Biomaterials 35 (2014) 9177e9186
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Phenformin-loaded polymeric micelles for targeting both cancer cells and cancer stem cells in vitro and in vivo Sangeetha Krishnamurthy, Victor W.L. Ng, Shujun Gao, Min-Han Tan, Yi Yan Yang* Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669, Singapore
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 June 2014 Accepted 16 July 2014 Available online 5 August 2014
Conventional cancer chemotherapy often fails as most anti-cancer drugs are not effective against drugresistant cancer stem cells. These surviving cancer stem cells lead to relapse and metastasis. In this study, an anti-diabetic drug, phenformin, capable of eliminating cancer stem cells was loaded into micelles via self-assembly using a mixture of a diblock copolymer of poly(ethylene glycol) (PEG) and ureafunctionalized polycarbonate and a diblock copolymer of PEG and acid-functionalized polycarbonate through hydrogen bonding. The phenformin-loaded micelles, having an average diameter of 102 nm with narrow size distribution, were stable in serum-containing solution over 48 h and non-cytotoxic towards non-cancerous cells. More than 90% of phenformin was released from the micelles over 96 h. Lung cancer stem cells (side population cells, i.e. SP cells) and non-SP cells were sorted from H460 human lung cancer cell line, and treated with free phenformin and phenformin-loaded micelles. The results showed that the drug-loaded micelles were more effective in inhibiting the growth of both SP and non-SP cells. In vivo studies conducted in an H460 human lung cancer mouse model demonstrated that the drug-loaded micelles had greater anti-tumor efficacy, and reduced the population of SP cells in the tumor tissues more effectively than free phenformin. Liver function analysis was performed following drug treatments, and the results indicated that the drug-loaded micelles did not cause liver damage, a harmful side-effect of phenformin when used clinically. These phenformin-loaded micelles may be used to target both cancer cells and cancer stem cells in chemotherapy for the prevention of relapse and metastasis. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Phenformin Polymeric micelles Lung cancer Cancer stem cells In vivo efficacy
1. Introduction The number of cancer-related deaths worldwide is in the increase in spite of the cutting-edge advances made in drug discovery and drug delivery [1e3]. Various treatment strategies like surgical resection, radiotherapy and systemic therapies are being used. However, the response to the treatments is low, and there is a high rate of recurrence and metastasis [4]. The capability of cancers to recur and metastasize is attributed to the presence of a side population of cancer cells (SP cells, or cancer stem cells) with the ability to self-renew, enhance drug resistance and initiate tumor [5e7]. Cancer stem cells (CSCs) have been well studied and documented in different types of cancers, and they express various unique surface markers [8e10]. A number of studies have shown that cancer stem cells were more resistant to chemotherapeutic drugs, and worsened the overall survival [11e16]. Therefore, an effective treatment strategy should be able to target both cancer cells and CSCs, thereby
* Corresponding author. Tel.: þ65 6824 7106; fax: þ65 6478 9084. E-mail address: [email protected] (Y.Y. Yang). http://dx.doi.org/10.1016/j.biomaterials.2014.07.018 0142-9612/© 2014 Elsevier Ltd. All rights reserved.
preventing recurrence and metastasis, and enhancing the survival rate of patients. Most conventional anti-cancer drugs have little effect on CSCs. This is potentially due to the inherent resistance of CSCs, their over-active efflux pumps that remove the drugs out of the cells and enhanced DNA repair capability [17]. Interestingly, other classes of drugs like anti-psychotics [18], anti-diabetics [19] and anti-helminthics [20] have been found to be effective in eradicating CSCs. Biguanides are a class of anti-diabetic drugs, of which metformin and phenformin have been frequently studied [21]. Both of these drugs have recently been shown to exhibit an antineoplastic effect against several types of cancers especially cancer stem cells [16,22,23]. Metformin is a highly prescribed drug for Type-2 diabetes mellitus (T2DM), and is preferred over phenformin because of lesser incidence of lactic acidosis in treated patients [24]. Compared to metformin, phenformin is 50 times more potent than metformin against different cancer cell lines [25]. Like many other drugs, phenformin is metabolized in the liver. The drug was associated with increased incidences of lactic acidosis [26], and was subsequently withdrawn by FDA in 1976 as
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an anti-diabetic drug [27]. Nanoparticles have been used to deliver drugs and reduce non-specific toxicity towards healthy tissues by exploiting the enhanced permeability and retention (EPR) effect of nanoparticles at leaky tumor tissues [28]. Among various types of nanoparticles, polymeric micelles are most commonly explored for drug delivery due to their unique core/shell structure having a hydrophobic core for encapsulating the drug and a hydrophilic corona that prolongs drug circulation in the blood, and ease of installing functionalities to the core for enhancing interactions with the drug and increasing drug loading level. We hypothesized that formulating phenformin into polymeric micelles would enhance its anti-cancer efficacy and reduce the CSC population without causing toxicity to the liver. Recently, we reported functional micelles self-assembled from a mixture of poly(ethylene glycol) (PEG)/acid-functionalized polycarbonate diblock copolymer (PEG-b-PAC) and PEG/ureafunctionalized polycarbonate diblock copolymer (PEG-b-PUC) via hydrogen-bonding [29]. These micelles had excellent stability due to the presence of hydrogen-bond (ureaeurea and urea-acid), exhibiting preferable accumulation at tumor tissues based on the EPR effect [30]. In addition, they had high loading capacity for basic drugs such as doxorubicin [29e31]. Phenformin is a basic drug, and has two guanidine groups in the molecule, which can form hydrogen-bond with urea group and ionic interaction with acid group in the micellar core. In this study, PEG-b-PUC and PEGb-PAC were synthesized by organocatalytic ring-opening polymerization (ROP) using methoxy-PEG (Mn 10 kDa) as the macroinitiator. Phenformin was loaded into micelles via self-assembly of PEG-b-PUC and PEG-b-PAC mixture. The phenformin-loaded micelles were characterized for drug loading level, particle size, size distribution, zeta potential, in vitro drug release, and stability in serum-containing solution. According to World Health Organization's 2014 statistics, lung cancer is the leading cause of cancer deaths worldwide in both men and women with 27% of all cancer deaths. Lung cancer also has the lowest 5-year survival rate (16.6%) compared to all other high-incidence cancers like colon (64.2%), breast (89.2%) and prostate (99.2%) (National Cancer Institute. SEER Cancer Statistics Review, 1975e2010). In addition, lung cancer is commonly metastatic, predominantly metastasizing to adrenal gland, bone, brain, liver and the other lung [32]. Therefore, in this study, lung cancer was chosen as a model cancer using H460 human lung adenocarcinoma cell line as an example. Cytotoxicity of phenformin and phenformin-loaded micelles against H460 cells was evaluated by MTS, and their non-specific cytotoxicity was also examined against human dermal fibroblasts (HDFs) and WI-38 human fibroblast like fetal lung cell line. SP (CSCs) and non-SP cells were sorted from H460 cells by flow cytometry, and identified by immuno-staining with anti-CD133-PE antibody. Inhibitory effect of free phenformin and phenforminloaded micelles on SP and non-SP cells was investigated, and their anti-cancer efficacy was studied in an H460 xenograft mouse model. The population of CSCs in the tumor tissue after treatment with free phenformin and phenformin-loaded micelles was also analyzed. 2. Materials and methods 2.1. Materials Phenformin-HCl, Hoechst 33342 and all other reagents were purchased from SigmaeAldrich (St. Louis, MO, U.S.A.) unless otherwise specified. CellTiter 96® AQueous non-radioactive cell proliferation assay kit (MTS) was obtained from Promega (Madison, WI, U.S.A.). Anti-CD133-PE was bought from Miltenyi Biotec (Auburn, CA, U.S.A.). CytoSelect™ 24-well cell invasion assay kit was purchased from Cell Biolabs (CA, U.S.A.). Lactic acid was assayed using L-lactate assay kit (Cayman Scientific, Michigan, U.S.A.). H460, human dermal fibroblast (HDF) cell line and human fetal lung cell line (WI38) were obtained from ATCC (Manassas, VA, U.S.A.). Cells were cultured in
RPMI-1640 medium (Lonza, Singapore) supplemented with 10% fetal bovine serum (FBS, Thermo Scientific Inc, MA, U.S.A.), 100 U/mL penicillin and 100 mg/mL streptomycin (Invitrogen, CA, U.S.A.) at 37 C in 5% CO2 atmosphere. Dulbecco's modified Eagle's medium (DMEM) was purchased from Lonza, Singapore. 2.2. Polymer synthesis The details for organocatalytic ring-opening polymerization (ROP) of ureafunctionalized (MTC-urea) and benzyl-protected acid-functionalized (MTC-OBn) carbonate monomers using methoxy poly(ethylene glycol) (PEG, Mn 10,000 g/mol, PDI 1.04) are provided below. All polymerizations were carried out in a glove-box under nitrogen atmosphere. MTC-urea and MTC-OBn monomers were synthesized according to a protocol reported previously [29]. Diblock copolymer of PEG and urea-functionalized polycarbonate (PEG-b-PUC): To a mixture of MTC-urea (242 mg, 0.75 mmol), PEG (500 mg, 0.05 mmol) and thiourea catalyst (TU) (18.5 mg, 0.05 mmol) dissolved in dichloromethane (5 mL) was added sparteine (11.5 mL, 0.05 mmol) to initiate the ROP. The homogeneous reaction mixture was stirred for 18 h at room temperature (~22 C). The polymerization was quenched by addition of benzoic acid (25 mg; ~4 eqv.), and the crude polymer was subsequently precipitated in 50 mL of diethyl ether. The white solid was collected after centrifugation, dried briefly and re-dissolved in dimethyl sulfoxide (2 mL)/dichloromethane (2 mL). The crude product was re-precipitated in diethyl ether four times to ensure complete removal of impurities and dried under vacuo to give a white solid (85%). PDI 1.14. 1H NMR (400 MHz, DMSO-d6, 22 C): d 8.53 (s, br, 10H, PhNHe), 7.36 (m, 20H, PhH), 7.18 (m, 20H, PhH), 6.86 (m, 10H, PhH), 6.20 (s, br, 10H, eCH2NHe), 4.05e4.25 (overlapping peaks, 60H, MTCeCH2e and eCOOCH2e), 3.50 (s, 906H, H of mPEG), 3.33 (s, 20H, eCH2NHCOe), 1.13 (s, 30H, eCH3). Diblock copolymer of PEG and benzyl-protected acid-functionalized polycarbonate (PEG-b-P(MTC-OBn): In a similar manner, DBU (7.5 mL, 0.05 mmol) was added to a mixture of MTC-OBn (188 mg, 0.75 mmol) and PEG (500 mg, 0.05 mmol) dissolved in dichloromethane (5 mL) for initiating the ROP. The mixture was stirred for 3 h at room temperature and subsequently quenched by the addition of benzoic acid (25 mg; ~4 eqv.). Identical purification protocol was employed using dichloromethane/diethyl ether as the precipitation solvent combination for PEG-bPUC polymer. Yield, 80%; PDI 1.13. 1H NMR (400 MHz, CDCl3, 22 C): d 7.31 (m, 60H, PhH), 5.13 (m, 24H, PhCH2e), 4.27 (m, 48H, MTCeCH2e), 3.64 (s, 906H, H of mPEG), 3.37 (s, 3H, CH3 of mPEG), 1.21 (m, 36H, eCH3). Benzyl groups in PEG-b-P(MTC-OBn) were deprotected to yield a diblock copolymer of PEG and acid-functionalized polycarbonate (PEG-b-PAC). Briefly, a mixture of PEG-b-P(MTC-OBn) (0.5 g) and PdeC (10% w/w, 0.2 g) in THF/methanol (7.5 mL each) was reacted with dry hydrogen (7 atm) overnight. After removal of hydrogen, the mixture was filtered through celite wetted with THF. To ensure complete transfer of the deprotected polymer, additional THF and methanol washings were carried out. The filtrate was then collected, and solvents evaporated. The resultant residue was re-dissolved in THF and precipitated with diethyl ether before being freeze-dried under vacuo. The final product PEG-b-PAC was obtained as a powdery white solid (yield: >90%). 2.3. Gel permeation chromatography (GPC) GPC analysis of acid- and urea-functionalized block copolymers was performed using a Waters HPLC system with a 2690D separation module and two Styragel HR1 and HR4E (THF) 5 mm columns (size: 300 7.8 mm) in series and a Waters 410 differential refractometer detector. THF was used as the mobile phase and a flow rate of 1 mL/min was fixed. A calibration curve was obtained from a series of polystyrene standards of molecular weight between 1350 and 151,700. Polydispersity indices and molecular weights of the block copolymers were calculated based on the standard curve. 2.4. Loading of phenformin into micelles Phenformin was loaded into polymeric micelles using a modified sonication method as reported previously [33]. Briefly, 10 mg of phenformin-HCl was dissolved in 100 mL of dimethyl sulfoxide (DMSO), and neutralized with 3-mol excess of triethylamine. PEG-b-PUC (4.5 mg) and PEG-b-PAC (5.5 mg) (urea: acid ¼ 1:1 M ratio) were added to the solution, which was then added dropwise into 10 mL of deionized (DI) water on ice, while sonicating at 130 W for 2 min using a probe-based sonicator (Vibra Cell VCX 130, Connecticut, U.S.A.). Subsequently, unloaded drug and individual polymer molecules were removed by filtering through a centrifugal concentrator (Vivaspin® Turbo 15, Sartorius, U.K.) with molecular weight cut-off of 10,000 Da and washing 3 times with DI water. The resulting solution was lyophilized to harvest phenformin-loaded micelles. 2.5. Characterization of phenformin-loaded micelles The particle size and size distribution of freshly prepared micelle solution (1 mg/mL) was determined by dynamic light scattering (scattering angle: 90 ) using DTS Zetasizer 3000 HAS (Malvern Instruments, Worcestershire, U.K.). The zeta potential of the micelles was also measured using the same instrument. The drug loading level of the micelles was analyzed by high performance liquid
S. Krishnamurthy et al. / Biomaterials 35 (2014) 9177e9186 chromatography (HPLC) (C18 reversed phase column, 0.5 mL/min, 100 mL injection, acetonitrile/water gradient elution) at 253 nm. The standard curve of phenformin was constructed using a concentration range of 7e500 mg/mL (where it shows linearity), and used to determine the drug content in micelles. The drug loading was calculated as the fraction of mass of drug-loaded in the micelles to the total mass of drug-loaded micelles. All measurements were done in triplicates. 2.6. In vitro release The in vitro release of phenformin from the micelles was determined in phosphate-buffered saline (PBS, pH 7.4) at 37 C. Briefly, phenformin-loaded micelles were dispersed in PBS (1 mg/mL, 2 mL) and placed in a dialysis membrane bag having a molecular weight cut-off of 2000 Da. The bag was immersed in 20 mL of PBS while being shaken at 100 rpm. The solution outside the dialysis bag (1 mL) was collected at predetermined time intervals and was replaced with the same volume of fresh buffer. Phenformin content was determined using HPLC as described in Section 2.5. 2.7. Sorting and identification of SP cells H460 cells were sorted by side population (SP) assay as previously described [34]. Briefly, confluent cells were trypsinized and filtered through a 40 mm cell strainer to get a single cell suspension. The cells were then centrifuged and resuspended at a density of 1 106 cells/mL in the pre-warmed RPMI-1640 medium containing 2% FBS. The cells were stained with Hoechst 33342 (5 mg/mL in PBS) with or without the addition of 100 mM verapamil hydrochloride for 120 min at 37 C, with intermittent shaking. The dye is actively effluxed in a specific population of cells, while the cells treated with verapamil retain the stain as verapamil blocks the drug-effluxing P-glycoprotein pumps. The cells were then centrifuged at 4 C at 1000 rpm for 5 min and re-suspended in ice-cold PBS at a density of 1 106 cells/mL, and stained with propidium iodide at a concentration of 1 mg/mL for dead cell discrimination. The side population was defined as the population of cells on the flow cytometry profile, which disappears due to verapamil treatment. Cell analysis and sorting were performed on a FACSDiva (Becton Dickinson, San Jose, CA, U.S.A.) by using a dual-wavelength analysis (blue, 420e470 nm; red, 660e680 nm). Propidium iodide-positive dead cells were excluded from the analysis. For identifying the cell-surface phenotype of SP cells, the sorted SP cells were immuno-stained with anti-CD133-PE antibody (Miltenyi Biotec, Germany) at 4 C for 20 min in PBS. The samples were then washed 3 times with cold PBS and resuspended in 300 mL of cold PBS. Dead cell discrimination was done using propidium iodide. Flow cytometry was performed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA, U.S.A.). Side scatter and forward scatter profiles were used to eliminate cell doublets.
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2.11. Oxygen consumption rate measurement Oxygen consumption rate of H460 cells was measured using Seahorse Bioscience XF24 analyzer. Cells were seeded at 30,000 cells/well in a 96-well cartridge provided with the analyzer, pre-treated free phenformin or phenformin-loaded micelles and incubated for 24 h before the analysis. Each experimental condition was analyzed in four replicates. Before measurements, the cells were washed, and 100 mL of sodium carbonate-free pH 7.4 DMEM was added to each well. After incubating the plate at 37 C without CO2 for 1 h, it was transferred to the analyzer. A total of 60 mL of reagents were injected into each well sequentially at 23, 50 and 76 min, and the measurements were automatically taken every 5e7 min. The reagents for internal control cells and drug-treated cells are oligomycin (20 mL), FCCP (20 mL) and rotenone (20 mL), and sodium carbonate-free pH 7.4 DMEM (60 mL) for baseline control. Oxygen consumption rates were recorded throughout.
2.12. In vitro invasion assay Invasion assay was done using CytoSelect™ 24-well Cell Invasion Assay (colorimetric) according to the manufacturer's instructions. A 24-well plate insert coated evenly with basement membrane matrix solution was provided in the kit. Sorted SP cells (as explained in Section 2.7) were serum starved for 6 h and seeded into the insert (100,000 cells/insert) along with 300 mL of serum-free culture medium containing different drug formulations and control without any drug. The inserts were then placed in the accompanying 24-well plate. The outside of the chamber was filled with 500 mL of 10% FBS containing RPMI-1640, which served as chemo-attractant to draw the invasive cells across the matrix layer. After 24 h of incubation at 37 C, the medium inside the insert was removed and the insert was gently lifted and placed in extraction solution provided in the kit for 10 min at room temperature with orbital shaking to extract the invasive cells. The percentage of invasive CSCs was quantified by transferring 100 mL of the above solution to a 96well plate in triplicates and measuring absorbance at 560 nm.
2.13. In vitro migration assay The effect of free phenformin and drug-loaded micelles on CSC migration was evaluated by a scratch assay. Initially, PDMS strips with uniform sizes were washed with ethanol thrice and then with PBS and fixed in the centre of a 24-well microplate. Sorted SP cells (as explained in Section 2.7) were seeded (100,000 cells/well) in the plate. After incubating for 24 h, the PDMS strip was removed using sterile forceps to form a scratch in the monolayer. Then the cells were exposed to different conditions (growth medium as control, free drug and drug-loaded micelles at a drug concentration of 35 mg/mL). The cells under different conditions were imaged at 0 h and 24 h using an inverted microscope (Olympus, Japan).
2.8. MTS assay
2.14. In vivo anti-tumor efficacy
To study the inhibitory effect of phenformin and phenformin-loaded micelles on various cell types, the cells were seeded into 96-well plates (H460 at 3000 cells/ well, WI38 at 20,000 cells/well, HDF at 20,000 cells/well) and grown for 24 h at 37 C in the presence of 5% CO2. Various concentrations of free phenformin and phenformin-loaded micelles were then added to the cells and incubated for 72 h. The viability of cells was determined by using MTS assay [CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI, U.S.A.)] according to the manufacturer's instructions. Briefly, 20 mL of Aqueous One Solution was added to each well (100 mL of medium) and incubated for 1e2 h at 37 C to allow color development due to formation of soluble formazan. The absorbance was recorded at 490 nm. Cell viability was calculated as [A490 of drug-treated wells/A490 of untreated control wells] 100, where A490 is the absorbance at 490 nm.
Female BALB/c nude mice were obtained from Singapore Biological Research Centre (BRC) at 20 weeks of age (20e26 g). All animal studies adhered to protocols approved by the Singapore BRC's Institutional Animal Care and Use Committee. H460 cells (1 107) were suspended in saline and injected subcutaneously into the right flank of mice. When the tumor volume reached about 200 mm3 on the 7th day, the mice were randomly divided into 4 groups of 10 animals each. Group 1 was saline-injected control, Group 2 was injected with blank micelles, Group 3 with 8 mg/kg of free phenformin and Group 4 with micelles equivalent to 8 mg/kg of phenformin. The formulations were administered to the mice by intravenous injection (tail vein) at day 7, 11 and 14. At the predetermined time points, mice were weighed and tumor size was measured with a digital Vernier caliper. The tumor volume was calculated using the formula: Tumor volume ¼ length width2/2. At the end of the study, blood was collected by cardiac puncture from all mice and serum was separated. The serum (0.1 mL) was used for testing the liver function enzymes such as alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP). The tumors were also excised from all the mice and fixed in 4% paraformaldehyde and embedded in paraffin. The extent of apoptosis in the tumor tissues was analyzed by terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL). Apoptotic bodies were then quantified in individual sections by counting the number of TUNEL-positive nuclei in ten random fields and averaging them.
2.9. Effect of free phenformin and phenformin-loaded micelles on SP cells To investigate the inhibitory effect of free phenformin and phenformin-loaded micelles on SP cells, H460 cells were seeded into 6-well plates at 2 105 cells/ well and grown at 37 C in the presence of 5% CO2 for 24 h. The cells were then exposed to different concentrations of either free phenformin or phenformin-loaded micelles. Control cells were not exposed to any drugs. After 72 h of incubation, the number of SP cells was enumerated and compared in each treatment using Hoescht 33342 staining and flow cytometry as described in Section 2.7.
2.15. Analysis of CD133-positive CSCs in tumor tissues 2.10. Effect of free phenformin and phenformin-loaded micelles on sorted SP and non-SP cells SP and non-SP cells were sorted from H460 cells using FACSDiva (Becton Dickinson, San Jose, CA, U.S.A.) as described in Section 2.7. The sorted SP and non-SP cells were then seeded separately into 96-well plates at a density of 3000 cells/well. After 24 h of incubation, different concentrations of free phenformin or phenforminloaded micelles were added to both SP and non-SP cells. The cell viability was then analyzed after 48 h using MTS assay as described in Section 2.8.
At the end of the treatments, the tumors were collected from the mice, and washed with PBS. The tumors were then cut into small pieces of about 1 mm3, and subjected to enzymatic digestion using collagenase type 3 at 37 C for 2 h. The cell suspension was then filtered through a 40 mm cell strainer to obtain single cells. The cells were then washed thrice with PBS and re-suspended in 50 mL of ice-cold PBS, and stained with anti-human CD133-PE antibody at 4 C for 20 min. It was further subjected to three washes with cold PBS, and the percentage of CSCs was analyzed by flow cytometry.
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2.16. Statistical analysis The normality of the populations was initially tested. The mean values of the different treatment groups were then statistically compared to that of the control group using ANOVA for normally distributed populations, and KruskaleWallis ANOVA for populations that were not normally distributed with OriginPro 8.1. P < 0.05 was considered statistically significant.
3. Results and discussion 3.1. Polymer synthesis Both MTC-OBn and MTC-urea monomers were obtained by direct functionalization and ring closing of commercially available 2,2-bis(methylol)propionic acid [29]. The synthesis of PEG-b-PAC and PEG-b-PUC was achieved by organocatalytic ROP of MTC-OBn and MTC-urea respectively using PEG as the macroinitiator (Fig. 1A). The simplicity and attractiveness of this methodology is that it involves no toxic heavy metal catalysts, and the sideproducts can be easily removed by precipitation. The degree of polymerization repeating units (DP) of MTC-OBn and MTC-Urea were determined to be 12 and 10 (relative to PEG), respectively, from 1H NMR analyses. In addition, narrow monomodal polydispersity indices for both polymers (
Contents lists available at ScienceDirect
Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Phenformin-loaded polymeric micelles for targeting both cancer cells and cancer stem cells in vitro and in vivo Sangeetha Krishnamurthy, Victor W.L. Ng, Shujun Gao, Min-Han Tan, Yi Yan Yang* Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669, Singapore
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 June 2014 Accepted 16 July 2014 Available online 5 August 2014
Conventional cancer chemotherapy often fails as most anti-cancer drugs are not effective against drugresistant cancer stem cells. These surviving cancer stem cells lead to relapse and metastasis. In this study, an anti-diabetic drug, phenformin, capable of eliminating cancer stem cells was loaded into micelles via self-assembly using a mixture of a diblock copolymer of poly(ethylene glycol) (PEG) and ureafunctionalized polycarbonate and a diblock copolymer of PEG and acid-functionalized polycarbonate through hydrogen bonding. The phenformin-loaded micelles, having an average diameter of 102 nm with narrow size distribution, were stable in serum-containing solution over 48 h and non-cytotoxic towards non-cancerous cells. More than 90% of phenformin was released from the micelles over 96 h. Lung cancer stem cells (side population cells, i.e. SP cells) and non-SP cells were sorted from H460 human lung cancer cell line, and treated with free phenformin and phenformin-loaded micelles. The results showed that the drug-loaded micelles were more effective in inhibiting the growth of both SP and non-SP cells. In vivo studies conducted in an H460 human lung cancer mouse model demonstrated that the drug-loaded micelles had greater anti-tumor efficacy, and reduced the population of SP cells in the tumor tissues more effectively than free phenformin. Liver function analysis was performed following drug treatments, and the results indicated that the drug-loaded micelles did not cause liver damage, a harmful side-effect of phenformin when used clinically. These phenformin-loaded micelles may be used to target both cancer cells and cancer stem cells in chemotherapy for the prevention of relapse and metastasis. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Phenformin Polymeric micelles Lung cancer Cancer stem cells In vivo efficacy
1. Introduction The number of cancer-related deaths worldwide is in the increase in spite of the cutting-edge advances made in drug discovery and drug delivery [1e3]. Various treatment strategies like surgical resection, radiotherapy and systemic therapies are being used. However, the response to the treatments is low, and there is a high rate of recurrence and metastasis [4]. The capability of cancers to recur and metastasize is attributed to the presence of a side population of cancer cells (SP cells, or cancer stem cells) with the ability to self-renew, enhance drug resistance and initiate tumor [5e7]. Cancer stem cells (CSCs) have been well studied and documented in different types of cancers, and they express various unique surface markers [8e10]. A number of studies have shown that cancer stem cells were more resistant to chemotherapeutic drugs, and worsened the overall survival [11e16]. Therefore, an effective treatment strategy should be able to target both cancer cells and CSCs, thereby
* Corresponding author. Tel.: þ65 6824 7106; fax: þ65 6478 9084. E-mail address: [email protected] (Y.Y. Yang). http://dx.doi.org/10.1016/j.biomaterials.2014.07.018 0142-9612/© 2014 Elsevier Ltd. All rights reserved.
preventing recurrence and metastasis, and enhancing the survival rate of patients. Most conventional anti-cancer drugs have little effect on CSCs. This is potentially due to the inherent resistance of CSCs, their over-active efflux pumps that remove the drugs out of the cells and enhanced DNA repair capability [17]. Interestingly, other classes of drugs like anti-psychotics [18], anti-diabetics [19] and anti-helminthics [20] have been found to be effective in eradicating CSCs. Biguanides are a class of anti-diabetic drugs, of which metformin and phenformin have been frequently studied [21]. Both of these drugs have recently been shown to exhibit an antineoplastic effect against several types of cancers especially cancer stem cells [16,22,23]. Metformin is a highly prescribed drug for Type-2 diabetes mellitus (T2DM), and is preferred over phenformin because of lesser incidence of lactic acidosis in treated patients [24]. Compared to metformin, phenformin is 50 times more potent than metformin against different cancer cell lines [25]. Like many other drugs, phenformin is metabolized in the liver. The drug was associated with increased incidences of lactic acidosis [26], and was subsequently withdrawn by FDA in 1976 as
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an anti-diabetic drug [27]. Nanoparticles have been used to deliver drugs and reduce non-specific toxicity towards healthy tissues by exploiting the enhanced permeability and retention (EPR) effect of nanoparticles at leaky tumor tissues [28]. Among various types of nanoparticles, polymeric micelles are most commonly explored for drug delivery due to their unique core/shell structure having a hydrophobic core for encapsulating the drug and a hydrophilic corona that prolongs drug circulation in the blood, and ease of installing functionalities to the core for enhancing interactions with the drug and increasing drug loading level. We hypothesized that formulating phenformin into polymeric micelles would enhance its anti-cancer efficacy and reduce the CSC population without causing toxicity to the liver. Recently, we reported functional micelles self-assembled from a mixture of poly(ethylene glycol) (PEG)/acid-functionalized polycarbonate diblock copolymer (PEG-b-PAC) and PEG/ureafunctionalized polycarbonate diblock copolymer (PEG-b-PUC) via hydrogen-bonding [29]. These micelles had excellent stability due to the presence of hydrogen-bond (ureaeurea and urea-acid), exhibiting preferable accumulation at tumor tissues based on the EPR effect [30]. In addition, they had high loading capacity for basic drugs such as doxorubicin [29e31]. Phenformin is a basic drug, and has two guanidine groups in the molecule, which can form hydrogen-bond with urea group and ionic interaction with acid group in the micellar core. In this study, PEG-b-PUC and PEGb-PAC were synthesized by organocatalytic ring-opening polymerization (ROP) using methoxy-PEG (Mn 10 kDa) as the macroinitiator. Phenformin was loaded into micelles via self-assembly of PEG-b-PUC and PEG-b-PAC mixture. The phenformin-loaded micelles were characterized for drug loading level, particle size, size distribution, zeta potential, in vitro drug release, and stability in serum-containing solution. According to World Health Organization's 2014 statistics, lung cancer is the leading cause of cancer deaths worldwide in both men and women with 27% of all cancer deaths. Lung cancer also has the lowest 5-year survival rate (16.6%) compared to all other high-incidence cancers like colon (64.2%), breast (89.2%) and prostate (99.2%) (National Cancer Institute. SEER Cancer Statistics Review, 1975e2010). In addition, lung cancer is commonly metastatic, predominantly metastasizing to adrenal gland, bone, brain, liver and the other lung [32]. Therefore, in this study, lung cancer was chosen as a model cancer using H460 human lung adenocarcinoma cell line as an example. Cytotoxicity of phenformin and phenformin-loaded micelles against H460 cells was evaluated by MTS, and their non-specific cytotoxicity was also examined against human dermal fibroblasts (HDFs) and WI-38 human fibroblast like fetal lung cell line. SP (CSCs) and non-SP cells were sorted from H460 cells by flow cytometry, and identified by immuno-staining with anti-CD133-PE antibody. Inhibitory effect of free phenformin and phenforminloaded micelles on SP and non-SP cells was investigated, and their anti-cancer efficacy was studied in an H460 xenograft mouse model. The population of CSCs in the tumor tissue after treatment with free phenformin and phenformin-loaded micelles was also analyzed. 2. Materials and methods 2.1. Materials Phenformin-HCl, Hoechst 33342 and all other reagents were purchased from SigmaeAldrich (St. Louis, MO, U.S.A.) unless otherwise specified. CellTiter 96® AQueous non-radioactive cell proliferation assay kit (MTS) was obtained from Promega (Madison, WI, U.S.A.). Anti-CD133-PE was bought from Miltenyi Biotec (Auburn, CA, U.S.A.). CytoSelect™ 24-well cell invasion assay kit was purchased from Cell Biolabs (CA, U.S.A.). Lactic acid was assayed using L-lactate assay kit (Cayman Scientific, Michigan, U.S.A.). H460, human dermal fibroblast (HDF) cell line and human fetal lung cell line (WI38) were obtained from ATCC (Manassas, VA, U.S.A.). Cells were cultured in
RPMI-1640 medium (Lonza, Singapore) supplemented with 10% fetal bovine serum (FBS, Thermo Scientific Inc, MA, U.S.A.), 100 U/mL penicillin and 100 mg/mL streptomycin (Invitrogen, CA, U.S.A.) at 37 C in 5% CO2 atmosphere. Dulbecco's modified Eagle's medium (DMEM) was purchased from Lonza, Singapore. 2.2. Polymer synthesis The details for organocatalytic ring-opening polymerization (ROP) of ureafunctionalized (MTC-urea) and benzyl-protected acid-functionalized (MTC-OBn) carbonate monomers using methoxy poly(ethylene glycol) (PEG, Mn 10,000 g/mol, PDI 1.04) are provided below. All polymerizations were carried out in a glove-box under nitrogen atmosphere. MTC-urea and MTC-OBn monomers were synthesized according to a protocol reported previously [29]. Diblock copolymer of PEG and urea-functionalized polycarbonate (PEG-b-PUC): To a mixture of MTC-urea (242 mg, 0.75 mmol), PEG (500 mg, 0.05 mmol) and thiourea catalyst (TU) (18.5 mg, 0.05 mmol) dissolved in dichloromethane (5 mL) was added sparteine (11.5 mL, 0.05 mmol) to initiate the ROP. The homogeneous reaction mixture was stirred for 18 h at room temperature (~22 C). The polymerization was quenched by addition of benzoic acid (25 mg; ~4 eqv.), and the crude polymer was subsequently precipitated in 50 mL of diethyl ether. The white solid was collected after centrifugation, dried briefly and re-dissolved in dimethyl sulfoxide (2 mL)/dichloromethane (2 mL). The crude product was re-precipitated in diethyl ether four times to ensure complete removal of impurities and dried under vacuo to give a white solid (85%). PDI 1.14. 1H NMR (400 MHz, DMSO-d6, 22 C): d 8.53 (s, br, 10H, PhNHe), 7.36 (m, 20H, PhH), 7.18 (m, 20H, PhH), 6.86 (m, 10H, PhH), 6.20 (s, br, 10H, eCH2NHe), 4.05e4.25 (overlapping peaks, 60H, MTCeCH2e and eCOOCH2e), 3.50 (s, 906H, H of mPEG), 3.33 (s, 20H, eCH2NHCOe), 1.13 (s, 30H, eCH3). Diblock copolymer of PEG and benzyl-protected acid-functionalized polycarbonate (PEG-b-P(MTC-OBn): In a similar manner, DBU (7.5 mL, 0.05 mmol) was added to a mixture of MTC-OBn (188 mg, 0.75 mmol) and PEG (500 mg, 0.05 mmol) dissolved in dichloromethane (5 mL) for initiating the ROP. The mixture was stirred for 3 h at room temperature and subsequently quenched by the addition of benzoic acid (25 mg; ~4 eqv.). Identical purification protocol was employed using dichloromethane/diethyl ether as the precipitation solvent combination for PEG-bPUC polymer. Yield, 80%; PDI 1.13. 1H NMR (400 MHz, CDCl3, 22 C): d 7.31 (m, 60H, PhH), 5.13 (m, 24H, PhCH2e), 4.27 (m, 48H, MTCeCH2e), 3.64 (s, 906H, H of mPEG), 3.37 (s, 3H, CH3 of mPEG), 1.21 (m, 36H, eCH3). Benzyl groups in PEG-b-P(MTC-OBn) were deprotected to yield a diblock copolymer of PEG and acid-functionalized polycarbonate (PEG-b-PAC). Briefly, a mixture of PEG-b-P(MTC-OBn) (0.5 g) and PdeC (10% w/w, 0.2 g) in THF/methanol (7.5 mL each) was reacted with dry hydrogen (7 atm) overnight. After removal of hydrogen, the mixture was filtered through celite wetted with THF. To ensure complete transfer of the deprotected polymer, additional THF and methanol washings were carried out. The filtrate was then collected, and solvents evaporated. The resultant residue was re-dissolved in THF and precipitated with diethyl ether before being freeze-dried under vacuo. The final product PEG-b-PAC was obtained as a powdery white solid (yield: >90%). 2.3. Gel permeation chromatography (GPC) GPC analysis of acid- and urea-functionalized block copolymers was performed using a Waters HPLC system with a 2690D separation module and two Styragel HR1 and HR4E (THF) 5 mm columns (size: 300 7.8 mm) in series and a Waters 410 differential refractometer detector. THF was used as the mobile phase and a flow rate of 1 mL/min was fixed. A calibration curve was obtained from a series of polystyrene standards of molecular weight between 1350 and 151,700. Polydispersity indices and molecular weights of the block copolymers were calculated based on the standard curve. 2.4. Loading of phenformin into micelles Phenformin was loaded into polymeric micelles using a modified sonication method as reported previously [33]. Briefly, 10 mg of phenformin-HCl was dissolved in 100 mL of dimethyl sulfoxide (DMSO), and neutralized with 3-mol excess of triethylamine. PEG-b-PUC (4.5 mg) and PEG-b-PAC (5.5 mg) (urea: acid ¼ 1:1 M ratio) were added to the solution, which was then added dropwise into 10 mL of deionized (DI) water on ice, while sonicating at 130 W for 2 min using a probe-based sonicator (Vibra Cell VCX 130, Connecticut, U.S.A.). Subsequently, unloaded drug and individual polymer molecules were removed by filtering through a centrifugal concentrator (Vivaspin® Turbo 15, Sartorius, U.K.) with molecular weight cut-off of 10,000 Da and washing 3 times with DI water. The resulting solution was lyophilized to harvest phenformin-loaded micelles. 2.5. Characterization of phenformin-loaded micelles The particle size and size distribution of freshly prepared micelle solution (1 mg/mL) was determined by dynamic light scattering (scattering angle: 90 ) using DTS Zetasizer 3000 HAS (Malvern Instruments, Worcestershire, U.K.). The zeta potential of the micelles was also measured using the same instrument. The drug loading level of the micelles was analyzed by high performance liquid
S. Krishnamurthy et al. / Biomaterials 35 (2014) 9177e9186 chromatography (HPLC) (C18 reversed phase column, 0.5 mL/min, 100 mL injection, acetonitrile/water gradient elution) at 253 nm. The standard curve of phenformin was constructed using a concentration range of 7e500 mg/mL (where it shows linearity), and used to determine the drug content in micelles. The drug loading was calculated as the fraction of mass of drug-loaded in the micelles to the total mass of drug-loaded micelles. All measurements were done in triplicates. 2.6. In vitro release The in vitro release of phenformin from the micelles was determined in phosphate-buffered saline (PBS, pH 7.4) at 37 C. Briefly, phenformin-loaded micelles were dispersed in PBS (1 mg/mL, 2 mL) and placed in a dialysis membrane bag having a molecular weight cut-off of 2000 Da. The bag was immersed in 20 mL of PBS while being shaken at 100 rpm. The solution outside the dialysis bag (1 mL) was collected at predetermined time intervals and was replaced with the same volume of fresh buffer. Phenformin content was determined using HPLC as described in Section 2.5. 2.7. Sorting and identification of SP cells H460 cells were sorted by side population (SP) assay as previously described [34]. Briefly, confluent cells were trypsinized and filtered through a 40 mm cell strainer to get a single cell suspension. The cells were then centrifuged and resuspended at a density of 1 106 cells/mL in the pre-warmed RPMI-1640 medium containing 2% FBS. The cells were stained with Hoechst 33342 (5 mg/mL in PBS) with or without the addition of 100 mM verapamil hydrochloride for 120 min at 37 C, with intermittent shaking. The dye is actively effluxed in a specific population of cells, while the cells treated with verapamil retain the stain as verapamil blocks the drug-effluxing P-glycoprotein pumps. The cells were then centrifuged at 4 C at 1000 rpm for 5 min and re-suspended in ice-cold PBS at a density of 1 106 cells/mL, and stained with propidium iodide at a concentration of 1 mg/mL for dead cell discrimination. The side population was defined as the population of cells on the flow cytometry profile, which disappears due to verapamil treatment. Cell analysis and sorting were performed on a FACSDiva (Becton Dickinson, San Jose, CA, U.S.A.) by using a dual-wavelength analysis (blue, 420e470 nm; red, 660e680 nm). Propidium iodide-positive dead cells were excluded from the analysis. For identifying the cell-surface phenotype of SP cells, the sorted SP cells were immuno-stained with anti-CD133-PE antibody (Miltenyi Biotec, Germany) at 4 C for 20 min in PBS. The samples were then washed 3 times with cold PBS and resuspended in 300 mL of cold PBS. Dead cell discrimination was done using propidium iodide. Flow cytometry was performed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA, U.S.A.). Side scatter and forward scatter profiles were used to eliminate cell doublets.
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2.11. Oxygen consumption rate measurement Oxygen consumption rate of H460 cells was measured using Seahorse Bioscience XF24 analyzer. Cells were seeded at 30,000 cells/well in a 96-well cartridge provided with the analyzer, pre-treated free phenformin or phenformin-loaded micelles and incubated for 24 h before the analysis. Each experimental condition was analyzed in four replicates. Before measurements, the cells were washed, and 100 mL of sodium carbonate-free pH 7.4 DMEM was added to each well. After incubating the plate at 37 C without CO2 for 1 h, it was transferred to the analyzer. A total of 60 mL of reagents were injected into each well sequentially at 23, 50 and 76 min, and the measurements were automatically taken every 5e7 min. The reagents for internal control cells and drug-treated cells are oligomycin (20 mL), FCCP (20 mL) and rotenone (20 mL), and sodium carbonate-free pH 7.4 DMEM (60 mL) for baseline control. Oxygen consumption rates were recorded throughout.
2.12. In vitro invasion assay Invasion assay was done using CytoSelect™ 24-well Cell Invasion Assay (colorimetric) according to the manufacturer's instructions. A 24-well plate insert coated evenly with basement membrane matrix solution was provided in the kit. Sorted SP cells (as explained in Section 2.7) were serum starved for 6 h and seeded into the insert (100,000 cells/insert) along with 300 mL of serum-free culture medium containing different drug formulations and control without any drug. The inserts were then placed in the accompanying 24-well plate. The outside of the chamber was filled with 500 mL of 10% FBS containing RPMI-1640, which served as chemo-attractant to draw the invasive cells across the matrix layer. After 24 h of incubation at 37 C, the medium inside the insert was removed and the insert was gently lifted and placed in extraction solution provided in the kit for 10 min at room temperature with orbital shaking to extract the invasive cells. The percentage of invasive CSCs was quantified by transferring 100 mL of the above solution to a 96well plate in triplicates and measuring absorbance at 560 nm.
2.13. In vitro migration assay The effect of free phenformin and drug-loaded micelles on CSC migration was evaluated by a scratch assay. Initially, PDMS strips with uniform sizes were washed with ethanol thrice and then with PBS and fixed in the centre of a 24-well microplate. Sorted SP cells (as explained in Section 2.7) were seeded (100,000 cells/well) in the plate. After incubating for 24 h, the PDMS strip was removed using sterile forceps to form a scratch in the monolayer. Then the cells were exposed to different conditions (growth medium as control, free drug and drug-loaded micelles at a drug concentration of 35 mg/mL). The cells under different conditions were imaged at 0 h and 24 h using an inverted microscope (Olympus, Japan).
2.8. MTS assay
2.14. In vivo anti-tumor efficacy
To study the inhibitory effect of phenformin and phenformin-loaded micelles on various cell types, the cells were seeded into 96-well plates (H460 at 3000 cells/ well, WI38 at 20,000 cells/well, HDF at 20,000 cells/well) and grown for 24 h at 37 C in the presence of 5% CO2. Various concentrations of free phenformin and phenformin-loaded micelles were then added to the cells and incubated for 72 h. The viability of cells was determined by using MTS assay [CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI, U.S.A.)] according to the manufacturer's instructions. Briefly, 20 mL of Aqueous One Solution was added to each well (100 mL of medium) and incubated for 1e2 h at 37 C to allow color development due to formation of soluble formazan. The absorbance was recorded at 490 nm. Cell viability was calculated as [A490 of drug-treated wells/A490 of untreated control wells] 100, where A490 is the absorbance at 490 nm.
Female BALB/c nude mice were obtained from Singapore Biological Research Centre (BRC) at 20 weeks of age (20e26 g). All animal studies adhered to protocols approved by the Singapore BRC's Institutional Animal Care and Use Committee. H460 cells (1 107) were suspended in saline and injected subcutaneously into the right flank of mice. When the tumor volume reached about 200 mm3 on the 7th day, the mice were randomly divided into 4 groups of 10 animals each. Group 1 was saline-injected control, Group 2 was injected with blank micelles, Group 3 with 8 mg/kg of free phenformin and Group 4 with micelles equivalent to 8 mg/kg of phenformin. The formulations were administered to the mice by intravenous injection (tail vein) at day 7, 11 and 14. At the predetermined time points, mice were weighed and tumor size was measured with a digital Vernier caliper. The tumor volume was calculated using the formula: Tumor volume ¼ length width2/2. At the end of the study, blood was collected by cardiac puncture from all mice and serum was separated. The serum (0.1 mL) was used for testing the liver function enzymes such as alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP). The tumors were also excised from all the mice and fixed in 4% paraformaldehyde and embedded in paraffin. The extent of apoptosis in the tumor tissues was analyzed by terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL). Apoptotic bodies were then quantified in individual sections by counting the number of TUNEL-positive nuclei in ten random fields and averaging them.
2.9. Effect of free phenformin and phenformin-loaded micelles on SP cells To investigate the inhibitory effect of free phenformin and phenformin-loaded micelles on SP cells, H460 cells were seeded into 6-well plates at 2 105 cells/ well and grown at 37 C in the presence of 5% CO2 for 24 h. The cells were then exposed to different concentrations of either free phenformin or phenformin-loaded micelles. Control cells were not exposed to any drugs. After 72 h of incubation, the number of SP cells was enumerated and compared in each treatment using Hoescht 33342 staining and flow cytometry as described in Section 2.7.
2.15. Analysis of CD133-positive CSCs in tumor tissues 2.10. Effect of free phenformin and phenformin-loaded micelles on sorted SP and non-SP cells SP and non-SP cells were sorted from H460 cells using FACSDiva (Becton Dickinson, San Jose, CA, U.S.A.) as described in Section 2.7. The sorted SP and non-SP cells were then seeded separately into 96-well plates at a density of 3000 cells/well. After 24 h of incubation, different concentrations of free phenformin or phenforminloaded micelles were added to both SP and non-SP cells. The cell viability was then analyzed after 48 h using MTS assay as described in Section 2.8.
At the end of the treatments, the tumors were collected from the mice, and washed with PBS. The tumors were then cut into small pieces of about 1 mm3, and subjected to enzymatic digestion using collagenase type 3 at 37 C for 2 h. The cell suspension was then filtered through a 40 mm cell strainer to obtain single cells. The cells were then washed thrice with PBS and re-suspended in 50 mL of ice-cold PBS, and stained with anti-human CD133-PE antibody at 4 C for 20 min. It was further subjected to three washes with cold PBS, and the percentage of CSCs was analyzed by flow cytometry.
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2.16. Statistical analysis The normality of the populations was initially tested. The mean values of the different treatment groups were then statistically compared to that of the control group using ANOVA for normally distributed populations, and KruskaleWallis ANOVA for populations that were not normally distributed with OriginPro 8.1. P < 0.05 was considered statistically significant.
3. Results and discussion 3.1. Polymer synthesis Both MTC-OBn and MTC-urea monomers were obtained by direct functionalization and ring closing of commercially available 2,2-bis(methylol)propionic acid [29]. The synthesis of PEG-b-PAC and PEG-b-PUC was achieved by organocatalytic ROP of MTC-OBn and MTC-urea respectively using PEG as the macroinitiator (Fig. 1A). The simplicity and attractiveness of this methodology is that it involves no toxic heavy metal catalysts, and the sideproducts can be easily removed by precipitation. The degree of polymerization repeating units (DP) of MTC-OBn and MTC-Urea were determined to be 12 and 10 (relative to PEG), respectively, from 1H NMR analyses. In addition, narrow monomodal polydispersity indices for both polymers (
