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Poly lactic-co-glycolic acid controlled delivery of disulfiram to target liver cancer stem-like cells

Disulfiram (DS), an anti-alcoholism drug, shows very strong cytotoxicity in many cancer types. However its clinical application in cancer treatment is limited by the very short half-life in the bloodstream. In this study, we developed a poly lactic-co-glycolic acid (PLGA)-encapsulated DS protecting DS from the degradation in the bloodstream. The newly developed DS-PLGA was characterized. The DS-PLGA has very satisfactory encapsulation efficiency, drug-loading content and controlled release rate in vitro . PLGA encapsulation extended the half-life of DS from shorter than 2 minutes to 7 hours in serum. In combination with copper, DS-PLGA significantly inhibited the liver cancer stem cell population. CI-isobologram showed a remarkable synergistic cytotoxicity between DS-PLGA and 5-FU or sorafenib. It also demonstrated very promising anticancer efficacy and antimetastatic effect in liver cancer mouse model. Both DS and PLGA are FDA approved products for clinical application. Our study may lead to repositioning of DS into liver cancer treatment.

Primary liver cancer is the fifth most common neoplasm and the third most common cause of cancer-related death worldwide. 1 Hepatocellular carcinoma (HCC) accounts for 70%-80% of cases of primary liver cancer. 2 The incidence of HCC is high in Asia and increasing in the Western world in the past decade. Although the therapeutic outcomes in many cancers have been improved significantly, the prognosis of advanced HCC remains very dismal. 3 Chemoresistance and metastasis are the major hindrances for the treatment of advanced HCC. HCC cells are resistant to all currently available anticancer drugs. Sorafenib (Nexavar) is the sole drug showing marginal efficacy in HCC with only approximately 3 month improvement for overall survival. 4 Therefore, development of efficacious chemotherapeutic drugs is of significant clinical importance for treatment of the advanced HCC. HCC is a highly heterogenic disease containing a small population of cancer stem-like cells (CSCs). HCC CSCs can be identified by detection of the expression of stem cell markers e.g. ALDH, CD133, CD90, CD44, EpCAM, and CD13. 5 Previous studies suggest that CSCs are maintained by the hypoxic milieu, 6,7 quiescent and highly resistant to currently available chemotherapeutic agents. Conventional anticancer drugs eliminate bulk of cancer cells but could not eradicate CSCs, which become the source of the cancer recurrence and metastasis. Therefore, development of efficacious CSC-targeting drugs may improve the therapeutic outcomes of HCC.
New drug development is a time and cash consuming procedure. This leads to the current interest in drug repositioning. 8 Previous studies demonstrate that disulfiram (DS), a commercially available anti-alcoholism drug, 9 is highly cytotoxic to a wide range of cancer cell lines [10][11][12][13] and enhances conventional anticancer drug-induced apoptosis in colon, breast and brain cancer cell lines. 10,[14][15][16] Importantly, DS is highly cancer specific and specifically inhibits the activity of aldehyde dehydrogenase (ALDH), a functional marker of CSCs and reactive oxygen species (ROS) scavenger 17,18 to eliminate CSCs.
Although the anticancer activity of DS has been reported for almost 3 decades, its translation into clinical cancer treatment is severely limited by its very short half-life in the bloodstream. 19 The anticancer activity of DS is copper (Cu), zinc and some other divalent transitional metal elements dependent. 11,[20][21][22] DS is quickly reduced to diethyldithiocarbamate (DDC), a strong chelator of copper(II). The reaction between DDC and Cu(II) generates ROS which are highly toxic to cancer cells. Due to the extremely short lifespan of ROS in human tissues, 23 the extracellular ROS can target cancer cells only when the reaction takes place within the cancer tissues. 22,24 DDC-Cu, the final product of the DS and Cu reaction, can also penetrate into cancer cells and induce apoptosis. 22 The sulfhydryl group of DDC is essential (Figure 1, A) for chelation of Cu(II) by DDC. The currently available oral version of DS is quickly reduced to DDC and the sulfhydryl group of DDC is promptly destroyed by glucuronidation, methylation and degradation in the bloodstream of the portal vein. 25 This may introduce the discrepancy between the anticancer activity of DS in vitro, in vivo and in patients. In order to translate DS into cancer therapeutics, intact sulfhydryl group of DS or DDC must be protected in the bloodstream and delivered to cancer tissues where it reacts with Cu to generate ROS and form DDC-Cu (Figure 1, A). We previously showed that encapsulation of DS into liposome extends the half-life of DS and improves its in vivo anticancer efficacy in breast cancer mouse model. 6 The purpose of this study is to develop a long circulating poly lactic-co-glycolic acid (PLGA) encapsulated DS (DS-PLGA) to achieve longer circulating time and improve the anticancer efficacy of DS.

Preparation and characterization of DS-PLGA nanoparticles
The DS loaded nanoparticles were prepared using an emulsion-solvent evaporation method. PLGA (200 mg) and DS (20,40,50, 100 or 150 mg) were dissolved in 10 mL of dichloromethane, and then mixed with 20 mL of 2.5% PVA aqueous solution. This mixture was homogenized for 1 min by vortex and then sonicated using a microtip probe sonicator set at 70% power output (XL 2002 Sonicator® ultrasonic liquid processor) for 3, 4 or 5 min to produce the oil-in-water emulsion. The organic phase was evaporated for 5 h at room temperature. The nanoparticles were recovered by centrifugation (10,000 rpm, 20 min, Hitachi). The nanoparticles were washed twice with water. The purified nanoparticles were freeze-dried in 5% sucrose.
The size distribution and zeta potential of DS-PLGA were measured by DLS using a Zetasizer (ZS90, Malvern, U.K.) with a scatter angle of 90°at 25°C. The morphology of nanoparticles was observed by scanning electron microscopy (SEM) (JEOL JSM T330A). A drop of the nanoparticle suspension was placed on a metallic surface. After drying under vacuum, the sample was coated with a gold layer.  and the flow rate was set at 1 ml/min. Separation was achieved using a Phenomenex C18 column (250 mm × 4.6 mm, 5 μm). The amount of DS entrapped in the nanoparticles was determined after their dissolution in dichloromethane. After evaporation of dichloromethane at room temperature, DS was dissolved in pure ethanol. The supernatants were passed through a membrane filter (pore size 0.22 μm, Millipore) before HPLC measurements. The cumulative release profile of DS-PLGA was measured in 0.1 M phosphate buffer solution (PBS, pH 7.4) with 0.5% Tween 80 at 37°C. In brief, 10 mg DS-PLGA was suspended in 25 ml PBS-Tween80 solution with continuously shaking (100 rpm min −1 ) at 37°C. At indicated time intervals, 500 μl of the release media was collected and centrifuged at 14,000 rpm for 10 min at room temperature to remove the PLGA nanoparticles with unreleased DS. The DS concentrations in the supernatant were measured using HPLC and equal volume of fresh media was replenished. The drug loading content (DLC) and encapsulation efficiency (EE) of DS in the PLGA nanoparticles were calculated using the following equations.

Â100%
To examine the half-life of DS in vitro, free DS or DS-PLGA (100 μl at a concentration of 3 mg/ml) was added into an Eppendorf tube containing 300 μl of horse serum with shaking at 37°C. The Eppendorf tubes were collected and protein precipitated by adding 300 μl of absolute methanol at different time intervals. The PLGA nanoparticles with unreleased DS were removed by centrifugation at 14,000 rpm for 10 min at room temperature. The DS in the supernatant was determined by HPLC measurement.

In vitro cytotoxicity of DS-PLGA to HCC cells
The PLC/PRF/5 and Huh7 HCC cell lines were cultured in DMEM supplemented with 10% FCS, 50 units/ml penicillin, and 50 μg/ml streptomycin. For in vitro cytotoxicity assay, the overnight cultured cells (5000/well) in 96-well flat-bottomed microtiter plates were exposed to drugs for 72 h in normoxic or hypoxic condition and subjected to a standard MTT assay. 16 Analysis of the combinational effect of 5-FU + DS-PLGA/Cu and sorafenib + DS-PLGA/Cu The HCC cells were cultured at 5000 cells/well in 96-well plates overnight and then exposed to various concentrations of 5-FU, sorafenib, DS-PLGA/Cu 1μM, 5-FU or sorafenib in combination with DS-PLGA/Cu 1μM at a constant ratio of 5-FU:DS-PLGA and sorafenib:DS-PLGA of 1000:1 and 100:1 respectively. The cells were exposed to different drug combinations for 72 hours subjected to MTT analysis. 16 The IC 50s from one and two-drug-treated cells were determined. The combinational cytotoxicity was analyzed by CI-isobologram program using CalcuSyn software (Biosoft, Cambridge, UK). 26 The combination index (CI) was determined by mutually exclusive equations.

Flow cytometric detection of ALDH activity and CD133 expression
The ALDH positive population was detected by ALDEFLUOR kit (StemCell Tech., Durham, NC, USA) following the supplier's instruction. The cells (2.5 × 10 5 ) were analyzed after stained in ALDH substrate containing assay buffer for 30 min at 37°C. Diethylaminobenzaldehyde (DEAB), a specific ALDH inhibitor, treated cells were used as a positive control.
To detect the expression of CD133 positive cells, the cells (2.5 × 10 5 ) were incubated with CD133 antibody (BD Pharmingen, Oxford, UK) for 30 min at 4°C. Unbound antibodies were washed off with 2% FCS HBSS (Sigma). After staining for no longer than 1 h, the positively stained population (10,000 events) was detected using a BD Accuri™ C6 flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) with 488-nm blue laser and standard FITC 530/30 nm bandpass filter.

Flow cytometric detection of apoptosis
DNA content was used to detect the apoptotic cells. Briefly, cells (1 × 10 6 ) were exposed to drugs and harvested by trypsinization. The cells were fixed in 70% ethanol and then incubated with RNase A (100 μg/ml) and propidium iodide (2.5 mg/ml) for 30 min. The DNA content data from 10,000 cells in each sample was collected by a BD Accuri™ C6 flow cytometer and analyzed using a CellQuest software (BD Biosciences, Oxford, UK).

In vitro HCC sphere cell culture
To culture the HCC spheres, cells were cultured in poly-HEMA coated ultra-low adherence flasks or plates to prevent cell adhesion. The spheres were cultured, at a density of

In vitro migration and invasion assay
Cell invasion and migration assays were performed using cell culture inserts (Millipore) coated with or without Matrigel (BD Bioscience, Bedford, MA, USA), respectively. Cells (5 × 10 4 ) were resuspended in 200 μl serum-free DMEM and placed in upper chamber of the insert. The lower chamber was filled with medium containing 10% FCS. After 48 h incubation at 37°C, migrated cells were fixed by methanol and stained in 0.5% crystal violet. Cells in six random microscopic fields were counted.  Table 2) and is more tolerable by animal. The animals were treated 3 times/week for successive 3 weeks. The xenograft size was recorded trice per week. The tumor volume was calculated by the following formula: V = (L × W 2 ) × 0.5, where L is the length and W is the width of the tumor. After 3 weeks, the animals were sacrificed. The tumors were removed, photographed and subjected to further analysis.

HCC xenograft and in vivo metastasis experiments
The in vivo lung metastasis mouse model was established by injecting 5 × 10 6 single-cell suspension of PLC/PRF/5 cells (in 200 μl of PBS) into the tail vein. After 2 weeks, the mice were treated 3 times/week with 10 mg/kg DS-PLGA i.v. and CuGlu 6 mg/kg p.o. for 4 successive weeks. The mice were sacrificed two days after the last treatment. The lungs were removed and formalin-fixed for paraffin embedment and H&E staining.

Statistical analysis
The statistical significance of treatment outcomes was assessed using the Student's ttest and one-way ANOVA; P b 0.05 was considered statistically significant in all analyses.

Physicochemical characterization of DS loaded PLGA nanoparticles
The influence of PLGA:DS ratio and sonication/homogenizing time on the EE, DLC and nanoparticle size were examined. The PLGA:DS ratio of 2:1 (w/w) and 4 min of sonication provided us with the most optimum nano-characteristics with most narrow size (131.8 ± 6.9 and 136.2 ± 6.2 nm) and satisfactory zeta potential (−22.3 ± 0.8 and −21.7 ± 0.96 mV) distribution between the empty and DS loaded PLGA nanoparticles (Table 1 and Supplementary Tables S1  and S2). The SEM image showed that the DS-PLGA was in analogous spherical shapes (Figure 1, B). DS-PLGA showed high EE (78.92 ± 2.16%) with satisfactory DLC (27.67 ± 3.47%).
To determine the stability and release of DS in DS-PLGA, the DS-PLGA was incubated in 1%, 5% and 10% BSA solutions at 37°C. The particle size was increased in a time and BSA concentration-dependent manner (Figure 1, C). The zeta potential of the DS-PLGA was also examined after 24 h incubation in BSA solution. At a physiological concentration of BSA (5%), the DS-PLGA maintained a satisfactory particle size (162 nm) and zeta potential (−15.8 mV) for at least 8 and 24 h, respectively (Figure 1, D). The DS-PLGA showed very good dispersibility.

In vitro cumulative release and half-life of DS in serum
The cumulative drug release was performed over a period of 7 days in a 0.5% Tween 80 PBS solution (pH 7.4) at 37°C. The DS concentrations at different time lengths were examined. Results demonstrate a very steady and sustained release of DS over 7 days. Around 20% of DS was released within 24 h and reached a 40% release at day 4, which was maintained until day 7  ( Figure 1, E). To determine the half-life of DS in vitro, the free DS and DS-PLGA (100 μg/ml) were dispersed into horse serum at 37°C. The free DS was rapidly degraded to undetectable levels within 30 seconds (Figure 1, F). In contrast, DS commenced its release from PLGA at 2 min, equivalent to 34.23 ± 3.38% of the initial drug loading and peaked in 4 min at

In vitro cytotoxicity of DS-PLGA NPs in HCC cells
The cytotoxicity of DS-PLGA and free DS in PLC/PRF/5 and Huh7 HCC cell lines was compared by MTT assay. CuCl 2 (1 μM) was supplemented in all the experiments. The in vitro cytotoxicity of DS-PLGA is slightly lower than that of the free DS but no significant difference between the IC 50s of DS-PLGA (Huh7, 30.69 ± 3.83 nM; PLC/PRF/5, 49.33 ± 4.11 nM) and free DS (Huh7, 25.34 ± 3.21; PLC, 45.25 ± 3.52 nM) (P N 0.05) (Figure 2, A and B). After exposure to DS-PLGA (50 nM) and CuCl 2 (1 μM) for 4 h, the apoptotic cells (sub-G1 population) were identified by DNA content assay using flow cytometry analysis. Background levels of spontaneous apoptotic cells were detected in untreated (2.47% and 3.31%) and PLGA empty nanoparticles plus CuCl 2 treated (2.58% and 3.14%) Huh7 and PLC/PRF/5 cell lines respectively. In contrast, massive apoptotic cells (15.68% and 44.47%) were detected in DS-PLGA/CuCl 2 treated Huh7 and PLC/PRF/5 cell lines, respectively (Figure 2, C).

Hypoxia induced CSC traits and resistance to conventional anticancer drugs
To determine the effect of hypoxia on CSC population, two CSC markers, ALDH and CD133, were examined in hypoxia and normoxia cultured HCC cell lines. The enhanced ALDH activity and elevated expression of CD133 indicated that the CSC population in PLC/PRF/5 and Huh7 cell lines was significantly enlarged by hypoxic environment (Figure 3, A  and B). The specificity of ALDH detection was confirmed by DEAB inhibition (Figure 3, C). Furthermore, we examined the chemosensitivity of HCC cell lines at hypoxic condition. When cultured at 1% O 2 , the HCC cell lines were significantly resistant to 5-FU and sorafenib (Figure 3  DS-PLGA alone had no effect on CSC marker expression (Figure 4, A). Furthermore, the combinational effect of DS-PLGA/Cu and 5-FU or sorafenib was examined using CI-isobologram assay. In combination with DS-PLGA, the in vitro cytotoxicity of 5-FU and sorafenib was significantly enhanced. CI-isobologram analysis indicates very significant synergistic combinational effect among 5-FU, sorafenib and DS-PLGA/Cu in a wide range of IC values (IC 50 − IC 90 ) ( Table 2, Figures S1 and S2).

DS-PLGA inhibited expression of CSC markers in spheroid culture and eradicated sphere-forming and clonogenic abilities
CSCs can form spheres in stem cell culture conditions. Sphere-forming ability is widely accepted as a golden standard for in vitro analysis of stemness of CSCs. 27 The effect of different treatments on sphere-forming ability was examined. A significantly higher percentage of ALDH and CD133 positive CSC population was detected in spheroid cells (Figure 4, B). The ALDH and CD133 positive CSC population in the spheres was significantly reduced after exposure to DS-PLGA (20 nM) and Cu (1 μM) for 4 h but not affected by other treatments. The sphere-forming ability was completely abolished after exposure to DS-PLGA (20 nM) plus Cu (1 μM) for 6 h. No inhibiting effect was observed in 5-FU and sorafenib treated cells (Figure 4, C and D). The DS-PLGA/Cu treated HCC CSCs underwent apoptosis (Figure 4, E and F). We next performed clonogenic assay, a traditional method to determine the self-renewal capacity, cancer cell regeneration and CSC traits, 28 to examine the ability of DS-PLGA/Cu to induce the 'reproductive death' in HCC cell lines. The monolayer-cultured PLC/PRF/5 and Huh7 cells were exposed to 5-FU (200 μM), sorafenib (20 μM), CuCl 2 (1 μM), DS-PLGA (50 nM) or DS-PLGA (50 nM) plus CuCl 2 (1 μM) for 6 h. The treated cells were collected and cultured in 6-well plates containing drug-free medium at a cell density of 500 cells/well for another 10 days. There was no significant difference in colony numbers when the control group was compared with 5-FU, sorafenib and CuCl 2 treated groups. In contrast, the colonies in DS-PLGA/ Cu treated group were completely eradicated in both cell lines (Figure 4, G and H).

DS-PLGA blocked migration and invasion ability of hypoxic and sphere-cultured cells in vitro
CSCs have high metastatic ability, another key event responsible for the very poor prognosis of the advanced HCC patients. Because DS-PLGA/Cu shows very strong inhibiting effect on CSC traits in HCC cell lines, we further examined its effect on cell migration and invasion in vitro. The hypoxia ( Figure 5, A and B) and CSC ( Figure 5, C and D) induced migration and invasion was significantly inhibited by DS-PLGA/ Cu at an extremely low concentration (5 nM) which is only 1/8 to 1/10 of the IC 50 concentrations for Huh7 and PLC/PRF/5 respectively. At this concentration, no cytotoxicity was observed in both cell lines.

In vivo anti-HCC efficacy of DS-PLGA in mouse xenografts
To assess anti-tumor activity in vivo, 2.5 mg/kg, 5 mg/kg and 10 mg/kg DS-PLGA was intravenously injected into the tail veins of the HCC xenograft bearing mice. In comparison with the control group, 10 mg/kg DS-PLGA significantly inhibited tumor size and tumor weight (Figure 6, A and B). An equivalent oral dose of DS/ Cu or Lipo-DS i.v./oral-Cu, had no effect on the xenografts. This result indicates that the PLGA encapsulation protected DS from degradation in the bloodstream and delivered the intact DS to cancer tissues. DS-PLGA/Cu treatment inhibited the expression of NF-κB p65 and ALDH in the HCC xenografts (Figure 6, C). No significant mouse body weight difference between the control and treated groups and no cytotoxic effect was observed in the vital organs e.g. lung, liver and kidney ( Figure 6, D and E).

DS-PLGA and copper blocks HCC lung metastasis in vivo
The very encouraging in vitro anti-migration and invasion data prompted us to examine the anti-metastatic effect of

Discussion
Only 5-25% of new oncology drugs in clinical development are actually reaching the market. 8,29 This has led to an increasing appreciation of the potential of repurposing known drugs for cancer treatment. DS is highly cytotoxic in several types of cancer cells in vitro, 6,10,15,16,[30][31][32] whereas oral administration of free DS does not inhibit tumor growth in vivo. 33 Recent publication suggests that the intact sulfhydryl group is essential for the anticancer function of DS. 22,24 The degradation of DS in the bloodstream destroys the sulfhydryl group making DS lose its cancer targeting ability. The degradation of DS in the serum kinetically results from a covalent interaction of this drug with the free sulfhydryl of serum albumin. The half-life of DS pH 7.4 is 1-1.5 minutes. 19,34,35, 36 ,37 This may introduce the discrepancy between the anticancer activities in vitro, in vivo and in clinic (ClinicalTrials.gov: NCT00256230, NCT00742911, NCT01118741). Nanomedicine can protect and deliver drugs in the bloodstream. 38 The in vivo anticancer efficacy of DS can be improved by liposomal encapsulation (half-lifẽ 25 min). 6,33 In this study, we successfully extended the half-life of DS to~7 h by encapsulation of DS into PLGA (Figure 1, G). We achieved very satisfied EE, DLC, nano-particle size and dispersibility ( Table 1). The DS encapsulated nanoparticles are stable in physiological concentrations of BSA and the release of DS was steadily controlled at 37°C. Importantly, after 8 h incubation, the DS concentration in the serum still remained at 40 μM (12 μg/ml, Figure 1, F), which is markedly higher than the IC 50s of DS in a wide range of cancer types (~50-500 nM). 6   ( Figure 2, A and B). Therefore, nanomedicine may be a novel strategy for protection of the sulfhydryl group in DS and translation of DS into cancer treatment.
HCC is resistant to all currently available anticancer drugs with only very marginal response to sorafenib. 4 It has been widely accepted that CSCs display significant resistance to cytotoxic drugs with different resistant mechanisms. 39 Hypoxia is a common feature of HCC. The median partial pressure of O 2 in HCC is 6 mmHg as compared to 30 mmHg in normal liver tissues. 40 Emerging evidence suggests that hypoxia plays pivotal roles in chemoresistance. 41 The hypoxic condition in the stem cell niche is essential for maintaining the stemness in CSCs. 42 Therefore inhibition of hypoxia-induced CSCs may sensitize cancer cells to conventional anticancer drugs. Previous studies indicate that DS targets CSCs and sensitizes several types of cancers to conventional anticancer drugs. 6,10,16,30,32,43,44 Our results demonstrated that hypoxia increased CSC population in HCC cell lines. The hypoxic cells were highly resistant to 5-FU and Sorafenib (Figure 3). DS-PLGA/Cu synergistically enhanced the cytotoxicity of 5-FU and sorafenib in HCC cells ( Table 2 and Figures S1 and S2). At a very low concentration (20 nM), DS-PLGA/Cu significantly inhibited the ALDH and CD133 positive CSC population in both hypoxia-induced and spheroid cultured HCC cell lines (Figure 4, A and B). The sphere forming and clonogenic ability in HCC cell lines was completely abolished by very low concentration of DS-PLGA/Cu. In contrast, 5-FU and sorafenib had no effect on CSC population. DS-PLGA/Cu also very strongly in vivo inhibited NF-κB and ALDH activity which are highly responsible for the stemness of CSCs 6,16,30 and efficaciously targeted HCC xenografts at an equivalent to 1/5 of the human antialcoholism dose. The effect of DS-PLGA/Cu on HCC xenografts was dose dependent (Figure 6, A and B). In consistence with our previous study, 6 no toxicity was observed in the vital organs of the DS-PLGA/Cu treated animals ( Figure 6, E).
HCC has been shown the hypoxia-induced epithelialto-mesenchymal transition (EMT), a typical character of CSCs involving in metastasis. 45,46 Liver transplantation is carried out to treat HCC, especially in the early stage. However, due to the extrahepatic organ micrometastasis, the incidence of postoperative recurrence and metastasis is very high (60-100%). 47 Lung remains the most frequent site for HCC metastasis. At the time of diagnosis, most patients have multiple pulmonary or systemic metastasis. 48 There are still no effective systemic therapies available for the metastatic HCC. Therefore development of a drug with antimetastatic effect will improve HCC treatment. 49,50 DS-PLGA/Cu showed very strong anti-metastatic effect on HCC in vitro ( Figure 5) and in vivo (Figure 7), highly in line with our in vivo metastatic lung cancer data (unpublished data). Therefore, DS-PLGA is potentially an effective anti-metastatic drug for HCC treatment.
In conclusion, we showed that the DS-PLGA has very satisfactory EE, DLC, in vitro controlled release and half-life. DS-PLGA and copper showed very strong inhibiting effect on CSCs and synergistic cytotoxicity with 5-FU and sorafenib. It also manifests very promising anticancer efficacy and anti-metastatic activity in HCC mouse model. Our results suggest that nano-controlled delivery system may lead to fast translation of DS into liver cancer treatment.