Co-delivery of 2-Deoxyglucose and a Glutamine Metabolism Inhibitor V9302 via a Prodrug Micellar Formulation for Synergistic Targeting of Metabolism in Cancer

Abstract

The unique metabolic demand of cancer cells suggests a new therapeutic strategy targeting the metabolism in cancers. V9302 is a recently reported inhibitor of ASCT2 amino acid transporter which shows promising antitumor activity by blocking glutamine uptake. However, its poor solubility in aqueous solutions and tumor cells’ compensatory metabolic shift to glucose metabolism may limit the antitumor efficacy of V9302. 2-Deoxyglucose (2-DG), a derivative of glucose, has been developed as a potential antitumor agent through inhibiting glycolysis in tumor cells. In order to achieve enhanced antitumor effect by inhibiting both metabolic pathways, a 2-DG prodrug-based micellar carrier poly-(oligo ethylene glycol)-co-poly(4-((4-oxo-4-((4-vinylbenzyl)oxy)butyl)disulfaneyl)butanoic acid)-(2-deoxyglucose) (POEG-p-2DG) was developed. POEG-p-2DG well retained the pharmacological activity of 2-DG in vitro and in vivo, More importantly, POEG-p-2DG could self-assemble to form micelles that were capable of loading V9302 to achieve co-delivery of 2-DG and V9302. V9302-loaded POEG-p2DG micelles were small in sizes (~10nm), showed a slow kinetics of drug release and demonstrated targeted delivery to tumor. In addition, V9302 loaded POEG-p-2DG micelles exhibited improved anti-tumor efficacy both in vitro and in vivo. Interestingly, 2-DG treatment further decreased the glutamine uptake when combined with V9302, likely due to inhibition of ASCT2 glycosylation. These results suggest that POEG-p2DG prodrug micelles may serve as a dual functional carrier for V9302 to achieve synergistic targeting of metabolism in cancers.

Graphical Abstract

1. Introduction

Cancer cells exhibit a pronounced metabolic reprogramming to support their abnormal proliferation[1–4]. One of the most well-studied metabolic alteration in cancer cell is the increase in and the dependency on anaerobic glycolysis pathway for ATP generation despite availability of sufficient oxygen to oxidize glucose completely. This is known as the “Warburg Effect”, which is regarded as a metabolic hallmark of aggressive tumors[5, 6]. Accordingly, targeting abnormal glycolysis for the treatment of cancer has been explored previously as a therapeutic approach[7]. Of all the glycolysis inhibitors that were evaluated, 2-deoxyglucose (2-DG) is the one that has been best characterized in animal model studies[8–10] and human clinical trials[11–13]. 2-DG is a glucose analog and is converted by hexokinase to phosphorylated 2-DG, which is trapped inside the cell. The phosphorylated 2-DG can inhibit the production of glucose-6-phosphate from glucose. As a direct consequence of 2-DG treatment, intracellular ATP is depleted, which ultimately suppresses cell proliferation. However, normal cell also requires glycolysis to produce energy. The rapid clearance of 2-DG and its indiscriminative nature in tissue distribution has hindered its further clinical application. Serious side effects were observed in 2-DG clinical trial, such as hypoglycemia, QTc prolongation and cardiac arrest. Most importantly, preclinical and clinical studies indicate that 2-DG treatment, when provided as a single agent, provides limited therapeutic benefits[14, 15]. This suggests the necessity of developing a combination therapy as well as a strategy for tumor-selective delivery[16].

Apart from glucose, emerging researches demonstrate a critical role for glutamine in energy generation, biosynthesis and cell homeostasis of cancer cells[17]. Glutamine is transported into cell largely through solute carrier family 1 neutral amino acid transporter member 5 (SLC1A5; also known as ASCT2)[18]. After being transported into cell, glutamine itself can contribute to nucleotide biosynthesis. It can also be involved in protein trafficking and folding through its contribution to the synthesis of uridine diphosphate N-acetylglucosamine. Alternatively, it can be converted to glutamate by mitochondria glutaminase (GLS). Glutamate can contribute to the synthesis of glutathione, which is critical for the maintenance of cell homeostasis, or more importantly, be converted to α-ketoglutarate (αKG) by glutamate dehydrogenase or aminotransferase. αKG is the central metabolite of glutamine metabolism that can enter the TCA cycle, through which glutamine pathway and glucose pathway are bridged and highly incorporated with each other[19–21]. Because of Warburg Effect, most glucose-derived carbons are secreted as lactate instead of entering TCA cycle as pyruvate. This leads to glutamine anaplerosis that utilizes glutamine-derived αKG to compensate the carbon source in TCA cycle and refill the TCA functions to guarantee the survival and proliferation of cancer cells. The alteration of glutamine demanding is also known as “glutamine addicted”—that the cancer cell is highly glutamine-consuming for maintaining the survival or proliferation compared to normal cell[3, 22]. Based on this feature, several compounds have been developed for targeting the abnormal dependency on glutamine of cancer cell[23, 24]. For example, CB-839 is a GLS inhibitor that is under phase II clinical trial[25, 26]. Also, V9302, a recently reported inhibitor of glutamine transporter ASCT2, showed promising antitumor response by inhibiting glutamine uptake in preclinical studies[18, 27]. However, similar to enhanced glutamine metabolism upon inhibition of glycolysis, interference of glutamic metabolism leads to upregulation of glucose metabolism.[27–29]. Although the mechanism is not clearly understood, the compensatory shift between glucose and glutamine metabolism suggests the importance of developing a combination strategy to target both metabolic pathways[30].

The major limitations with combination of 2-DG and V9302 are their rapid elimination from blood and the potential toxicity towards normal cells. Development of a strategy to achieve selective codelivery of the two drugs to tumor represents an attractive solution to these problems. However, engineering the two drugs into a single carrier is challenging as 2-DG is highly water-soluble and V9302 is highly water-insoluble. Alternatively, prodrug micelle provides an attractive strategy to co-deliver two drugs with different physiochemical properties[31–34]. Prodrug polymers with amphiphilic properties, while maintaining the pharmacological activity of the conjugated drug, could self-assemble into micelles to load another drug to achieve synergistic effect[4, 35–37]. Furthermore, polymeric prodrug carriers can realize controlled release of both conjugated drugs and encapsulated drugs in response to a particular stimulus via introducing stimuli-responsive linkages between the drug and the polymer backbone[38–40].

Here, we reported a strategy of synergistic targeting of cancer metabolism through codelivery of V9302 via a 2-DG-based prodrug polymeric carrier poly-(oligo ethylene glycol)-co-poly(4-((4-oxo-4-((4-vinylbenzyl)oxy)butyl)disulfaneyl)butanoic acid)-(2-deoxyglucose) (POEG-p-2DG). POEG-p-2DG polymer can serve as a depot system allowing the release of active 2-DG over a prolonged period of time. Moreover, POEG-p-2DG polymer can self-assemble to from micelles for codelivery of V9302. Also, through introducing self-immolative disulfide linkage, we could realize the redox-responsive release of 2-DG and V9302. We hypothesize that selective codelivery of V9302 and 2-DG via POEG-p-2DG-based nanocarrier can improve the overall antitumor activity through targeting of both glucose and glutamine metabolism simultaneously.

2. Experimental Section

2.1. Materials

V9302 was synthesized and purified following a published paper[41]. 4-Vinylbenzyl chloride, 4,4-dithiodibutyric acid, triethylamine, 4-Cyano-4-(phenyl-carbonothioylthio)pentanoic acid, poly(Ethylene glycol)methyl ether methacrylate (average Mn = 950), 2,2-Azobis (isobutyronitrile)(AIBN), Dulbecco’s Modified Eagle’s Medium (DMEM), RPMI 1640 Medium, trypsin-EDTA solution, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (MO, U.S.A). 2-deoxy-D-glucose was purchased from AK Scientific Inc. (CA, U.S.A.). Antibody against ASCT2 was purchased from Cell Signaling Technology, Inc. (MA, U.S.A). SuperSignal™ West Fento Maximum Sensitivity Substrate Kit and Pierce™ RIPA buffer were purchased from Thermo Scientific (MA, U.S.A). Fetal bovine serum (FBS), penicillin-streptomycin solution and Trizol lysis reagent were purchased from Invitrogen (NY, U.S.A.). QuantiTect Reverse Transcription Kit was purchased from Qiagen (MD, U.S.A). EnzyChrom Glutamine Assay kit was purchased from BioAssay Systems (CA, USA), Glucose Colorimetric/Fluorometric Assay Kit and Lactate Colorimetric/Fluorometric Assay Kit were purchased from BioVision Inc. (CA, USA). ALT/SGPT Liqui-UV Test Kit and AST/SGOT Liqui-UV Test Kit were purchased from Stanbio Laboratory (TX, USA).

2.2. Method

2.2.1. Synthesis of VBSS monomer

Vinylbenzyl chloride (305.2 mg, 2 mM), 4,4’-Dithiodibutyric acid (2.38 g, 10 mM) and K₂CO₃ (1.38 g 10 mM) were mixed in 10 mL DMF and reacted at 50°C under stirring. After 16h, the mixture was cooled down to room temperature and 90mL methylene dichloride is added to extract the product. The mixture was centrifuged at 4500 rpm for 10 min. The precipitate was washed with methylene dichloride twice and all the supernatant was collected, washed with brine for three times and then dried with anhydrous sodium sulfate. The VBSS monomer was purified by column chromatography with ethyl acetate/petroleum ether (v/v=1/3 to 1/1) as the elution buffer.

2.2.2. Synthesis of POEG-p-2DG polymer

4-Cyano-4-(thiobenzoylthio)pentanoic acid (15.2 mg, 0.0545 mmol), AIBN (7.2 mg, 0.0446 mmol), VBSS monomer (880 mg, 2.79 mmol), OEG950 monomer (880 mg, 0.924 mmol) and dried tetrahydrofuran (3.2 mL) were added into a Schlenk tube. The Schlenk tube was placed in oil bath and stirred at 80°C with N₂ protection overnight after deoxygenation with three free pump-thawing cycles. The product (POEG-p-VBSS) is weighted and dissolved in DMSO. In 5 mL DMSO solution, POEG-p-VBSS (30 mg), 2-Deoxyglucose (2DG) (60 mg, 0.365 mmol), 3-(ethyliminomethylideneamino)-N,N-dimethylpropan-1-amine;hydrochloride (EDC·HCl) (100 mg, 0.522 mmol), Hydroxy benzotriazole (HOBt) (60 mg, 0.444 mmol), and 100 μL N,N-Diisopropylethylamine (DIPEA) were mixed and reacted in 37°C for 72h. Dialysis and lyophilization is followed to purify and render the final POEG-p-2DG product.

2.2.3. Characterization of the synthesized monomer and polymers

¹H NMR spectrum was examined on a Varian-400 FT-NMR spectrometer at 400.0 MHz with d6-DMSO as the solvent. Molecular weight (Mn and Mw) and distribution (Mw/Mn) of the synthesized polymers were measured by gel permeation chromatography (GPC) performed on a Waters 515 HPLC pump and a Waters 717 Plus Autosampler equipped with a Waters 2414 refractive index detector. Tetrahydrofuran (THF) was used as the eluent with a flowing rate of 1.0 mL/min at 35°C. A series of commercial polystyrene standards with narrow molecular weight distribution were applied to calibrate the GPC elution traces.

2.2.4. Preparation and Characterization of Blank or V9302-loaded POEG-p-2DG micelles

V9302 solution (10 mg/mL in methylene chloride) was mixed with POEG-p-2DG polymers (20 mg/mL in methylene chloride) at different carrier/drug weight ratios. The solvent was removed by nitrogen flow to produce a thin film of carrier/drug mixture, which was further dried in vacuum for 2h to remove any remaining solvent. Then the thin film was hydrated and gently vortexed in PBS to form V9302-loaded POEG-p-2DG micelles.

The average diameter and the size distribution of POEG-p-2DG micelles were assessed via a Zetasizer (DLS). The morphology of POEG-p-VBSS polymer backbone, POEG-p-2DG blank micelles and drug-loaded micelles was observed by transmission electron microscopy (TEM).

The CMC of POEG-p-2DG and POEG-p-VBSS polymer was determined by using Nile red as a fluorescence probe as described previously[35]. The CMC of V9302 loaded POEG-p-2DG was determined via a DLS-based method [42].

The V9302 concentration was determined by Waters e2695 HPLC system equipped with a Waters 2489 UV detector. Phosphoric acid (0.1%) /Methanol (v/v=45/55) was used as eluent with the flow rate of 1mL/min at 30°C. V9302 was detected by UV at 276 and 227 nm. The drug loading efficiency (DLE) was calculated as the ratio between the amount of drug in the micelles and the amount of input drug. The drug loading capacity (DLC) was calculated as the percentage of the drug amount incorporated in the micelles versus the total amount of input materials.

The kinetics of V9302 in vitro release from V9302/POEG-p-2DG was studied using a dialysis method. Briefly, 1 mL of V9302/ POEG-p-2DG micelles containing 6mg of V9302 were placed in a clamped dialysis bag (MWCO 3.5 kDa) and immersed in 25 mL of 0.1M PBS buffer solution containing 0.5% (w/v) Tween 80 with or without 10mM GSH. The experiment was performed in an incubation shaker at 37 °C at 100 rpm. At selected time intervals, both 10 μL V9302/ POEG-p-2DG micelles solution in the dialysis bag and 1mL medium outside the dialysis bag were withdrawn while same amount of fresh medium was added for replenishment. For comparison, free V9302 dissolved in 2% DMSO was included as free diffusion control. The V9302 release from micelles was also measured by Waters e2695 HPLC system equipped with a Waters 2489 UV detector. Chromatography condition is the same as above part.

The kinetics of 2-DG in vitro release from POEG-p-2DG was also studied by using a dialysis method. 1mL of 60mg POEG-p-2DG was placed in dialysis bag and immersed in 25mL of 0.1M PBS buffer solution with 10 μM GSH, 10mM GSH or without GSH. The experiment was performed in an incubation shaker at 37°C at 100 rpm. At selected time intervals, 10 μL solution in the dialysis bag and 1mL medium outside the dialysis bag were withdrawn while same amount of fresh medium was added for replenishment. Outside medium was freeze-drying and add water to 10μL. All sample were then derivatized by using sugar-PMP(1-phenyl-3-methyl-5-pyrazolone) derivatization method to detect the 2-DG. [43, 44] Briefly, 10 μL sample solution was added in 50 μL 0.3M NaOH and then 50 μL 0.5 mol/L PMP solution is added. The mixture was incubated under 70°C for 1.5h. 0.4M HCl was then added to the mixture followed by adding chloroform to wash the excessive PMP. 100 μL upper layer liquid was taken out to add 100 μL HPLC acetic acid buffer mobile phase to prepare the final sample. The 2-DG derivative was measured by Waters e2695 HPLC system equipped with a Waters 2489 UV detector. 0.1M acetic ammonium /Acetonitrile (v/v=78:22) was used as eluent with the flow rate of 0.8 mL/min at 30 °C. 2DG-PMP derivative was detect by UV at 248 nm.

2.2.5. In Vitro Cytotoxicity Assay

Cytotoxicity assay was performed on 4T1.2 mouse breast cancer cell lines, CT26 mouse colon cancer cell line, 3LL mouse lung cancer cell line, PANC-2 mouse pancreatic cancer cell line, MCF-7 human breast cancer cell line, MDA-MB-231 human breast cancer cell line, HCT116 human colon cancer cell line and A549 human lung cancer cell line. Cells were seeded in 96-well plates at various densities respectively (1×10³ cells per well for 4T1.2, CT26, 3LL, PANC-2, HCT116, A549 and 5×10³ cells per well for MCF-7, MDA-MB-231) followed by 24 h of incubation in DMEM or RPMI 1640 with 10% FBS and 1% streptomycin/penicillin.

To evaluate the combinational effect of 2-DG and V9302, cells were treated with various concentrations of free 2-DG, free V9302, and the combination of both respectively for 48h. To examine the cytotoxicity of drug-loaded POEG-p-2DG micelles, free V9302, blank POEG-p-2DG and V9302/POEG-p-2DG (w/w ratio POEG-p-2DG:V9302=20:1) were incubated with cells for 48h before MTT assay was performed.

MTT assay and the calculation of cell viability were performed as described before. The anti-proliferation data for single drug and combination treatment were fitted to an inhibitory, normalized dose-response model with variable slope (Y = 100/ (1 + 10^((LogEC50-X) *HillSlope) (GraphPad Prism, San Diego, CA).

2.2.6. Real-time PCR

Real-time PCR studies were performed on 4T1.2 mouse breast cancer cell line, CT26 mouse colon cancer cell line, MDA-MB-231 human breast cancer cells and HCT116 human colon cancer cell line. 4T1.2, CT26 and HCT116 (2×10⁴ cells/well) or MDA-MB-231 (6×10⁴ cells/well) cells were seeded in 6-well plates followed by 24h of incubation in DMEM (4T1.2, MDA-MB-231) or RPMI 1640 (CT26, HCT116) containing 10% FBS and 1% streptomycin/penicillin. After 24 hours, the culture medium was replaced with the medium with 2% FBS containing free V9302, blank POEG-p-2DG, V9302/POEG-p-2DG, free 2-DG (0.5 mM) of the same amount of 2-DG as in POEG-p-2DG as well as free 2-DG of much higher concentration (10 mM). After 48 hours, total cellular RNA was extracted using the TRIzol lysis reagent. cDNA was generated from the purified RNA using QuantiTect Reverse Transcription Kit according to the manufacturer’s instructions. The cDNAs for different enzymes examined were amplified by PCR using the specific primers respectively (Table S1). Quantitative real-time PCR was performed using SYBR Green Mix on a 7900HT Fast Real-time PCR System. Relative target mRNA levels were analyzed using delta-delta-Ct calculations and normalized to GAPDH.

2.2.7. Analysis of Glucose and Glutamine Metabolism

The glutamine metabolism is determined by measuring the glutamine concentration intracellularly. The cells were first cultured under glutamine-deprived medium for 6h to remove all the endogenous glutamine. Then different drug treatments were applied, along with normal RPMI medium. After 24h, cells were trypsinized, gently washed three times with ice-cold PBS, and then resuspended in cold PBS. Afterwards, the cell suspension was sonicated on ice for 60s with an amplitude of 50%, and the resulting cell debris was removed by centrifugation at 14,000 rpm for 10 min at 4°C. The cell supernatant was transferred to a new microtube for immediate measurements. Glutamine measurement was determined using the EnzyChrom Glutamine Assay kit according to manufacturer’s protocol. The protein concentrations of cell extracts were measured using BCA protein assay. Intracellular glutamine levels were expressed as glutamine concentration normalized to protein concentration and control. The experiment was performed in triplicate.

The glucose metabolism is determined by measuring the glucose and lactate concentration in the cell culture medium. Cells for each experimental group were cultured in RPMI 1640 for 48h with different treatments. Then cell culture medium of each group was collected and determined by using Glucose Colorimetric/Fluorometric Assay Kit and Lactate Colorimetric/Fluorometric Assay Kit according to manufacturer’s protocol. Total protein amount of cell extracts was measured using BCA protein assay. Glucose uptake and lactate production were calculated by comparing with the glucose or lactate amount in fresh medium and then normalized to protein concentration and control. The experiment was performed in triplicates.

2.2.8. Western Blot

Western blotting was performed to evaluate the ASCT2 glycosylation in MDA-MB-231 and HCT116 cells. Cells grown in six-wells plates with 80% confluency were treated with free V9302, blank POEG-p-2DG micelles, V9302/POEG-p-2DG, and 10mM 2-DG respectively for 24h. Cells were washed twice with pre-cooled PBS and lysed in Pierce™ RIPA buffer for 40 min in 4°C. Protein concentrations were determined by BCA method, and equal amounts of total protein lysate were resolved on a 10% SDS-PAGE and subsequently transferred to a nitrocellulose membrane. Membranes were blocked with 5% nonfat milk in PBS for 1h prior to incubation with rabbit anti-ASCT2 primary antibody dissolved in PBST (DPBS with 0.1% Tween 20) overnight at 4°C. The blots were washed with PBST and then probed with goat antirabbit IgG for 1h at room temperature followed by chemiluminescence detection by SuperSignal™ West Fento Maximum Sensitivity Substrate. β-Actin or GAPDH was used as a loading control.

2.2.9. In vivo Biodistribution with Near-Infrared Fluorescence Imaging

DiR-loaded POEG-p-2DG micelles were similarly prepared as V9302-loaded POEG-p-2DG micelles at a POEG-p-2DG to DiR ratio of 20/1 (w/w). DiR-loaded POEG-p-2DG micelles were injected to 4T1 tumor bearing mice at a DiR dose of 1 mg/kg. At 4 hours and 24 hours, the mice were imaged by IVIS 200 system (Perkin Elmer, USA) at a 60s exposure time with excitation at 730 nm and emission at 835 nm. The mice were then sacrificed and perfused. Then major organs were dissected and subjected to ex vivo imaging.

For plasma PK study, DiR/POEG-p-2DG formulation was prepared as above. DiR-loaded POEG-p-2DG micelles were injected to Balb/c mice at a DiR dose of 1 mg/kg. Blood was withdrawn from retro-orbital plexus at 3 min, 10 min, 30 min, 1h, 2h, 4h, 8h, 12h post injection. Blood was immediately centrifuged at 12500rpm, 10min, 4°C and plasma was collected for imaging as described above.

2.2.10. In Vivo Therapeutic Study

The in vivo antitumor efficacy of the V9302-loaded POEG-p-2DG micelles was tested in a syngeneic 4T1.2 mouse breast cancer model. 4T1.2 cells (2×10⁵ in 20 μL PBS) were inoculated s.c. at the right mammary fat pad of female BALB/c mice. When the tumor volume reached ~ 50 mm3, mice were randomly divided into 6 groups (n=5), and treated via tail vein injection with DPBS, blank POEG-p-2DG micelles, V9302-loaded POEG-p-2DG micelles (Polymer: V9302 = 20:1(wt/wt), V9302: 60 mg/kg) and V9302/2-DG free drug combination (V9302: 60 mg/kg, 2DG: 120 mg/kg) through i.p. or i.v. injection respectively, at a V9302 dose of 60 mg/kg. The treatments were first conducted every two days for 2 times and then every 3 days for the rest 3 times. Tumor sizes were measured with the digital caliper every three days following the initiation of the treatment and calculated by the formula: (L×W²)/2, where L is the longest and W is the shortest in tumor diameters (mm). Body weights were also monitored for the indication of toxicity. On day 17 post injection, all mice were sacrificed, and tumor tissues were collected for weight, photography and H&E staining. All animal protocols were approved by Animal Use and Care Administrative Advisory Committee at the University of Pittsburgh and all animal studies were performed according to the guidelines approved by the Ethics Committee of University of Pittsburgh.

2.2.11. Histochemical Staining

After completion of the in vivo therapy study, tumor tissues and liver tissue were excised and fixed in PBS solution containing 10% formaldehyde, followed by embedment in paraffin. The paraffin-embedded tumor samples were sectioned into slices at 5 μm using an HM 325 Rotary Microtome. The slices were then stained with hematoxylin and eosin (H & E) for histopathological examination or ki67 immunostaining for cell proliferation by using Zeiss Axiostar plus Microscope (PA, USA).

2.2.12. ALT/AST and Creatinine Assessment

Mouse blood was collected after sacrifice and centrifuged (12500 rpm, 4°C), and serum was collected for blood biochemical assessment. ALT/AST were measured by ALT/SGPT liqui-UV assay kit or AST/SGPT liqui-UV assay kit following manufacturer ‘ s protocol. Creatinine was measured by QuantiChrom™ Creatinine Assay Kit according to manufacturer’s protocol.

2.2.13. Statistical Analysis

Statistical analysis was performed with two-tailed Student’s t-test between two groups and one-way analysis of variance (ANOVA) for multiple groups. p < 0.05 was considered statistically significant, and p < 0.01 was considered highly statistically significant. All analyses and graphs were generated with GraphPad Prism 7.

3. Result

3.1. Synergistic effect of V9302 and 2-DG on cancer cell growth

In order to assess the synergistic effect between V9302 and 2-DG, the proliferation inhibitory activity of V9302 and 2-DG, alone or in combination, was examined on breast cancer cell lines and colon cancer cell lines. As shown in Fig. 1, V9302 or 2-DG alone showed a concentration-dependent inhibition of proliferation in 4T1 breast cancer cell line(A). It is also apparent that the combination of the two drugs could result in a significant improvement in the level of inhibition of cell growth. Similar synergistic effects between 2-DG and V9302 were also found in CT26(B), MDA-MB-231(C), MCF-7(D) and HCT116(E) cell lines. Combination Index was then calculated to further assess the potential synergistic effect between V9302 and 2-DG through the equation CI = (d1/D₅₀1) +(d2/D₅₀2), with D₅₀1 or D₅₀2 being the IC50 of 2-DG or V9302 monotherapy respectively, while d1 being the concentration of V9302 required to achieve 50% killing effect of combination treatment at d2(2-DG) =0.6mM. The CI values of all tested cell lines were calculated to be less than one, an indication of the synergistic effect between 2-DG and V9302

Fig. 1.

Synergistic effect between V9302 and 2-DG in inhibiting the proliferation of tumor cells. (A-E) 4T1.2, CT26, MDA-MB-231, MCF-7, HCT116 cells were treated with various concentrations of free V9302 and free 2-DG. After 48 h, the cytotoxicity was determined by MTT assay. The experiments were performed in triplicate and repeated three times. Data are presented as means ± SD.

3.2. Synthesis and Characterization of the POEG-p-2DG micelles

The synthesis route of POEG-p-2DG is shown in the synthesis scheme 1. The structures of POEG-p-VBSS backbone and POEG-p-2DG were confirmed by ¹H-NMR (Fig. S1). The numbers of repeated POEG unit and VBSS unit were determined based on the peaks at area 6.0ppm-7.5ppm(d), 3.35ppm(a) and the conversion of two monomers at the end of polymerization(the monomer conversion was calculated based on the disappeared percentage of double bond peak of the monomer)[45]. The post-conjugation of 2-DG number was confirmed by the disappearance of hydrogen of carboxyl group (12.2 ppm, s, 1H) and was quantitatively determined by the ratio of hydrogens on benzene ring peaks (6.0ppm-7.5ppm, broad,4H) and those on newly appeared 2-DG peak (h, 4.34 ppm, m, 0.28H, h, 4.07 ppm, m, 0.36H; i, 1.27, m, ppm 0.62H). The post conjugation was also verified by GPC by comparing the molecular weight of POEG-p-VBSS and POEG-p-2DG (Fig. S2, Table S1). Based on NMR and GPC, each POEG-p-2DG molecule was calculated to contain an average 8 units of OEG₉₅₀ and 21 units of VBSS with an average of 7 units of 2-DG conjugated. The 2-DG accounts for around 10% molecular weight of POEG-p-2DG polymer.

Scheme 1.

Synthesis Scheme of POEG-p-2DG Conjugate

Blank POEG-p-2DG micelles or V9302-loaded POEG-p-2DG were then prepared by simple film hydration method. The hydrodynamic sizes of p-VBSS backbone micelles, blank POEG-p-2DG micelles or V9302-loaded POEG-p-2DG micelles were examined by dynamic light scattering as shown in Fig. 2A–2C. The polymer backbone POEG-p-VBSS without 2-DG conjugation formed micelles with a size around 100nm (Fig 2A), which is consistent with previous study[45]. However, after the conjugation of 2-DG to the POEG-p-VBSS polymer backbone, the major size peak of the resulting POEG-p-2DG micelles was drastically decreased to around 10–15 nm (Fig. 2B). This might be because the conjugation of highly water-soluble molecule 2-DG increases the hydrophilicity of the polymer and greatly facilitates the size reduction. We also observed another peak with a size of 120 nm, suggesting the presence of two populations. Interestingly, loading of V9302 into POEG-p-2DG micelles led to disappearance of the large-sized peak (Figure. 2C). This is likely because the hydrophobic interaction between V9302 and POEG-p-2DG polymer might help the micelle to form a more compact structure. TEM further shows the spherical morphology with homogenous size distribution for POEG-p-VBSS, blank POEG-p-2DG or V9302-loaded POEG-p-2DG micelles, which is consistent with the result of DLS analysis. The CMCs of POEG-p-VBSS, POEG-p-2DG and V9302/POEG-p-2DG were determined by Fluorescence or DLS (Fig. 2D–2F). The CMCs were in the order of POEG-p-VBSS> POEG-p-2DG> V9302/POEG-p-2DG. This is likely due to fact that POEG-p-VBSS formed a looser micelle structure compared to POEG-p-2DG and that incorporation of V9302 helped to facilitate the formation of an even more compact structure through hydrophobic-hydrophobic interaction between V9302 and POEG-p-2DG polymer. The CMC data were consistent with the data of size measurement. The relatively low CMC of V9302/POEG-p-2DG shall help to maintain the good stability of the micelles upon being diluted in blood stream after intravenous administration and prevent the early release before reaching to the tumor site.

Fig. 2.

In vitro Physicochemical characterization of Blank POEG-p-2DG and V9302 loaded POEG-p-2DG micelle. Size characterization of p-VBSS backbone micelles(A), (B) Blank POEG-p-2DG micelles and (C) V9302-loaded POEG-p-2DG micelles using TEM and Dynamic Light Scattering. (D-F) Critical micelle concentration of POEG-p-VBSS backbone, POEG-p-2DG and V9302 loaded POEG-p-2DG. (G) Cumulative V9302 release profile from POEG-p-2DG micelles and free V9302 for 48h. PBS was used as the release medium. V9302 concentration was fixed at 6 mg/mL (H) Cumulative 2-DG release profile from POEG-p-2DG micelles under different GSH concentration. POEG-p-2DG concentration was 60mg/mL. Values reported are the means ± SD for triplicate samples. Release curve is fitted by non-linear fit one phase decay model in GraphPad Prism 7.

3.3. In vitro Drug Release Study

The release profile of V9302 and 2-DG from V9302-loaded POEG-p-2DG micelles was investigated by using a dialysis method with PBS containing varying concentrations of GSH (0, 10mM). As shown in Fig. 2G, free V9302 was quickly diffused outside the dialysis bag. Around 70% of free V9302 was diffused out rapidly in 2 h and around 90% of V9302 was found to be outside of dialysis bag after 12 h. Meanwhile, the V9302 release from POEG-p-2DG micelles was significantly slower. In the absence of GSH treatment, only less than 20% of V9302 loaded in POEG-p-2DG was released out after 2 h, and the slow kinetics of release was extended for 48 h. However, upon exposure to 10 mM GSH, the release of V9302 from the POEG-p-2DG micelles was greatly accelerated: 20% more V9302 was released compared with the group without GSH. POEG-p-2DG was stable in PBS or upon exposure to low concentration (10 μM) of GSH, only small amounts of released 2-DG were detected (Fig. 2H). However, treatment of 10 mM GSH led to significant acceleration in the release of 2-DG from POEG-p-2DG. Release of 2DG from POEG-p-2DG may render formation of more loose micelles, which, in return, facilitate the release of V9302.

3.4. In vitro Cytotoxicity of V9302 loaded POEG-p-2DG Micelles

The in vitro cytotoxicity of V9302-loaded POEG-p-2DG micelles (micelles to drug ratio: 20:1 (w/w)) was tested in several cell lines. As shown in Fig. 3, V9302 inhibited the proliferation of 4T1 cancer cells in a concentration-dependent manner (A). Blank POEG-p-2DG micelles alone also showed modest cytotoxicity towards the cancer cells (A). However, V9302-loaded POEG-p-2DG showed decreased IC50 when compared to V9302 alone. Similar results were also observed in other types of cell lines (B-E). These data indicated that POEG-p-2DG might sensitize the response of cancer cells towards V9302 and achieve a synergistic effect (Table. S2), which is consistent with data from free drug combination (Fig. 1). This is likely attributed to the release of active 2-DG following the uptake of the micelles and the subsequent cleavage of self-immolative linker[35], resulting in synergistic interaction with V9302.

Fig. 3.

Cytotoxicity of V9302-loaded POEG-p-2DG micelles in 4T1.2 (A), MDA-MB-231 (B), CT26 (C), HCT116 (D), PANC-2 (E) and A549 (F) cell lines after 48 h treatment. Data are presented as the means ± SD for triplicate samples.

3.5. Effect of V9302-loaded POEG-p-2DG micelle on glucose and glutamine metabolism

It has been previously reported that the glucose consumption in tumor cells was significantly increased when glutamine metabolism was interfered[27–29]. We also observed a similar increase in glucose uptake after V9302 treatment in both 4T1 and CT26 cell lines. We then went on to examine whether V9302/POEG-p-2DG micelles could block the increased glucose metabolism induced by V9302. As expected, V9302 treatment led to a significant increase in both glucose uptake and lactate production (Fig. 4). The upregulation in glucose metabolism induced by V9302 was inhibited when V9302 was loaded in POEG-p-2DG micelles. However, POEG-p-2DG itself did not have significant impact on the basal levels of glucose uptake and lactate production. As a positive control, free 2-DG (10 mM) significantly inhibited the lactate production. Treatment with free 2-DG (10 mM) also led to a slight decrease in glucose uptake but this is not statistically significant. The more dramatic effect of POEG-p-2DG micelles on glucose uptake/lactose production upon co-treatment with V9302 might be due to increased susceptibility of the tumor cells to the inhibition by 2-DG when the glucose metabolism was upregulated.

Fig. 4.

V9302/POEG-p-2DG have inhibition effect on glucose metabolism and glutamine metabolism. V9302/POEG-p-2DG could downregulate compensatory glucose metabolism from (A) glucose uptake and (B) lactate production. (C) V9302/POEG-p-2DG can further inhibit the glutamine uptake. (D) POEG-p-2DG could downregulate the glycosylation of ASCT2. Data are presented as the means ± SD for triplicate samples. P values were generated by one-way ANOVA using the Tukey test for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001.

Then glutamine metabolism was examined by measuring glutamine uptake (Fig. 4C). In consistent with previous study, the uptake of glutamine was significantly inhibited by V9302. Interestingly, treatment with V9302/POEG-p-2DG led to a further decrease in glutamine uptake. We also noticed slight decreases in the uptake of glutamine following treatment with POEG-p-2DG or free 2-DG; however, this was not statistically significant. To understand the potential mechanism involved in the impact of POEG-p-2DG or 2-DG on the uptake of V9302, we examined the ASCT2 glycosylation following various treatments as glycosylated ASCT2 is more active than the non-glycosylated counterpart in glutamine transport. As shown in Fig. 4D, significant inhibition of ASCT2 glycosylation was observed following treatment with POEG-p-2DG, V9302/POEG-p-2DG, or free 2-DG, particularly the free 2-DG at a high concentration.

3.6. Biodistribution study

A preliminary PK study on DiR/POEG-p-2DG micelles was first performed in tumor-free C57BL/6 mice by examining the fluorescence intensity of plasma at different times following tail vein injection. DiR was loaded into POEG-p-2DG to form mixed micelles with small size (10–15 nm) and then i.v. injected into mice with free DiR as a control. Plasma was collected and then subjected to ex vivo imaging (Figure. S4) followed by quantitative measurements of fluorescence intensity (Fig. 5A). It is apparent that plasma from mice treated with DiR/POEG-p-2DG micelles had significantly higher fluorescence intensity compared to free DiR group at all time points examined.

Fig. 5.

Biodistribution and Plasma PK of POEG-p-2DG formulation. (A) The plasma fluorescence intensity at different times following i.v. injection of free DiR or DiR-loaded POEG-p-2DG micelles. (B) The whole-body fluorescence image at 24 h after intravenously injection of free DiR and DiR loaded in POEG-p-2DG micelle. (C) The organ fluorescence image at 24 h after intravenously injection of free DiR and DiR loaded in POEG-p-2DG micelle. (D-E) Quantified fluorescence intensity of different organ at 4h and 24h after intravenously injection of free DiR (D) and DiR loaded in POEG-p-2DG micelle (E). Scale Bar: 6.41×10⁸ to 1.14×10¹⁰. Values reported are the means ± SEM, n = 3.

The in vivo biofluorescence imaging was then used for the assessment of the biodistribution of DiR/POEG-p-2DG micelles in tumor-bearing mice after systemic administration. Whole body imaging shows that DiR loaded into POEG-p-2DG micelles was mainly concentrated at tumor site while free DiR was largely found in the area of liver (Fig. 5B). Fig. 5C shows the ex vivo imaging of tumors and other major organs that were removed from the mice after the completion of whole-body imaging. In consistent with whole body imaging, strong signals were observed in tumor tissues treated with DiR/POEG-p-2DG micelles, significantly higher than those in other normal orangs and tissues. We also observed significant uptake of the nanoparticles by liver and spleen. The fluorescence signals in tumors and other organs were significantly lower following injection of free DiR. In addition, the signals were mainly concentrated in the liver (Fig. 5D, E). All these data suggest that free DiR was quickly eliminated while the DiR loaded into POEG-p-2DG micelles exhibited extended half-life in the circulation and were preferentially accumulated at tumor sites.

3.7. In vivo Therapeutic Study

The in vivo tumor growth inhibitory activity of V9302/POEG-p-2DG was investigated in a highly aggressive syngeneic murine breast cancer model 4T1.2 (s.c. at mammary fat pad). As Fig. 6 shows, blank POEG-p-2DG showed minimal antitumor activity, this might be due to the limited dose of POEG-p-2DG that was used to deliver V9302. I.V. injection of V9302 and 2-DG in combination also exhibited limited improvement of antitumor efficacy. I.P. injection of V9302 alone showed modest antitumor efficacy and combination of V9302 with 2-DG via i.p. route led to an enhancement in antitumor activity although it was not statistically significant. Among all treatment groups, V9302/POEG-p-2DG group was most effective in inhibiting the tumor growth. The weights of tumor tissues that were collected at the end of the experiment were shown in Fig. 6B, which was consistent with data of tumor growth curves. Also, the ki67 staining further confirmed the tumor growth inhibition effect of V9302/POEG-p-2DG micelles (Fig. 6C).

Fig. 6.

Antitumor activity of blank POEG-p-2DG micelles, V9302, Free drug combination of V9302 and 2-DG via i.p. or i.v. injection and V9302 loaded POEG-p-2DG micelles in a syngeneic murine breast cancer model (4T1.2). Five injections were given on day 1, 3, 5, 8 and 11. (B) Weights of tumors collected from different groups at the end of experiment. (C) Histological analyses of tumor tissues collected from different groups at the end of in vivo therapeutic experiment using ki67 staining. Scale bar = 50 μm. Ki67 positive cells were quantified by using ImageJ software. (D) Changes of body weight in mice receive different treatment. Values reported are the means ± SEM, n = 5. P values were generated by one-way ANOVA using the Tukey test for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001 (vs control).

Body weights were also monitored together with the tumor growth (Fig. 6D). Treatment with blank POEG-p-2DG or V9302/POEG-p-2DG had no obvious effect on body weights compared to control group. However, significant decreases in body weights were observed in mice treated with V9302&2-DG free drug combination via i.p. route. We also noticed significant decreases in body weights after three injections of V9302&2-DG free drug combination via i.v. route. But, after increasing the injection interval, the body weights of these mice grew back to normal range while the body weights continued to decrease in mice receiving combination treatment via i.p. injection.

4. Discussion

The metabolic reprogramming plays an important role in cancer initiation and progression, and suggests an opportunity for cancer targeted therapy and precision medicine[46]. It is well known glucose and glutamine are two most important nutrients used by cancer cells for their proliferation and growth. However, monotherapy targeting each metabolism alone has proven to be of limited effectiveness in controlling the cancer growth [47]. One possible reason is that the complicated metabolic network in cancer provides a mechanism of compensation when one metabolic pathway is inhibited [28, 48]. Therefore, a combination strategy that simultaneously targets multiple metabolic pathways shall represent a more effective treatment for cancer.

V9302 is a selective and potent small molecule antagonist of glutamine transporter ASCT2, which was reported to have significant impact on glutamine metabolism and tumor growth. However, its poor aqueous solubility may hinder its clinical development. Also, in consistent with previous reports[27–29], we overserved that inhibition of glutamine metabolism by V9302 resulted in compensatory upregulation of glucose metabolism, such as increases in glucose uptake (Fig. 4) and the mRNA expression levels of several glucose metabolism-related genes (Fig. S5). Hence, combination of V9302 with an inhibitor of glycolysis shall represent an effective strategy to improve the therapeutic outcome.

2-DG is a glucose analog that is widely used for glycolysis inhibition. Our study showed a synergistic effect with the combination of V9302, which might be attributed to the blockade of the compensatory upregulation of glucose metabolism induced by V9302 treatment. The underlying mechanism for the compensatory metabolic reprogramming is unclear and requires more studies in the future. Interestingly, besides glucose metabolism inhibition, 2-DG appeared to be able to further decrease the glutamine uptake when combined with V9302. One of the possible mechanisms is that 2-DG can block the glycolysis pathway, which can further interfere with the glycosylation process of ASCT2. Without sufficient glycosylation, the glutamine uptake function of ASCT2 is likely to be undermined [49]. Nonetheless, the inhibitory effect of 2-DG on glutamine uptake was only observed in the presence of V9302 co-treatment. This might be due to the fact that the decreased glycosylation following 2-DG alone is not sufficient to compromise the glutamine uptake due to the existence of compensatory mechanisms such as upregulation of ASCT2 expression[48, 49]. On the other hand, inhibition of ASCT2 by V9302 may lead to sensitization of cells to 2-DG treatment with respect to its impact on glutamine uptake through inhibition of ASCT2 glycosylation.

Recently, nanocarriers have been employed to improve the delivery of metabolism inhibitors to tumors to improve the efficacy. Amira Elgogary et al. reported the use of PEG-PLGA nanoparticle for the delivery of BETES, a prototype GLS1 inhibitor. Moreover, further improvement in antitumor efficacy was demonstrated in combination with free metformin that inhibits glycolysis [26]. However, no reports have been published on codelivery of glucose and glutamine inhibitors to tumors. It has been challenging to co-formulate the two different inhibitors as one is often water soluble and the other one is water insoluble.

As a strategy to facilitate the co-delivery of the highly water-soluble 2-DG and the water-insoluble V9302, we have developed a 2-DG prodrug-based dual-functional polymeric carrier, POEG-p-2DG that is capable of codelivery of V9302. One common issue with the pro-drug carrier is the ineffective release of the parent drug from the conjugated polymer. In this study, we introduced 4,4′-dithiodibutyric acid as the self-immolative linker between 2-DG and polymer backbone to facilitate the redox-responsive release of 2-DG in the cytoplasm[35]. Moreover, this hydrophobic linker could facilitate the loading of V9302 through hydrophobic interaction. POEG-p-2DG could readily form micelles via a simple film hydration method. Interestingly, coupling of 2-DG to the polymer backbone led to a drastic decrease in the particle size from 100 to 15 nm. Loading of V9302 into the micelles led to formation of more compact and more homogeneously distributed nanoparticles. The very small-sized drug-loaded micelles shall greatly facilitate extravasation from leaky tumor vasculature and accumulation at tumor site through EPR effect. Furthermore, after reaching tumor site, the ultra-small sizes shall facilitate the penetration of the micelles into hypoxic core of tumor[45, 50, 51]. It should be noted that tumor cells in hypoxic environment might have better response to 2-DG due to increased dependence on anaerobic glycolysis[52], which warrants more studies in the future.

POEG-p-2DG retains the pharmacological activity of the parent 2-DG as evident from its activity in inhibiting the basal level of glucose metabolism and in reversing the V9302-induced upregulation of glucose metabolism. More importantly, POEG-p-2DG could also inhibit glycosylation of ASCT2 and further decrease the glutamine uptake when combined with V9302. The more dramatic impact of POEG-p-2DG on glucose or glutamine metabolism that is seen in presence of V9302 co-treatment might be due to sensitization of tumor cells to POEG-p-2DG treatment when the function of ASCT2 is inhibited.

POEG-p-2DG-formulated V9302 demonstrated significantly enhanced antitumor activity both in vitro and in vivo. Particularly, codelivery of V9302 via POEG-p-2DG led to effective growth inhibition of tumor in an aggressive murine breast cancer model (4T1.2), much more effectively than V9302 monotherapy or V9302 & 2-DG free drug combination. 2-DG could be effectively released from POEG-p-2DG following delivery to tumor cells and the synergistic effect between the released 2-DG and V9302 shall contribute to the improved antitumor activity. The improved delivery of both 2-DG and V9302 via POEG-p-2DG nanocarrier shall also play an important role.

Conclusion

We have developed a well-characterized POEG-p-2DG prodrug-based micellar nanocarrier for efficient delivery of V9302, a water-insoluble inhibitor of glutamine uptake transporter. POEG-p-2DG well retained the pharmacological activity of 2-DG. V9302 loaded in POEG-p-2DG micelles exhibited slow release kinetics in vitro. Combination of V9302 with POEG-p-2DG led to inhibition of the compensatory metabolic shift to glucose metabolism that was induced by V9302 treatment. It also led to more effective inhibition of glutamine uptake likely due to POEG-p-2DG-mediated inhibition of ASCT2 glycosylation. V9302/POEG-p-2DG showed more cytotoxicity towards cultured cancer cells than V9302 alone. More importantly, systemic administration of V9302 formulated in POEG-p-2DG micelles resulted in significantly improved antitumor activity.

Table 1.

Synergistic Antiproliferative Activity of V9302 and 2-DG in Cancer Cells

	d1(mM)	D501 (mM)	d2(μ M)	D502(μ M)	CI
MCF-7	0.6	9.02±0.78	9.48±0.89	11.83±0.75	0.868
MDA-MB-231	0.6	2.65±0.60	6.44±0.81	18.64±0.53	0.572
CT26	0.6	11.05±1.03	7.66±0.46	10.09±0.44	0.816
4T1.2	0.6	3.99±0.53	12.10±0.59	16.79±0.56	0.871
HCT116	0.6	15.47±1.37	11.09±0.41	12.69±0.31	0.913

Combination Index (CI) of simultaneous treatment of V9302 and 2-DG in MCF-7, MDA-MB-231, CT26, 4T1.2, HCT116 cells. Cells were treated with a combination of 2-DG and V9302 and cell viability was determined by MTT assay. The CI was calculated by the formula: CI=(d1/D₅₀1) +(d2/D₅₀2), where D₅₀1 is the concentration of 2-DG required to produce 50% effect alone, and d1 is the concentration of 2-DG required to produce the same 50% effect in combination with d2. D₅₀2 is similarly the concentration of V9302 required to produce 50% effect alone, and d2 is the concentration of V9302 required to produce the same 50% effect in combination with d1. The CI values are interpreted as follows: <1.0, synergism; 1.0, additive; and >1.0, antagonism. Each experiment was done in triplicate

Table 2.

Physicochemical properties table of blank POEG-p-2DG micelle and V9302 loaded in to POEG-p-2DG micelles

Micelle	Number of 2DG units	Mass Ratio	CMC (mg/mL)	Size (nm) b	PDI	DLE (%) c	DLC (%) d
POEG-p-VBSS	-	-	0.102	94.84±14.23	0.270±0.009	-	-
POEG-p-2DG	7a	-	0.020	19.08±0.14	0.461±0.012	-	-
V9302/POEG-p-2DG	7a	20 : 1	0.008	14.28±0.25	0.157±0.032	95.4±3.3	4.6±0.2

^aCalculated according to ¹H NMR and GPC

^bAccording to Measurement data of dynamic scattering particle sizer.

^cDLE: V9302 loading efficiency

^dDLC: V9302 loading capacity

Values reported are the means ± SD for triplicate samples

Statement of Significance.

Unique cancer cell’s metabolism profile denotes a new therapeutic strategy. V9302 is a recently reported glutamine metabolism inhibitor that shows promising antitumor activity. However, its poor waster solubility and tumor cell’s compensatory metabolic network may limit its potential clinical application. 2-Deoxyglucose(2-DG) is a widely used glycolysis inhibitor. However, its clinical application is hindered by low efficacy as monotherapy. Thus, in this study, we developed a redox-sensitive, 2-DG-based prodrug polymer, as a dual-functional carrier for co-delivery of V9302 and 2-DG as a combination strategy. V9302 loaded POEG-p-2DG micelle showed significantly improved antitumor activity through synergistic targeting of both glutamine and glycolysis metabolism pathway. More interestingly, POEG-p-2DG itself further facilitates inhibition of glutamine metabolism, likely through inhibition of ASCT2 glycosylation.