginations of the

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plasma membrane) and is involved in various cellular functions such as signal transduction, lipid metabolism, cell growth, survival and apoptosis[19, 20]. The biological importance of caveolae stems from the difference in the caveolae density and expression levels of CAV1 in different cell types and tissues. For instance, reduced expression of CAV1 has been observed in ovarian, lung, head and neck carcinomas [21], while high expression of CAV1 was found to be associated with the progression of colon and prostate cancers. Importantly, low expression of CAV1 occurs in normal, healthy cells, making it an ideal pharmacological target [22, 23]. Thus far, main aim of present study was to increase the therapeutic efficacy of OL in CRCs by loading it in a SMAL carrier. Based on the simple premise, it was hypothesized that OL-loaded SMAL NPs (SMAL-OL) could be specifically taken up by cancer cells without the need for cellspecific targeting ligands. This could be realized based on the specificity of the SMAL NPs towards the caveolae, which are overexpressed in CRC, while it can inherently discriminate the healthy cells due to the low expression of CAV1 (Figure 1).

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2. MATERIALS AND METHODS 2.1. Materials OL was purchased from TCI Co., LTD (Tokyo, Japan). 1,3-bis(N-2 (hydroxyethyl) lauroyl amino)-2-hydroxy propane (SMAL102), 1,3-bis (N-2-(hydroxyethyl) palmitoyl amino)-2hydroxy propane (SMAL104) and 1,3-bis (N-2-(hydroxyethyl) tetramide amino)-2-hydroxy propane (SMAL108) were purchased from Macrocare Tech., LTD (Ochang, South Korea). Primary antibodies caveolin1 (CAV1), poly (ADP-ribose) polymerase-1(PARP1); caspase-3; caspase-9; mTOR; phospho-mTOR; phospho-p70S6K; pAKT; p53; p21; cyclin A; cyclin B; cyclin E; pCDC2; Bcl-xl; Bax; Bid and glyceraldehyde-3-phosphate dehydrogenase (GADPH) and the corresponding secondary antibodies for western blotting were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA). MTT reagent, sucrose, methyl-betacyclodextrin (M-β-CD), propidium iodide (PI), and ribonuclease were obtained from SigmaAldrich (St. Louis, MO, USA). 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2dipalmitoyl-sn-glycero-3-phosphatidylglycerol

(DPPG)

and

1,2-dioleoyl-sn-glycero-3-

phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (rhodamine B) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). SMAL 102, 104 and 108 was stored at room temperature. DPPC and DPPG were stored at -20°C. All the materials were stable during preparation and storage conditions. All other chemicals were of reagent grade and used without further purification. 2.2. Preparation of NPs SMAL-based lipid NPs were prepared by the thin-film hydration technique[24]. Briefly, SMAL102 (1 mg/mL), SMAL104 (1 mg/mL) and SMAL108 (1 mg/mL) were separately taken in a round bottom flask (RBF) and to each flask, DPPC (0.125 mg/mL) followed by DPPG

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(0.125 mg/mL) were added. The organic mixture (chloroform and methanol, 2:1) was added and the mixture was subjected to rotary evaporation to form a thin film for 1 h. The film was hydrated via the addition of 10 mM HEPES (pH 7.4) buffer containing OL (250 µg/ml), and incubated for 1 h at 60°C. The hydrated mixture was then subjected to bath sonication (continuous) (Cole-Parmer Instrument Company, Illinois, USA) at low intensity (20 W) for 10 min and the product was stored at 4ºC until further use. For the intracellular cellular uptake study, rhodamine-B was used as a fluorescent dye.

2.3. Characterization of drug-loaded NPs The mean particle size, polydispersity index (PDI) and zeta potential were measured using high performance dynamic light scattering (DLS) technique with a Malvern Zetasizer Nano ZS (Malvern Instrument, UK) at a fixed angle of 90°. The samples were suitably diluted in double distilled water and measurements were performed in triplicate. Morphological analysis of all the NPs was performed using a transmission electron microscope (TEM; H7600, Hitachi, Tokyo, Japan). A drop of SMAL NPs was deposited on the copper grid and counter stained with 2% (w/v) phosphotungstic acid and then air-dried. 2.4. Drug loading and entrapment efficiency During preparation, drug was loaded during the hydrating process followed by which formulations were sonicated. The drug-loaded NPs were separated from the free drug by ultrafiltration using Amicon YM-10 centrifugal filter devices (MWCO 10,000 Da, Millipore). The concentrations of OL in the filtrates and in the NPs were determined by high-performance liquid chromatography (HPLC). The mobile phase consisted of methanol 0.9 % w/v sodium

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chloride solution (80: 20, v/v). The flow rate was maintained at 1 mL/min and the eluent was detected at 240 nm. Loading efficiency (LE) % = Total amount of drug-Free drug in filtrate/Total amount of drug× 100 Loading capacity (LC) % = Total amount of drug-Free drug in filtrate/Mass of NP× 100 2.5. In vitro drug release study The release of OL from SMAL-OL was evaluated in phosphate-buffered saline (PBS, pH 7.4, 0.14 M NaCl) and acetate-buffered saline (pH 5.0, 0.14 M NaCl) using a dialysis method (Spectra/Por; 3,500 Da cutoff). At specific time intervals, the medium was completely withdrawn and replaced with fresh medium to maintain sink conditions. The concentration of the released drug was determined by HPLC, as described above. 2.6. Stability studies The stability of the SMAL-OL formulation was assessed via determination of the particle size after 10, 20, 40, and 60 days of storage in the refrigerator (4°C) in PBS and serum conditions. 2.7. Cell Culture HCT116, HT-29 human colon cancer cells and CCD-18Co human normal colon fibroblast cells (all obtained from the American Type Culture Collection, Manassas, VA, USA) were grown in Roswell Park Memorial Institute Media (RPMI) containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in a humidified atmosphere containing 5% CO2. 2.8. Cytotoxicity Assay The cytotoxicity of SMAL102 NPs, SMAL104 NPs, SMAL108 NPs, free OL and SMAL-OL to HCT116 and HT-29 cells was evaluated using the MTT assay. Cells were seeded onto 96-well plates (5000 cells/well) and grown for 24 h. The cells were then treated with various 8

concentrations of the formulations (0.1–2.5 µM) for 48 h. After treatment, MTT stock solution (5 mg/mL in PBS, 10 µL) was added to each well and incubated for 2 h at 37°C in a humidified incubator containing 5% CO2. The media was removed and 100 µL DMSO was added to dissolve the formazan blue crystals. The absorbance was measured using a microplate reader at 570 nm. For cytotoxicity assay, a concentration range of 0.1-2.5 µM was selected. The concentration employed in this study was remarkably less compared to that of marketed formulations (Eloxatin injection; 85 mg/m2). 2.9. Colony Formation Assay Briefly, 6-well plates were seeded with 100 to 200 viable cells per well and incubated for 48 h. Thereafter, cells were treated with free OL, SMAL NPs and SMAL-OL (0.1–2.5 µM) in RPMI containing 10% FBS. The media was changed every 3 days. After 10 to 12 days, the visible colonies were fixed with methanol and then stained with hematoxylin and eosin. The colonies were compared with untreated cells and counted. All treatments were conducted in triplicate. 2.10. CLSM observation HCT116 and HT-29 cells were seeded on cover slides in 6-well plates at a density of 4 × 105 cells/well in 2 mL of complete growth media and incubated for 24 h in a 5% CO2 atmosphere. The cells were then incubated with SMAL-rhodamine or SMAL-PL-rhodamine (rhodamine ~1 µg/mL) NPs in RPMI for 2 h at 37°C. After treatment, culture media was removed and the cells were washed with PBS to remove non-internalized NPs. Cells were then fixed with 4% paraformaldehyde for 5 min and slides were washed twice. Finally, cells were sealed with mounting media containing DAPI (Vectashield, Vector Laboratories, Burlingame, CA, USA) and examined by CLSM (Nikon A1+, Japan). 2.11. Quantitative analysis of cellular uptake by flow cytometry 9

HCT116 and HT-29 cells were seeded into 6-well plates at a density of 2 × 105 cells/well in 2 mL of complete growth media and incubated for 24 h at ambient conditions. Cells were then incubated with SMAL-rhodamine or SMAL-PL-rhodamine (rhodamine~1 µg/mL) NPs in RPMI for 2 h at 37°C for different time periods. The cells were washed twice with PBS to remove rhodamine-loaded NPs that were not ingested by the cells. The cells were then detached by trypsinization and centrifuged at 2000 rpm for 5 min. After the removal of the supernatants, cell pellets were resuspended in 500 µL of PBS and directly introduced into a FACS CaliburTM (BD Biosciences, NJ, USA). The mean fluorescence intensity (MFI) was analyzed at excitation (495 nm) and emission (519 nm) wavelengths to detect rhodamine. At least 10,000 cells were acquired and analyzed per sample. 2.12. Endocytosis inhibitor treatment HCT116 and HT-29 cells were seeded in slides on a 6-well plate at a density of 4 × 105 for CLSM analysis and 2 × 105 cells/well for FACS analysis in 2 mL of complete RPMI media and incubated for 24 h. To determine the SMAL NPs cellular uptake, cells were pretreated with two different endocytosis inhibitors at concentrations that were non-toxic to cells (10 mM M-β-CD and 0.45 M sucrose). Then, SMAL-PL-rhodamine NPs were added and incubated for 1 h. The subsequent steps were similar to those described in Section 2.10 and 2.11. 2.12. Cell cycle study HCT116 and HT-29 cells were seeded in 6-well plates (2 × 104 cells/well) and incubated for 24 h. Cells were treated with free OL, SMAL NPs, and SMAL-OL (0.1–2.5 µM) for 48 h. The cells were collected by trypsinization and fixed using 70% ice-cold ethanol on ice for 1 h. The cells were subsequently incubated with 10 µL of PI (10 mg/mL) and 5 µL of ribonuclease (10 mg/mL) for 30 min at 37ºC in the dark. The cell cycle distribution was assessed using a FACS Calibur

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analyzer (BD Biosciences) with 10,000 cells. Cell-cycle compartment and the percentage of cells at each phase was calculated using the CellQuest software. 2.13. Annexin V/PI- based apoptosis assay Apoptotic cell death was determined using an annexin V-FITC Apoptosis Detection Kit (BD Biosciences). Briefly, HCT116 and HT-29 cells (2 × 106) were exposed to free OL, SMAL NPs, and SMAL-OL (0.1–2.5 µM) for 48 h, according to the manufacturer’s protocols. The cells were stained with annexin V-FITC and PI for 15 min at room temperature and then enumerated via flow cytometry analysis using a FACS CaliburTM instrument (BD Biosciences). Cells that were annexin-V+ (in apoptotic cells, phosphatidylserine translocates from the inner to the outer leaflet of the plasma membrane, hence allowing annexin-V to bind to it) were early apoptotic cells and V-/PI- (with an intact cellular membrane) were live cells, whereas both annexin-V+/PI+ cells indicated late apoptosis or necrosis. 2.14. Western Blotting Cells were seeded and treated with the respective formulations. The cells were then lysed on ice for 60 min in RIPA buffer (Thermo Scientific, MA, USA). The whole-cell lysates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a polyvinylidene difluoride membrane (Millipore). Membranes were blocked with 5% skim milk and subsequently incubated with the specific primary antibodies overnight at 4°C. After incubation with the secondary antibody conjugate-horseradish peroxidase, blots were revealed using the ECL system (AbClon) for signal detection. Films were developed using a Kodak M35-A X-OMAT processor. 2.15. Statistical Analysis

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Data were analyzed by analysis of variance (ANOVA) or the Student’s t-test using IBM SPSS Statistics for Windows, Version 22.0 (IBM Corp., Armonk, NY, USA) to determine whether differences between test groups were statistically significant. Data are presented as the mean ± standard deviation of four independent experiments. A value of P < 0.05 was considered statistically significant.

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3. RESULTS AND DISCUSSION 3.1. Formulation and characterization of Oxaliplatin-loaded SMAL NPs Bioactive lipids such as sphingolipids play an important role in signal transduction pathways, especially growth arrest, cell proliferation and pro-apoptotic effects in cancer cells [25, 26]. Kim and coworkers recently reported on the drug-like pro-apoptotic SMAL and investigated its selfassembling properties, thermodynamic stability and biological responses [15]. The SMAL carrier itself exhibited a remarkable antitumor activity in CRC cells (HT-29 and HCT116) via apoptotic and autophagy pathways. To ascertain the therapeutic potential of SMAL NPs in cancer therapy. In the present study, SMAL NP was encapsulated with OL, the first line treatment for advanced or metastatic CRC. Among all the materials, we have selected SMAL 102 based on the particle size and its colloidal stability. Most importantly, we have studied the cytotoxic effect of 102, 104 and 108 NP in colorectal cancer cells. SMAL102 showed a marked cytotoxic effect in both CRC cell lines whereas SMAL104 and SMAL108 did not induce appreciable cytotoxic effect which was in turn attributed to its large particle size. Therefore, based on particle size and cytotoxic effect, we have selected SMAL102 for further studies (Table S1). Moreover, a surface charge of around -20 mV could contribute to its high colloidal stability where as SMAL104 and SMAL108 possessed almost neutral surface charge. At this stage, much influence of surface charge on the internalization pathways could not be derived as all the particles have either negative to neutral surface charge. The mean diameters of blank and OL-loaded SMAL NPs were determined by the DLS method. The physicochemical characteristics of all the NPs are summarized in Table 1. SMAL was able 13

to self-assemble into stable NPs with a mean diameter of 110±3.25 nm and an acceptable dispersity index of 0.264 (PDI). As expected, the addition of the phospholipid mixture led to a significant decrease in the NP size (85±2.46 nm). The decrease in NP diameter could be attributed to an increase in the rigidity and stability of the structure. The (SMAL-OL) exhibited a mean diameter of 96±2.71 nm with an excellent PDI of 0.125 and effective surface charge of 18.4 mV, which is sufficient to stabilize the particles against aggregation. The nanosized SMALOL are within the desired range for tumor targeting via the EPR effect [27]. A sub-100 nm particle is considered to be very effective in reducing non-specific uptake by the reticuloendothelial (RES) system [28, 29]. SMAL-OL showed a high entrapment efficiency of >90%, with an active drug loading of approximately 15%. Nanocarriers with high drug loading capacity are considered to possess clinical benefits [30]. The morphology of different NPs was characterized using TEM (Figure 2a). Consistent with the DLS observations, SMAL NPs exhibited polydispersed particles with an irregularly arranged spherical outfit. However, phospholipid-combined SMAL NPs revealed perfect spherically shaped particles with a uniform distribution. SMAL-OL also exhibited distinct and spherically shaped particles with a homogenous distribution. To verify the effectiveness of SMAL-OL for intracellular drug delivery, a release study was performed at pH 7.4 and 5.5, to simulate the physiological and tumor environments. As seen in Figure 2b, encapsulated OL was released in a controlled manner throughout the study period in both pH conditions. The data revealed the high stability of SMAL-OL in neutral and acidic pH conditions with no initial burst release. Approximately 25% of the OL was released from SMAL NPs in the first 24 h of the study period, while the drug was released in a constant slow manner thereafter. A slightly higher release in acidic conditions might be attributed to the disturbances in

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the structural integrity and quicker diffusion of drug. The slow and continuous release of encapsulated drug will be beneficial for the cancer treatment since a constant exposure of drug to the cancer cells will inhibit its proliferation [31]. In contrast, free drugs diffuse at once and will be having limited effect on the cancer cell inhibition. The ability of the carrier to release the anticancer drug efficiently at the desired site is an important feature of any delivery system. The long-term stability of SMAL-OL was studied in PBS and serum medium at 4°C. As shown in Figure 2c, particles remained stable, even when stored for 60 days. Furthermore, an insignificant difference in particle size (between day 0 and 60) was observed in PBS and serum conditions, with no tendency to aggregate or sediment. A slight increase in particle size in PBS was due to the presence of ions such as CO32−, PO42− and H+ in buffered media that formed the counter-ions shell on the NP surface and structure of water surrounding the system. In the case of serum media, physical adsorption of serum albumin on the NPs’ surfaces may increase the particle size. Nevertheless, drug-loaded NPs showed excellent stability throughout the study period, indicating their suitability for clinical applications [32]. 3.2. Effect of phospholipids on the cellular uptake of NPs SMAL NPs were prepared using a phospholipid mixture of DPPC and DPPG (SMAL-PL). To evaluate the effect of phospholipids on the cellular uptake, rhodamine B was used as a fluorescence probe and DAPI was used as a nuclear staining agent. The cellular uptake of NPs and localization of fluorescein in HCT116 and HT-29 cells were studied by CLSM. As shown in Figure 3a and b, SMAL-PL NPs exhibited remarkably higher fluorescence intensity in both the cancer cell lines than SMAL NPs. The results indicate the faster internalization of the PLcontaining NPs. It is worth noting that all the NPs accumulated in the cytoplasmic region,

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indicating a typical endocytosis-mediated cellular uptake. Bright fluorescence in the cytoplasm was possibly due to the fact that some of the fluorescein molecules diffused out of the SMAL NPs and distributed throughout the cytoplasmic region. To further quantitate the cellular uptake efficiency of SMAL (with and without PL), FACS was performed (Figure 3c and d). Consistent with the intracellular distribution patterns, SMAL-PL showed a manifold shift in the fluorescence intensity to higher values in both the cancer cell lines in a time-dependent manner. This time-dependent behavior may be due to the presence of an active endocytosis process within the system. These results were in accordance with our hypothesis that the internalization of NPs by cells is affected by the incorporation of phospholipids in SMAL NPs. The phospholipids phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI) and sphingomyelin (SM) are distributed throughout the cellular membrane and are important for endocytosis-mediated cellular uptake [33]. Therefore, the difference in the NPs cellular uptake was perhaps due to changes in the fluidity of biological membranes. This was attributed to changes in membrane-initiated signaling processes according to the phospholipid head group on the NPs’ surface. Differences in the membrane fluidity for the SMAL and SMAL-PL carrier systems might influence the mechanism of entry into the cells. Utilizing both CLSM and flow cytometry, the results provided enough proof of the improved cellular uptake efficiency of SMAL-PL. 3.3. Endocytosis inhibition and caveolae targeting Knowledge of intracellular delivery and cellular distribution of nanoparticulate systems is deemed necessary to selectively target drugs at a subcellular level. To elucidate the internalization mechanisms, M-β-CD and hypertonic sucrose were used as endocytosis inhibitors. CLSM images clearly revealed a sharp decrease in the fluorescence intensity surrounding the 16

cytoplasmic region in both the cancer cell lines (Figure 4a and b). Specifically, M-β-CD had a remarkable effect on the uptake of SMAL-OL, as evidenced by the marked decrease in the fluorescence intensity due to the blockade of NP entry. At the same time, sucrose also decreased the uptake of NPs in the cancer cells. Similar observations were made from the FACS analysis, where both endocytosis inhibitors decreased the cellular uptake (Figure 4c and d, Figure S1). This clearly indicates that the uptake of NPs is related to lipid rafts, cholesterol-dependent and CvME. Based on the prominent inhibitory effect of M-β-CD, we cautiously assume that SMALOL undergoes cytosolic delivery via CvME, a prominent internalization pathway. Because SMAL-OL is taken up through lipid rafts, or more precisely CvME, this suggests a possible role for the caveolae in endocytic transport of lipidic NPs [34]. Considering the fact that NP uptake is higher in HCT116 cells , and taking into account the finding that HCT116 cells have a higher CAV1 content than HT-29 cells [35], we have demonstrated that NPs with specific affinity for caveolae can be realized. It has been reported that designing a nanoparticulate system with special affinity towards caveolae (CvME) could potentially bypass the lysosome and therefore escape lysosomal degradation [36]. Earlier, we have reported low cellular uptake and nontoxicity in normal cells; consistently, others also reported a low expression of CAV1 in normal healthy cells [15, 21, 22]. Nanoparticles for chemotherapy need to be absorbable to cells with a sufficiently high rate and extent. It has been proposed that the size of the particles plays a key role in their adhesion to and interaction with the biological cells and determines the extent of cell uptake. It is generally believed that particles up to about 100–200 nm can be internalized by receptor-mediate endocytosis, while larger particles have to be taken up by phagocytosis. Based on this fact, we assumed that the difference in cellular uptake of SMAL (~180 nm) and SMAL-PL (~100 nm)

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was mainly attributed to the difference in the particles size. The small particle size of SMAL-PL resulted in higher cellular uptake in cancer cells than that of SMAL NP. Moreover, it has been reported that caveolae could effectively transport sub-100 nm particles. Based on all this fact, it can be assumed that small particle size of >100 nm allows the particle to be internalized in a better way. 3.4. Synergistic anticancer effect of SMAL and OL In this study, we introduced a phospholipid (PL) combination via the use of SMAL102 (lauramide derivative), SMAL104 (palmitamide derivative), and SMAL108 (stearamide derivative) in order to increase cellular uptake of the NPs. The antiproliferative effect of different nanomaterials was studied using the MTT assay and the results are presented in Figure S2. SMAL102 NPs exhibited a remarkable anticancer effect in both the cancer cell lines after 48 h of incubation. The SMAL102-based NPs showed cytotoxic effects at a much lower concentration (10 µg/mL) and killed almost 95% of cancer cells (HCT116 and HT-29). In contrast, SMAL104and SMAL108-based NPs did not elicit potent anticancer effects and larger fractions of cells were viable even at the maximum concentration tested. In our previous study, we showed the remarkable anticancer effect of SMAL102 in CRC cells and its induction of apoptotic cell death. Earlier, we theorized that SMAL102 NPs loaded with chemotherapeutic agents would lead to an increase in therapeutic efficacy in cancers. Based on our past and present findings, we loaded OL in SMAL102 and evaluated the synergistic anticancer effect (Figure 5a and b). At equivalent drug concentrations, SMAL-OL induced a remarkable effect on cell death when compared to free OL in both the cancer cell lines. Therefore, it is clear that the combination of SMAL and OL could induce a greater anticancer effect in CRC cells [37]. The IC50 values of OL, blank SMAL NPs and SMAL-OL were 2.44 µM, 0.78 µM and 0.47 µM, respectively, in HCT116 cells, while 18

in HT-29 cells they were 2.12 µM, 1.89 µM and 0.73 µM, respectively. The remarkable cytotoxic effect of SMAL NPs and SMAL-OL is mainly attributed to augmented caveolae-mediated endocytosis uptake, which increased the accumulation of therapeutic agent in the intracellular environment and resulted in greater cell death. However, free OL enters the cells via passive diffusion through the cell membrane and therefore, results in a low cytotoxic effect. These results indicate that use of SMAL could serve two functions. Firstly, it acts as a stable delivery carrier, which preserves the pharmacological activity of the encapsulated drug. Secondly, the carrier itself synergizes with the anticancer effect of the OL in cancer cells, while being non-toxic to normal healthy cells (Figure S3) [38, 39]. The superior anticancer effect of SMAL-OL was further confirmed by a colony formation assay (Figure 5c and d). Consistent with the MTT assay; OL, blank SMAL NPs and SMAL-OL decreased colony formation in a dose-dependent manner in HCT116 and HT-29 cancer cells (Figure S4). At an equivalent drug concentration, SMAL-OL showed significant inhibition of colony formation compared to that of free OL and blank SMAL NPs. In general, the degree of the anticancer effect correlates with the amount of OL bound to DNA. Our results suggest that the incorporation of OL into the SMAL nanocarrier improved its accumulation in cancer cells and induced a more potent signal transduction regulatory pathway leading to greater inhibition of colony formation and resulting in cell death [40, 41]. 3.5. Cell cycle analysis and cell apoptosis The checkpoints in the cell cycle control the process of cell division and distinguish whether the processes at each phase of the cell cycle have been accurately completed before entering into the next phase. In general, cell cycle distribution following DNA damage is controlled by

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checkpoints. The failure to repair the DNA could result in mitotic dysregulation and lead to cancer cell apoptosis. OL is known to cause DNA damage and, thereby, induces cell cycle arrest in rapidly dividing cells, eventually resulting in apoptosis. To understand the cell inhibitory effect, cell cycle distribution of HCT116 and HT-29 cells was studied after treatment with blank SMAL NPs, OL and SMAL-OL (Figure 6). Two different trends were observed for free OL and drug-loaded SMAL NPs, with both exhibiting dose-dependent cell cycle patterns. At 0.1 µM, all the formulations exhibited a G2/M phase arrest of cells, which was closely associated with the cell inhibition effect. Meanwhile, when the cells were treated with 1 µM or 2.5 µM, a substantial amount of cells were present in the sub-G1 phase. As expected, the result for SMAL-OL (2.5 µM) showed approximately 68% of cells in the sub-G1 phase compared to approximately 27% and 18% for blank SMAL NPs and OL, respectively, in HCT116 cells (Figure S5). Similar trends were observed in HT-29 cells with approximately 82%, 60%, and 12% of cells in the sub-G1 phase for SMAL-OL, blank SMAL NPs and OL, respectively (2.5 µM). This indicates that SMAL-OL decreases the percentage of cells in the G1 phase, along with an increase in those in the sub-G1 phase of the cell cycle, without the disruption of the S phase. Nevertheless, blank SMAL NPs induced greater cell cycle arrest than that of free OL in both the cancer cells. These results are in accordance with those of Voland et al. who showed that OL-induced G1 and G2/M arrest was associated with the blockage of DNA replication and mitosis and simultaneous downregulation of expression of genes involved in the regulation of these processes [42]. In summary, our findings clearly demonstrate that the growth of CRC cells was markedly inhibited by the combination of SMAL and OL, which accumulates preferentially in the intracellular environment owing to its specificity towards caveolae (CvME).

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The apoptosis of cancer cells with phosphatidylserine on the outer side of an intact cell membrane was evaluated by annexin-V/PI staining. As shown in Figure 7, early apoptosis (annexin-V+ and PI-) and late apoptosis (annexin-V+ and PI+) were the major mechanisms of cell death caused by all the formulations. At all concentrations (0.1 to 2.5 µM), SMAL-OL showed significantly higher apoptosis compared to that of free OL or blank SMAL NPs in both the cancer cell lines (Figure S6). For example, SMAL-OL (2.5 µM) induced apoptosis in approximately 80% of cells, which is significantly (P < 0.01) greater than the blank SMAL (~60%) and free OL (~14%) in the HCT116 cell line. Similarly, in HT-29 cells, SMAL-OL induced apoptosis in approximately 65% of cells compared to approximately 50% and 25% for blank SMAL NPs and free OL, respectively. Cell shrinkage and membrane blebbing were consistently observed in the SMAL-OL treated group (Figure S7). In summary, SMAL-OL inhibited the growth of CRC cells through cell cycle arrest at the G2/M phase and induces cell apoptosis. The superior apoptotic effect could be explained by the higher uptake by cancer cells through the CvME pathway, which actively transports the drug into cells. It is believed that the endocytosis pathway is much more effective in cell uptake than the passive diffusion pathway utilized by free OL solutions [43]. 3.6. Regulation of intrinsic apoptotic signaling pathway OL is known to bind to DNA, thus forming intrastrand adducts (OL-DNA) and causes DNA damage by activating the cell cycle checkpoints. This results in the activation of p53 and p21 in HCT116 and HT-29 cells. Consistent with the cytotoxicity and apoptosis analysis, SMAL-OL elicited a marked expression of p53 and p21 in these cells (Figure 8a). It has been reported that damaged DNA activates Chk1, which in turn targets phosphatase Cdc25a for degradation, ultimately leading to the failure of Cdk2 activation [44]. p53-mediated transcription is deemed 21

necessary to maintain the arrest and the p21 protein inhibits cyclin E-Cdk2 or cyclin D-Cdk4 complexes, thereby, suppressing the Rb/E2F pathway and cell death [45]. Our studies have shown that the antiproliferative effects of OL and SMAL-OL are attributed to the modulation of the cell cycle machinery. Therefore, it is important to understand the mechanism regulating the passaging of these cells, which controls the diverse cellular processes that lead to cell death. Cyclin A and B are key cell cycle regulators of G2 to mitosis (G2/M) progression, whereas cyclin E regulates the transition to the S phase. Due to their roles, these cyclins are intrinsically involved in the regulation of apoptosis [46]. The G2/M checkpoint ensures that cells do not enter mitosis and contact with chemotherapeutic drugs. Inability of cells to halt their progression at the G2 stage may, therefore, be fatal and result in death. Our immunoblot analysis indicated that the levels of cyclin A, cyclin B, cyclin E, and pCDC2 were significantly decreased when treated with a synergistic combination of OL and SMAL. Cyclin A and cyclin B, which are important regulatory proteins, were inhibited by SMAL-OL. Therefore, DNA synthesis was blocked and resulted in a shortened S-phase. These results implied that the anti-apoptotic function of these nanoformulations might depend on the function of cyclins. We further monitored the change in the expression of pro-apoptotic markers (Bax, caspase-3, caspase-9 and PARP-1) and anti-apoptotic markers (Bcl-2 and Bid). Figure 8b clearly shows the remarkable induction of pro-apoptotic Bax by SMAL-OL. Consistently, expression levels of caspase-3, caspase-9, and PARP were markedly downregulated. Furthermore, remarkable cleavage of caspase-3 from a 35-kDa polypeptide to a 17-kDa polypeptide was observed in the cells treated with OL-loaded SMAL compared to that of free OL. The cleavage of caspase-3 was further supported by the cleavage of PARP (an enzyme that is activated after DNA damage). Data revealed a cleavage of the PARP polypeptide from 116-kDa fragments into smaller 89-kDa

22

fragments. The cleavage of this enzyme was considered a reliable marker for the onset of cellular apoptosis. Similarly, SMAL-OL showed a significant decrease in the expression of Bcl-xl (from the Bcl-2 family) and Bid, an anti-apoptotic marker. Bcl-2 is one of the key regulators of apoptosis and may promote cell survival by interfering with activation of the cytochrome c/Apaf1 pathway through stabilization of the mitochondrial membrane [47]. The decreased expression of Bcl-xl and Bid protein might result in the formation of pores in the mitochondrial membranes. This leads to mitochondrial dysfunctions and cytochrome C release, induces binding to procaspase-9 and results in proteolytic processing and activation of pro-caspase-9 [48]. The active caspase-9 then directly cleaves initiating a cascade of additional caspase activation that culminates in apoptosis as mentioned above [49]. These results clearly emphasize that SMALOL was efficiently internalized via the CvME pathway and enhanced apoptotic cell death through higher accumulation of OL in cancer cells, in synergy with the activity of SMAL. 3.7. Effect of SMAL on PI3K/AKT pathway Studies have reported the possible role of the PI3K/AKT/mTOR pathway on tumorigenesis and resistance to chemotherapeutics [50]. Therefore, effects of free OL and SMAL-OL on the phosphorylation of AKT and mTOR in HCT116 and HT-29 cancer cells were tested. As shown in Figure 9, SMAL-OL markedly suppressed the phosphorylation of AKT, mTOR and p70S6K in CRC cells in a dose-dependent manner. Blank SMAL NPs showed higher downregulation of AKT, mTOR and p70S6K compared to free OL, indicating its possible role in enhancing the therapeutic potency of OL. These results suggest that SMAL-OL induces cell apoptosis and overcomes therapeutic resistance through the PI3K/AKT/mTOR signaling pathways in both CRC cell lines.

23

Overall, we reported the successful encapsulation of OL in a pro-apoptotic nanocarrier, SMAL. The drug-loaded SMAL NPs demonstrated enhanced cellular uptake via the CvME pathway, and increased the cytotoxicity towards tumor cells while mitigating its toxic effect towards normal healthy cells. Further studies are warranted to substantiate our hypothesis and to develop this strategy as an exciting means of drug delivery – a subject of ongoing research.

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4. CONCLUSION With the above attributes, we present evidence of the selective and substantial accumulation of OL in CRC cells when encapsulated in SMAL NPs via the caveolae-mediated endocytosis (CvME pathway). We demonstrated a new strategy to exploit the higher expression of caveolin1 (CAV1) in endothelial cells (in comparison with epithelial cells). This is a purely physiochemical approach for targeting NPs in the tumor cells, which will have major implications in the development of targeted therapeutics. This simple, efficient, novel strategy led to remarkable cancer cell death and induced a more potent signal transduction regulatory pathway. Our findings clearly revealed the superior anticancer effect of a synergistic combination of SMAL and OL. The combination successfully inhibited the growth of CRC cells via cell cycle arrest at the G2/M phase and induced cell apoptosis owing to its specificity towards caveolae. SMAL-OL showed a remarkable downregulation of anti-apoptotic markers (Bcl-xl and Bid) and upregulation of proapoptotic proteins (p53, P21, Bax, caspase-3, caspase-9 and PARP). Furthermore, it induced cell apoptosis through the PI3K/AKT/mTOR signaling pathways. Based on our in vitro findings, we propose OL-loaded SMAL NPs as an effective drug delivery system with promising potential in cancer therapy. Further studies are necessary to fully exploit these findings to achieve clinical translation.

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ACKNOWLEDGMENTS This research was supported by the Gachon Institute of Pharmaceutical Sciences Research Fund 2015, Gachon University and the Republic of Korea.

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FIGURE CAPTIONS Figure 1: Schematic illustration of preparation of oxaliplatin (OL)-loaded SMAL NP (SMALOL) and its effect on the cell signaling pathways in the intracellular environment. Figure 2: (a) Transmission electron microscope (TEM) of SMAL, SMAL-PL, and SMAL-OL nanoparticle system, respectively; (b) In vitro drug release profile of OL from SMAL-OL in PBS (pH 7.4) and ABS (pH 5.0) buffer conditions at 37°C; (c) Stability analysis of SMAL-OL in PBS and serum conditions. Stability studies were carried out at 4°C up to 60 days. Data are expressed as the mean ± SD (n= 3). Figure 3: In vitro cellular uptake of SMAL and SMAL-PL in colorectal cancer cells. CLSM was used to observe the cellular uptake of nanoparticles in HCT116 (a) and HT-29 (b) cancer cells. Rhodamine-B was used as a fluorescent probe and nucleus in each cell was stained with DAPI (blue). Scale bar is 10 µm. Time-dependent cellular uptake of SMAL and SMAL-PL in HCT116 (c) and HT-29 (d) cancer cells were studied using FACS. The mean fluorescence intensity was calculated and compared between two groups. Data are represented as the mean±S.D. *p<0.05 and **p<0.01. Figure 4: Effect of endocytosis inhibitors on the cellular uptake of nanoparticles. The dominant endocytic uptake mechanism in CRC was probed using various endocytosis inhibitors, M-β-CD (marker for caveolae-mediated uptake) and sucrose (marker for lipid raft-mediated endocytosis). CLSM (a,b) and FACS (c,d) were used to observe the cellular uptake of nanoparticles. in HCT116 and HT-29 cancer cells. Experiments were replicated at least twice. Figure 5: The antiproliferative effect of SMAL in CRC. The cytotoxic effect of different formulations in (a) HCT116 and (b) HT-29 cancer cells after 48 h incubation. The CRC were 34

incubated with increasing concentration (0.1-2.5 µM) of free OL, SMAL and SMAL-OL and cell proliferation was evaluated by MTT assay. Colony formation of HCT116 (c) and HT-29 (d). The cells were exposed with an equivalent drug concentration from 0.1 µm to 2.5 µm. Results are representative of 3 independent experiments and data were represented as the mean±S.D. *p<0.05 and **p<0.001 Figure 6: Effect of formulations on the cell cycle distributions of CRC. HCT116 (a) and HT-29 (b) cells were treated with free OL, SMAL and SMAL-OL at various concentrations and incubated for 48 h. A representative set of data from three independent experiments is shown. Cell cycle phases were analyzed using FACS. G0/G1, S and G2/M show the cell cycle phase, and subG1 refers to the proportion of apoptotic cell. Figure 7: Effect of formulations on the apoptosis of cancer cells. HCT116 (a) and HT-29 (b) cells were treated with free OL, SMAL and SMAL-OL in concentration-dependent manner for 48 h. The apoptotic cell death was assessed using annexin V/PI staining by FACS analysis. Lower left quadrant indicates viable cells (Annexin-V-/PI-), lower right quadrant indicates early apoptotic cells (Annexin-V+/PI-), upper left quadrant indicates necrotic cells (Annexin-V-/PI+), and upper right quadrant necrotic cells or late apoptotic cells (Annexin-V+/PI+). Figure 8: Western blot analysis of apoptotic and cell cycle related protein expressions. Lysates from HCT-116 and HT-29 cells were incubated with 1 and 2.5 µM of free OL, SMAL and SMAL-OL for 48 h and analyzed by western blotting for tumor suppressor protein p53 and p21 was upregulated, and cell cycle protein was suppressed such as cyclin A, B, E, and pCDC2 (a). Pro-apoptotic markers BAX and BID were upregulated and PARP, caspase-3 and caspase-9 were

35

cleaved and enhanced apoptosis. Finally, anti-apoptotic protein Bcl-xl was inhibited by SMALOL (b). Blots were replicated at least twice. Figure 9: SMAL affects PI3K/AKT/mTOR signaling pathway in colon cancer cells. Lysates from HCT-116 and HT-29 cells incubated with 1 and 2.5 µM of free OL, SMAL and SMAL-OL for 48 h. SMAL-OL treatment significantly suppressed the phosphorylation of AKT, mTOR, pmTOR and p70S6K proteins.

36

Figure.1

Figure.2

Figure.3

Figure.4

Figure.5

Figure.6

Figure.7

Figure.8

Figure.9

Graphical abstract

37

Table 1: Physicochemical characteristics of different nanoparticle system Size (nm)

PDI

ζ- Potential (mV) Encapsulation efficiency (%) Loading capacity (%)

SMAL NP

112.6±3.65

0.264±0.038

-14.5±2.65

-

-

SMAL-PL NP

85.2±2.43

0.152±0.029

-3.26±3.62

-

-

SMAL-OL NP

94.5±3.89

0.169±0.032

-18.4±3.38

92.36±4.65

14.58±3.64

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Statement of significance

In this work, we developed a novel self-micellizing anticancer lipid (SMAL)-based proapoptotic nanoparticles (NPs) that enhance the accumulation and chemotherapeutic efficacy of oxaliplatin (OL) in colorectal cancer cells. We demonstrated that NPs with special affinity to caveolae could be realized and based on this specificity, NPs effectively differentiated between endothelial cells (tumor cells) and epithelial cells, without the need for a cell-specific targeting moiety. In addition, oxaliplatin-loaded SMAL were efficiently endocytosed by the cancer cells and represent a significant breakthrough as an effective drug delivery system with promising potential in cancer therapy. We believe this work holds promising potential for the development of next generation of multifunctional nanocarriers for an exciting means of targeted delivery without the need for receptor-ligand-type strategies.