Carrier-Enhanced Anticancer Efficacy of Sunitinib-Loaded Green Tea

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Carrier-Enhanced Anticancer Efficacy of Sunitinib-Loaded Green Tea-Based Micellar Nanocomplex beyond Tumor-Targeted Delivery Nunnarpas Yongvongsoontorn, Joo Eun Chung, Shu Jun Gao,† Ki Hyun Bae, Atsushi Yamashita, Min-Han Tan,* Jackie Y. Ying,† and Motoichi Kurisawa* Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, #07-01, Singapore 138669 S Supporting Information *

ABSTRACT: Although a few nanomedicines have been approved for clinical use in cancer treatment, that recognizes improved patient safety through targeted delivery, their improved efficacy over conventional drugs has remained marginal. One of the typical drawbacks of nanocarriers for cancer therapy is a low drug-loading capacity that leads to insufficient efficacy and requires an increase in dosage and/or frequency of administration, which in turn increases carrier toxicity. In contrast, elevating drug-loading would cause the risk of nanocarrier instability, resulting in low efficacy and off-target toxicity. This intractable drug-to-carrier ratio has imposed constraints on the design and development of nanocarriers. However, if the nanocarrier has intrinsic therapeutic effects, the efficacy would be synergistically augmented with less concern for the drug-to-carrier ratio. Sunitinib-loaded micellar nanocomplex (SU-MNC) was formed using poly(ethylene glycol)-conjugated epigallocatechin-3-O-gallate (PEGEGCG) as such a carrier. SU-MNC specifically inhibited the vascular endothelial growth factor-induced proliferation of endothelial cells, exhibiting minimal cytotoxicity to normal renal cells. SU-MNC showed enhanced anticancer effects and less toxicity than SU administered orally/intravenously on human renal cell carcinoma-xenografted mice, demonstrating more efficient effects on anti-angiogenesis, apoptosis induction, and proliferation inhibition against tumors. In comparison, a conventional nanocarrier, SU-loaded polymeric micelle (SU-PM) comprised of PEG-b-poly(lactic acid) (PEG-PLA) copolymer, only reduced toxicity with no elevated efficacy, despite comparable drug-loading and tumortargeting efficiency to SU-MNC. Improved efficacy of SU-MNC was ascribed to the carrier−drug synergies with the highperformance carrier of PEG-EGCG besides tumor-targeted delivery. KEYWORDS: cancer therapy, nanocarrier, epigallocatechin-3-O-gallate, sunitinib, synergistic effect

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One of major drawbacks is their low drug-loading capacity, typically ∼10% (w/w)13,14 that results from a large amount of carrier, necessary to stably encapsulate the drug. The low drugloading capacity requires an increased dosage and/or frequency of administration to deliver enough drug amount at the specific sites. As most carriers are just excipients for delivering drugs and not therapeutically active, their use in large quantities can cause carrier-induced toxicity associated with their metabolism and elimination.5,15 Although nano-

umerous nanocarrier-based drug delivery systems have been developed for cancer treatment, enabling prolonged systemic circulation and targeted delivery to minimize the systemic side effects associated with conventional drug formulations.1−3 However, only a limited number of nanocarriers have been approved for clinical use. Although improvements in safety and morbidity have led to clinical approval, the effectiveness of many of these nanocarriers remains modest, offering only marginal improvements compared to conventional drugs.4−8 Despite research advancements in this area with various modifications on the nanocarrier platform to improve efficacy,9−12 achieving desirable effectiveness still remains an issue for clinical success. © XXXX American Chemical Society

Received: January 17, 2019 Accepted: July 1, 2019 Published: July 1, 2019 A

DOI: 10.1021/acsnano.9b00467 ACS Nano XXXX, XXX, XXX−XXX

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Figure 1. SU-MNC formed by self-assembly of SU and PEG-EGCG inhibits VEGF-dependent proliferation of endothelial cells sparing normal cells. (A) Schematic diagram of SU-MNC self-assembled from SU and PEG-EGCG. (B) 1H NMR spectra of SU (4 mg/mL), PEGEGCG (4 mg/mL), and a mixture of SU (4 mg/mL) and PEG-EGCG (1, 2, and 4 mg/mL, respectively) in D2O. (C) TEM image of SU-MNC (scale bar = 50 nm). (D) CMC determined by light scattering intensity of SU-MNC and empty MNC (PEG-EGCG alone) with varying concentrations (n = 3, mean ± SD). The dashed lines indicate CMC of SU-MNC and empty MNC, respectively. (E) Hydrodynamic diameters of SU-MNC upon addition of Triton X-100, Tween 20, urea, and NaCl (1−50 mM) (n = 3, mean ± SD). (F) Antiproliferative effect of SU-MNC and SU on HUVEC cultured in normal or VEGF-dependent growth condition (n = 5, mean ± SD). (G) Cytotoxicity of SU-MNC or SU on HRPTEC at various SU concentrations (n = 5, mean ± SD).

carriers can overcome the systemic toxicity of conventional drugs by their propensity for accumulation in specific sites, the carrier-induced toxicity would place additional hurdles for effective treatment. To elevate drug-loading, reduction/disuse

of the carrier has been attempted by prodrug or drug selfassembly systems,16−21 nevertheless, they have mostly led to problems of instability in the blood circulation, leading to low efficacy and off-target toxicity, and the limited applications of B


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anticancer efficacy and reduced toxicity, compared to SU administered orally (conventional treatment) or intravenously, demonstrating synergistically enhanced efficacy as well as tumor-targeted delivery facilitated by the carrier. Carrierenhanced efficacy was systematically analyzed in comparison with a conventional nanocarrier, SU-loaded PEG-PLA polymeric micelles (SU-PM), which enabled only reducing toxicity with no elevated efficacy despite their comparable drug-loading and tumor-targeted delivery. SU-MNC efficacy was also compared with free SU at equivalent intratumoral SU amounts to evince the carrier contribution toward improved efficacy. This report systematically and quantitatively investigates the drug−carrier synergistic efficacy and elucidates on histological and molecular mechanisms. This study shows that EGCG-based nanocarriers would provide an opportunity for potential improvement in therapeutic efficacy of the nanocarrier platform, beyond the improvements in patient safety presently conferred by tumor-targeted delivery.

covalent modifications of drugs. This intractable issue of drugto-carrier ratios has imposed constraints on the design and development of nanocarriers. These difficulties associated with the drug-to-carrier ratio could be challenged through the use of an innovative carrier that has intrinsic anticancer effects, which would augment the anticancer efficacy in combination with drugs besides the sitespecific accumulation. Such an approach would offer superior therapeutic outcomes and/or dosage reduction, where the drug-to-carrier ratio would be of less concern because all of the ingredients composing the system would display therapeutic activities in combination. In addition, the combinational mechanism of the drug and the carrier would improve efficacy against drug resistance and tumor heterogeneity, which hinder clinical outcomes when cancer treatments rely on a single therapeutic agent.22 It will be a distinguishing approach from the co-delivery or combination of multidrugs that still need the aid of the conventional carriers for stabilization and targeted delivery of drugs. To effectively realize the therapeutic carrier, the carrier should ideally have low toxicity on healthy cells/ organs and efficient binding properties to various drugs for general applicability. EGCG, a major component of green tea, has been shown to possess anticancer effects.23,24 The therapeutic effects of EGCG are attributed to its binding property to various bioactive molecules via noncovalent bonds.25−27 We have pioneered the design of nanocarriers comprised of EGCG derivatives for anticancer drug delivery.28 These micellar nanocarriers stably encapsulated anticancer proteins and achieved tumor-selective delivery, dramatically increasing anticancer efficacy from the combinational effects of the carrier and protein drugs, as compared to the drug alone. The complexation of EGCG moieties, the driving force that forms the micellar nanocomplex, was also applicable with the small molecular drugs such as doxorubicin achieving high drugloading and stability.29 We found that the EGCG-based carriers displayed varied interactions and combinational effects depending on the structural properties and the effects/toxicity mechanisms of the individual drugs. SU is a tyrosine kinase inhibitor used as the first-line treatment of advanced human renal cell carcinoma (HRCC), the most common type of adult kidney cancer with the most lethal urologic malignancy.30−32 SU displays anticancer effects through anti-angiogenesis and apoptosis induction by targeting tyrosine kinases receptors, including vascular endothelial growth factor (VEGF) receptors (VEGFRs) and plateletderived growth factor receptors (PDGFRs).33 However, SU treatment often needs to be reduced in dosage and discontinued due to severe systemic side effects.34 For this reason, effective and safe SU delivery systems are urgently required for SU treatment. In recent years, a few conventional nanocarriers have been attempted for SU delivery, but they did not show improved efficacy compared to SU.35,36 EGCG has been demonstrated to induce apoptosis and suppress the migration/invasion of HRCC cells37,38 and form precipitates with SU readily.39 These findings have further motivated us to establish the SU delivery system using the EGCG-based nanocarrier for HRCC treatment. Herein, we report SU-MNC formed by the self-assembly of SU and poly(ethylene glycol)-conjugated epigallocatechin-3-O-gallate (PEG-EGCG), wherein the SU and EGCG moiety favorably formed a core complex surrounded by a hydrated PEG shell (Figure 1A). The SU-MNC displayed significantly superior

RESULTS AND DISCUSSION SU-MNC Formation by the Self-Assembly of SU and PEG-EGCG. PEG-EGCG was synthesized through the nucleophilic addition reaction of thiol-functionalized PEG to the pyrogallol moiety of EGCG (Figure S1). The interaction between SU and PEG-EGCG was assessed by 1H NMR analysis. In the mixture of SU and PEG-EGCG, 1H signal of SU (H-2, δ = 2.2 ppm) was shifted downfield splitting into a doublet, as PEG-EGCG concentrations increased (Figure 1B), which was similarly observed in SU and EGCG mixtures (Figure S2). These results evinced proximal intermolecular interaction between SU and the EGCG moiety of PEG-EGCG. SU and PEG-EGCG self-assembled into the MNC through the SU−EGCG interaction by hydrating a film of SU and PEG-EGCG mixture. SU-MNC formation was systematically optimized by varying PEG-EGCG/SU (w/w) in feed and hydration temperature (Figure S3). Hydration temperatures had a significant influence on the size and drug-loading capacity of SU-MNC. Hydration at higher temperature resulted in smaller size and higher drug-loading, suggesting the formation of more compact structures with increased drugloading via enhanced interaction of SU−EGCG at higher temperature. The resulting SU-MNC showed nearly neutral surface charge, implying that a PEG shell covered a SU-loaded core. The SU-MNC formed with PEG-EGCG/SU (w/w) of 4 in feed at 45 °C was selected for further investigation. The structural characteristics and a transmission electron microscopy (TEM) image of the optimized SU-MNC are shown in Table S1 and Figure 1C, respectively. The critical micelle concentration (CMC) of SU-MNC was determined by comparing it with the empty MNC (PEGEGCG alone) through measuring the light scattering intensity.40 The CMC (16 μg/mL) of SU-MNC was 8-fold lower than that of the empty MNC (125 μg/mL) (Figure 1D). This finding illustrated that the strong intermolecular SU− EGCG interaction drove the SU-MNC formation with the encapsulation of SU and resulted in higher thermodynamic stability compared to empty MNC. SU-MNC was readily dissociated by Triton X-100 and Tween 20, but urea and NaCl did not affect its structure (Figure 1E), proving that hydrophobic interaction played a primary role in SU-MNC formation. SU release from SU-MNC was investigated in the presence of 10% mouse serum at 37 °C. The MNC released C


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Figure 2. SU-MNC shows enhanced anticancer efficacy and reduced toxicity on human kidney cancer cell-xenograft models. (A,C) Relative tumor volume and (B,D) relative body weight of (A,B) ACHN- and (C,D) A498-xenograft mice treated with saline (control, i.v., twice/ week), SU (5 mg/kg, i.v., twice/week), SU (40 mg/kg, p.o., daily), SU-MNC (5 mg/kg, i.v., twice/week), or a combination (i.v., twice/week) of SU (5 mg/kg) and empty MNC (equivalent PEG-EGCG) for 35 days (n = 10−12, mean ± SEM). (E) Tumor microvascular density, (F) tumor cell apoptosis induction, and (G) proliferation in ACHN-xenograft tumors harvested at day 56 (n = 3, mean ± SEM). (H,I) Biodistribution of SU and SU-MNC in (H) ACHN- and (I) A498-xenografted mice at 8 h post-injection (i.v.) with an equivalent SU dose (5 mg/kg) (n = 6, mean ± SEM). (J) ALT, (K) ALP, and (L) AMY concentrations of athymic nude mice treated with saline (control, i.v., twice/ week), SU (5 mg/kg, i.v., twice/week), SU (40 mg/kg, p.o., daily), or SU-MNC (5 mg/kg, i.v., twice/week) for 35 days (n = 5, mean ± SEM). *p < 0.05; **p < 0.01, ***p < 0.005; ****p < 0.001; N.S. (not significant) versus control group.

one of the significant challenges, as it may lead to disintegration and premature release of drugs during systemic circulation.41 SU-MNC maintained its size against 4000-fold immediate dilution in 10% mouse serum solution at 37 °C (Figure S4B), underlining its high stability for the prevention

SU in a sustained fashion, which reached 100% in 48 h (Figure S4A). For drug carrier systems formed by a noncovalent selfassembly, structural integrity against the severe dilution that would occur immediately upon intravenous administration is D


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HRCC-xenograft models: ACHN (wild-type von Hippel− Lindau (VHL)) and A498 (VHL mutations):47 saline (control, intravenous (i.v.), twice/week), SU (5 mg/kg, i.v., twice/ week), SU gavage (40 mg/kg, per os (p.o.), daily), SU-MNC (5 mg/kg of SU, i.v., twice/week), and a combination (i.v., twice/week) of SU (5 mg/kg) and empty MNC (equivalent PEG-EGCG). 40 mg/kg/day of SU (p.o.), a proven effective dose in various tumor-xenografted mice, was compared as a conventional treatment.48 The progression of ACHN-xenograft tumors was significantly inhibited by SU-MNC, whereas SU (i.v.) showed no efficacy at the same dose (Figure 2A). SU (p.o.) showed comparable efficacy to SU-MNC. However, severe weight loss was observed during the treatment with SU (p.o.) (Figure 2B), resulting in 25% of mice sacrificed due to the regulated termination criterion (>20% body weight loss) (Figure S8A). Also severe skin discoloration, one of major side effects reported for patients under conventional SU oral treatment,34 was observed over the whole bodies of all the mice during SU (p.o.) treatment. Taking account of the dosage and administration frequency, SU-MNC (5 mg/kg x 10 times) was evaluated to enable the attainment of comparable efficacy to SU (p.o.) (40 mg/kg x 35 times) with a 28-fold lower dose without any toxicity symptoms (body weight loss, tachypnoea/ dyspnoea, lethargy, diarrhea, rashes, discoloration, abnormal activity, or death). The empty MNC alone showed no efficacy at the equivalent dose (Figure S9A). For comparison, the combination of SU (i.v.) and EGCG (i.v.) showed no tumor size reduction but induced significant body weight loss (Figure S9). Anticancer efficacy of SU-MNC was further investigated on an A498-xenograft model harboring VHL mutations that lead to increased resistance to SU by upregulating VEGF and PDGF expression, resulting in highly vascularized tumors.47 SU-MNC significantly retarded tumor growth, while SU (i.v.) and SU (p.o.) failed to suppress the tumor progression (Figure 2C). Although SU (p.o.) with a 28-fold higher dose suppressed tumor growth comparably to SU-MNC during the treatment period, the tumor growth immediately accelerated after the treatment was halted. Also, major body weight loss was induced during the treatment period, which remained even after a halt in the treatment (Figure 2D). SU-MNC efficiently inhibited tumor growth without any toxicity symptoms even on an A498-xenograft tumor, where SU (p.o.) efficacy was attenuated compared to ACHN-xenograft tumors. As observed on the ACHN-xenograft model, the empty MNC alone showed no efficacy at the equivalent dose (Figure S10). To investigate the mechanism responsible for anticancer efficacy of SU-MNC, the tumors harvested at the end point (day 56) were histologically analyzed using CD34 (angiogenesis marker), TUNEL (apoptosis marker), and Ki67 (proliferation marker) (Figure S11). In ACHN tumors, SUMNC significantly decreased intratumoral microvascular density, whereas no decrease was observed by SU (i.v.) (Figure 2E). SU-MNC was found much more effective than SU (p.o.) with a 28-fold lower dose. Interestingly, the combination of SU and empty MNC also showed significantly higher effects than SU (p.o.) as well as SU (i.v.), implying that the carrier played an important role in SU-MNC efficacy. In addition, SU-MNC more effectively induced apoptosis and proliferation inhibition of tumor cells than SU (p.o.) and SU (i.v.) (Figure 2F,G). Similarly in A498 tumors, SU-MNC demonstrated superior effects on anti-angiogenesis, apoptosis induction, and proliferation inhibition against tumors over SU

of premature drug release by disassembly when diluted upon systemic injection or infusion. SU-MNC showed high colloidal stability in aqueous medium for the whole period of more than 4 months observation at 4 and 25 °C (Figure S4C) and preserved its structural integrity when reconstituted in 0.9% NaCl after lyophilization (Figure S4D). The lyophilized SUMNC exhibited high stability and maintained its size during the storage, when observed for up to 2 months at 25 °C and 1 year at 4 and −25 °C. The light scattering intensity (in kcps (kilo count-per-second)) monitored at the size measurements was also consistently retained, demonstrating that no precipitation occurred (Table S2). The excellent stability of SU-MNC in both liquid and lyophilized formulation was indicative of its advantages for long-term storage and preparation at high concentrations. Specific Inhibition of VEGF-Induced Proliferation of Endothelial Cells. We investigated the inhibitory effects of SU-MNC on the proliferation of human umbilical vein endothelial cells (HUVECs) in normal or VEGF-induced growth conditions to simulate the tumor-associated endothelial microenvironment.42 SU-MNC showed higher antiproliferative effects when VEGF-induced, compared to normal conditions, demonstrating that SU-MNC exerted a more robust antiproliferative effect when VEGF signaling pathways were activated (Figure 1F). SU also showed a similar propensity. However, the IC50 (1.17 μg/mL) of SU-MNC under normal conditions was higher than the IC50 of SU (0.68 μg/mL), implying less toxicity in VEGF-inactivated conditions, whereas IC50s in the VEGF-induced growth conditions were comparable for both SU-MNC (0.044 μg/mL) and SU (0.035 μg/ mL). Notably, PEG-EGCG alone showed antiproliferative effects when VEGF-induced but not under normal growth conditions, which was not observed by EGCG in the range of concentrations tested (Figure S5). The antiproliferative effects of PEG-EGCG upon VEGF activation were examined on a molecular mechanism by analyzing the gene expression and protein secretion of interleukin-8 (IL-8), a pro-angiogenic factor upregulated by VEGF activation, that plays a major role in the proliferation, migration, and tube formation of endothelial cells.43−46 PEG-EGCG effectively decreased mRNAs expression of IL-8 from VEGF-stimulated HUVECs in a concentration-dependent manner, whereas EGCG showed no effect in the range of tested concentrations (Figure S6A). IL-8 protein secretion from VEGF-stimulated HUVECs was also significantly suppressed by PEG-EGCG with greater effects than that of EGCG (Figure S6B). These results illustrated the superior inhibitory effects of PEG-EGCG on the VEGF-induced signaling pathway over EGCG. Moreover, SU-MNC exhibited minimal cytotoxicity on primary human renal proximal tubule epithelial cells (HRPTECs), while SU substantially killed cells at equivalent concentrations (Figure 1G). PEG-EGCG did not affect the cell viability of HRPTEC up to a concentration much higher than the equivalent concentrations where EGCG killed the cells severely (Figure S7). These results revealed that SU-MNC effectively inhibited VEGF-induced endothelial cell proliferation, which plays a critical role in angiogenesis, and displayed a significant reduction in cytotoxicity on human normal renal cells and human endothelial cells under normal growth conditions when compared to SU. Enhanced Anticancer Efficacy and Reduced Toxicity on HRCC-Xenograft Models. Anticancer efficacy and toxicity profiles of SU-MNC were investigated on two E


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Figure 3. SU-MNC achieves a wider therapeutic window than SU. (A,D) Relative tumor volume, (B,E) relative body weight, and (C,F) survival rate by treatment (i.v., twice/week) of (A−C) SU or (D−F) SU-MNC at various equivalent SU doses on ACHN-xenografted mice. Survival cutoff criteria included regulated euthanasia at >20% body weight loss, >2 cm tumor diameter, or tumor ulceration (n = 12, mean ± SEM). *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001; N.S. (not significant) versus control group.

tumor accumulation of SU-MNC was considered to facilitate the reduced systemic toxicity and play a part in improved anticancer efficacy over SU. Toxicity was further evaluated with treatments of saline (control, i.v., twice/week), SU (5 mg/kg, i.v., twice/week), SU (40 mg/kg, p.o., daily), and SU-MNC (5 mg/kg, i.v., twice/ week). Blood chemistry analysis at the end of the treatment (day 35) revealed that the mice treated with SU (p.o.) showed elevated levels of alanine aminotransferase (ALT), alkaline phosphatase (ALP), and amylase (AMY), indicating the toxicity of SU (p.o.) in the liver and pancreas (Figure 2J− L). This was in agreement with the abnormality observed in patients under the conventional SU oral treatment.49 Contrarily, the mice treated with SU-MNC showed comparable ALT, ALP, and AMY levels to those of healthy mice, demonstrating an improved safety compared with conventional SU treatment. Hematological studies and blood chemistry testing of the SUMNC treatment did not show any abnormality (Table S3). Improved Therapeutic Window. Anticancer efficacy and toxicity of SU (5−100 mg/kg) and SU-MNC (2−100 mg/kg of equivalent SU) were evaluated to determine the therapeutic window that allows effective treatment without toxic effects. For SU on the ACHN-xenograft mouse model, 100 mg/kg was found to be the minimum effective dose (MED) to achieve significant tumor growth inhibition (Figure 3A). However, this dose of SU caused severe body weight loss, increasing the number of sacrificed mice (Figure 3B,C). The maximum tolerated dose (MTD) was found to be 50 mg/kg, where body weight loss was insignificant but so was tumor regression. This

(p.o.) and SU (i.v.) (Figures S12 and S13). The combination of SU and empty MNC also exhibited greater anti-angiogenic effects and tumor cell apoptosis induction effects than SU (p.o.) and SU (i.v.), as observed in the ACHN tumors. Collectively, the anticancer efficacy of SU-MNC was considered to be attributed to its efficient activities of antiangiogenesis, apoptosis induction, and proliferation inhibition against tumors, wherein the carrier played a substantive role. Biodistribution of SU-MNC and SU (i.v.) was examined at 8 h post-injection on both ACHN- and A498-xenografted mice (Figure 2H,I). SU-MNC exhibited a 2.2-fold greater SU accumulation (4.9 ± 0.9% ID/g) in the ACHN-xenograft tumor compared to SU (2.2 ± 0.7% ID/g). In contrast, 0.4-, 0.1-, 0.3-, and 0.2-fold lower SU depositions of SU-MNC were observed in the lungs, stomach, skin, and muscles, respectively, compared to SU, suggesting that SU-MNC has the potential to attenuate the off-target toxicities in these organs. The improved tumor accumulation of SU-MNC was more prominent in the A498-tumor, exhibiting a 5.1-fold greater SU accumulation (10.8 ± 2.4% ID/g) compared to SU (2.1 ± 0.5% ID/g), likely because the enhanced permeability and retention (EPR) effect-mediated accumulation of SU-MNC was more pronounced due to its highly vascularized nature.42 This should be responsible for a more pronounced efficacy of SU-MNC on the A498-xenograft than the ACHN-xenograft, while SU (p.o.) efficacy was attenuated. On the other hand, SU-MNC induced a 0.5-, 0.7-, and 0.4-fold lower SU accumulation in the heart, lungs, and muscles, respectively, compared to SU on A498-xenografted mice. The preferential F


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Figure 4. SU-MNC shows higher anticancer efficacy than a conventional nanocarrier (SU-PM), despite comparably improved systemic circulation and SU accumulation in tumors. (A,B) Plasma pharmacokinetics of (A) SU and (B) N-desethyl sunitinib (active metabolite) after a single injection (i.v.) of SU, SU-MNC, or SU-PM (10 mg/kg of equivalent SU) on athymic nude mice (n = 3, mean ± SEM). (C) Kinetic distribution of SU in ACHN-xenograft tumors after a single injection (i.v.) of SU, SU-MNC, or SU-PM (5 mg/kg of equivalent SU) for 24 h (n = 6, mean ± SEM). (D) CTmax and AUCT of SU from (C). (E) Tumor growth inhibitory effects of SU, SU-MNC, and SU-PM (5 mg/kg of equivalent SU, i.v., twice/week for 35 days) on ACHN-xenografted mice at day 56 (n = 12, mean ± SEM). *p < 0.05; ***p < 0.005; ****p < 0.001 (SU versus SU-MNC), #p < 0.05; ##p < 0.01; ###p < 0.005 (SU-PM versus SU-MNC).

micelle formation and clinically approved for the paclitaxelloaded micelle formulation in Asia,50−52 was used to form SUPM that displayed a comparable size, ζ potential, and drug content to SU-MNC, in order to achieve comparable delivery efficiency (Table S4). Pharmacokinetic analysis revealed that SU-MNC and SU-PM markedly prolonged the systemic circulation of SU and N-desethyl sunitinib (SU12662, active metabolite) compared to SU (Figure 4A,B and Table S5), exhibiting their enhanced stability in systemic circulation by the encapsulation in nanocarriers. Tumor accumulation of SU, SU-MNC, and SU-PM in ACHN-xenografted mice for 24 h after a single i.v. administration exhibited that SU-MNC and SU-PM delivered a larger amount of SU into the tumors than SU (Figure 4C). The maximum SU concentration in tumors (CTmax) and the area under the curve (AUC) of tumor accumulation (AUCT) were calculated to analyze the peak level and the total amount of SU deposited in the tumors, respectively.53 SU-MNC showed greater CTmax and AUCT than those of SU, demonstrating an enhanced tumor accumulation (Figure 4D). The CTmax and AUCT of SU-MNC were comparable to those of SU-PM, indicating that SU-MNC and SU-PM delivered similar amounts of SU into the tumor. In the liver and kidneys, SU-MNC and SU-PM exhibited a significantly lower Cmax and AUC compared to SU (Figure

indicated no effective dose of SU within its tolerated doses. Contrarily, SU-MNC effectively inhibited tumor growth at a dose as low as 2 mg/kg (MED = 2 mg/kg: 50-fold lower than MED of SU) without body weight loss or death (Figure 3D− F). Surprisingly, SU-MNC had no toxic signs of body weight loss or death in up to a 100 mg/kg dosage (MTD > 100 mg/ kg), suggesting its safety and high tolerability. On the A498xenograft model, SU did not show tumor growth inhibition even at 100 mg/kg, which showed significant toxicity-induced body weight loss (Figure S14A−C). Contrarily, SU-MNC demonstrated significant anticancer efficacy with a MED of 20 mg/kg, and no toxic signs were observed up to 100 mg/kg (Figure S14D−F). Collectively, SU-MNC was shown to provide a significantly wider therapeutic window, demonstrating elevated anticancer efficacy and reduced systemic toxicity, which could enable an effective and safe treatment when compared to SU. Drug−Carrier Synergistic Anticancer Effects. Pharmacokinetics, biodistribution, and anticancer efficacy of SU-MNC were compared to a SU-loaded conventional polymeric micelle (SU-PM), wherein the carrier does not have an anticancer activity to elucidate the carrier impact on the anticancer activity of SU-MNC. PEG-block-poly(lactic acid) copolymer (PEG-PLA), the most widely used polymer for polymeric G


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Figure 5. PEG-EGCG of the carrier confers synergistic anticancer effects. (A) Tumor growth inhibitory effects of SU and SU-MNC as a function of CTmax (n = 12, mean ± SEM). (B) CTmaxs of PEG-EGCG and SU as a function of the SU-MNC dose injected (i.v., n = 6, mean ± SEM). (C) Tumor growth inhibitory effects of SU-MNC (5 mg/kg of equivalent SU, i.v.) as a function of the dosing frequency on ACHNxenografted mice (n = 12, mean ± SEM). (D) Tumor growth inhibitory effects and the intratumoral amounts of PEG-EGCG and SU at day 56 as a function of the dosing frequency (n = 12, mean ± SEM). *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001.

of SU-MNC (75.4%) were significantly higher than those of SU-PM (−8.3%) as well as SU (0.9%), despite the comparable CTmax and AUCT to those of SU-PM. SU-PM only showed just a comparable inhibition effect to that of SU. These results indicated that SU-PM was only capable of attenuating systemic toxicity with no significant improvement on the anticancer efficacy despite its prolonged systemic circulation and preferential tumor accumulation, suggesting that the anticancer activity of PEG-EGCG played an important role in achieving the enhanced anticancer efficacy of SU-MNC. Moreover, when the anticancer effects of SU-MNC and SU were compared on the basis of the intratumoral amount accumulated, SU-MNC showed prominently higher tumor growth inhibition (69%) even with a small amount of SU (CTmax = 0.2 μg/g), whereas SU showed only 6% of tumor growth inhibition at CTmax = 0.3 μg/g (Figure 5A). The tumor growth inhibition of SU was increased as CTmax increased. However, the CTmax-dependent propensity was not dominantly

S15), illustrating the propensity of SU-MNC and SU-PM to avoid elimination by the reticuloendothelial system and renal clearance. When anticancer efficacy and toxicity of SU-PM were investigated at varying doses (2−100 mg/kg of equivalent SU), SU-PM showed no significant tumor growth inhibition at doses up to 100 mg/kg on both the ACHN- and A498xenograft models (Figure S16). Although SU-PM failed to suppress tumor growth, it did not cause toxicity symptoms of body weight loss or death at doses up to 100 mg/kg. The histological analysis of both tumors treated by SU-PM demonstrated no effect on anti-angiogenesis, apoptosis induction, and proliferation inhibition against tumors when compared to untreated tumors (Figure S17). The tumor growth inhibitory effects of SU-MNC at day 56 were compared with those of SU-PM and SU administered with the equivalent dose for 35 days on ACHN-xenografted mice (Figure 4E). Notably, the tumor growth inhibitory effects H


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Table 1. Quantitation of Synergism and Dose-Reduction of SU and PEG-EGCG on the Tumor Growth Inhibitory Effect of SUMNCa time after start of treatment (day)

Fab

CIc

(Dx)SU (mg/kg)d

(Dx)PEG‑EGCG (mg/kg)d

(DRI)SUe

(DRI)PEG‑EGCGe

35 (end of treatment) 56

0.630 0.538

0.060 0.071

106.4 89.3

2468.0 2171.6

21.3 17.9

74.0 65.2

a

SU-MNC (SU = 5 mg/kg, PEG-EGCG = 33 mg/kg) was injected (i.v., twice/week for 35 days) to ACHN-xenografted mice (n = 12). bFa (fraction affected): Fa = x/100 at x% inhibition. cCI (combination index): CI < 1 (synergism), CI = 1 (additive effects), CI > 1 (antagonism). d (Dx)SU, (Dx)PEG‑EGCG: The dose of SU alone or PEG-EGCG alone to achieve x% inhibition eDRI (dose-reduction index): DRI > 1 (favorable dose-reduction), DRI = 1 (no dose-reduction), DRI < 1 (unfavorable dose-reduction). (DRI)SU, (DRI)PEG‑EGCG: DRI for SU or PEG-EGCG by SU-MNC.

PEG-EGCG still remained in the tumors, which increased with the increasing dosing frequency and was correlated to tumor growth inhibition observed at day 56. From these results, the prolonged and higher anticancer effects of SU-MNC with more frequent dosing were likely attributed to the anticancer activity of PEG-EGCG that remained in the tumor even after SU was cleared.

shown in the effects of SU-MNC. These results demonstrated that the higher anticancer effects of SU-MNC compared to SU were attributed to not only the tumor-targeted delivery that enabled higher accumulation in tumors but also to the combinational anticancer effects of SU and PEG-EGCG carrier. Indeed, CTmaxs of PEG-EGCG and SU correspondingly increased with increasing SU-MNC doses, demonstrating their intratumoral coaccumulation (Figure 5B). For quantitative analysis of the combinational effects of SU and PEG-EGCG carrier on the tumor growth inhibition of SUMNC (5 mg/kg of SU), the combination index (CI) and the dose-reduction index (DRI) were determined by the Chou− Talalay method54−56 from the tumor growth inhibitory effects of SU and PEG-EGCG at various doses on the ACHNxenograft mouse model (Figures 3A and S18A and Supporting Information section 12). The CI value was 0.060 at the end of treatment (day 35), demonstrating a strong synergism which persisted even after a halt of treatment (CI = 0.071 at day 56) (Table 1). Also, the DRI values indicated that SU-MNC enabled a 21.3- and 74.0-fold dose-reduction of SU and PEGEGCG, respectively, at day 35 through the strong synergism, and the highly favorable dose-reduction was still observed after a halt of treatment (DRI = 17.9 and 65.2 for SU and PEGEGCG, respectively, at day 56). The synergistic effects were investigated on an intracellular molecular mechanism by analyzing the gene expression and protein secretion of IL-8, a pro-angiogenic factor, upregulated through a VEGF-induced signaling pathway.43−46 SU and PEG-EGCG decreased the mRNAs expression and protein secretion of IL-8 from VEGF-stimulated HUVECs, demonstrating their inhibitory effects on the VEGF-induced signaling pathway. The inhibitory effects were significantly enhanced by SU and PEG-EGCG in combination, which was not observed for the combination of SU and PEG-PLA (the combined effects were comparable to SU alone) (Figure S19). From these results, it was considered that SU-MNC effectively inhibited VEGF-induced angiogenesis as a result of the synergistic effects of the SU and PEG-EGCG carrier. Prolonged efficacy of SU-MNC was further investigated by varying the dosing frequencies on ACHN-xenograft tumors. The effective tumor suppression at twice/week dosing still remained when the dosing frequency was reduced to once/ week, and even after a halt in the treatment (Figure 5C). A further decrease in the dosing frequency to once/2 weeks and a one-time injection of SU-MNC also showed significant tumor growth inhibition during treatment, but they allowed an attenuation of the tumor suppression effects after treatment halted. No toxic signs were observed in all treatments (Figure S20). When the intratumoral amounts of SU and PEG-EGCG at day 56 were analyzed, SU was not detected in the tumors, regardless of the dosing frequencies (Figure 5D). However,

CONCLUSION In conclusion, SU-MNC formed by utilizing the favorable interaction of the SU and EGCG moiety of PEG-EGCG demonstrated an improved anticancer effectiveness and reduced systemic toxicity, leading to a much wider therapeutic window on HRCC-xenograft models compared to conventional SU formulations. Notably, SU-MNC showed a greater efficacy than a conventional nanocarrier, SU-PM. Enhanced efficacy of SU-MNC was ascribed to the synergistic anticancer effects of SU and the carrier comprised of PEG-EGCG, as well as taking advantage of nanocarriers for tumor-targeted delivery. The high-performance drug carrier system of SU-MNC was considered to enable a more effective and safer strategy for cancer therapy, suggesting a scope for an improved nanomedicine formulation. EXPERIMENTAL SECTION Cell Culture. HUVECs were obtained from Lonza (Singapore) and cultured in the endothelial cell basal medium (EBM-2) supplemented with EGM-2 SingleQuots (Lonza). Primary HRPTECs were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in the renal epithelial cell basal medium (ATCC PCS-400-030) supplemented with the renal epithelial cell growth kit (ATCC PCS-400-040). HRCCs (ACHN and A498) were obtained from ATCC (Manassas, VA, USA), and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Inhibitory Effects on VEGF-Induced Cell Growth and Cytotoxicity on Normal Cells. HUVECs were seeded in 96-well plates (5 × 103 cells/well, n = 5) and cultured for 1 day for cell attachment. After the cells were starved in EBM-2 media containing 0.1% FBS for 1 day, they were treated with either SU-MNC or SU malate at various SU concentrations (0.04−1.99 μg/mL) in two different conditions: in regular media (EBM-2 media supplemented with 2% FBS) or VEGF-supplemented media (EBM-2 media supplemented with 0.1% FBS and 50 ng/mL of recombinant human VEGF165 (i-DNA, Singapore)). After 3 days, cell viability was measured by using AlamarBlue reagent (Life Technologies, USA) according to the manufacturer’s protocol. Briefly, the medium was replaced by 200 μL of phenol red-free media containing 10% AlamarBlue reagent. After incubation for 4 h at 37 °C, fluorescence intensity (λex = 549 nm and λem = 587 nm) was measured by a microplate reader (Tecan Group Ltd., Switzerland). Results were expressed as a percentage of viable cells relative to untreated cells. HRPTECs were seeded in 96-well plates (5 × 103 cells/well, n = 5) I


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AUC were calculated by a noncompartmental method using the PKSolver software. Tumor growth inhibition as a function of SU CTmax was measured at day 56 after treatment of SU and SU-MNC with different doses (2−100 mg/kg of equivalent SU, i.v. twice/week) for 35 days on ACHN-xenografted mice (n = 6), where CTmax of SU was determined after a single injection with the same dose. Statistical Analysis. All of the data were expressed as mean ± standard deviation (SD) unless otherwise stated. Statistical analysis was conducted by one-way analysis of variance (ANOVA), and survival analysis was conducted by the log-rank test using the SigmaStat 3.5 software (Systat Software Inc., USA).

and cultured for 1 day. The culture media was replaced by media containing either SU or SU-MNC at various SU concentrations (0.26−3.26 μg/mL). After 3 days, cell viability was determined by using AlamarBlue reagent as described above. Anticancer Effects on HRCC-Xenograft Mouse Models. Female athymic nude mice (CrTac:NCr-Foxn1nu, 5−6 weeks old, 18−20 g) were obtained from InVivos Pte. Ltd. (Singapore). The HRCC-xenograft mouse models were established by inoculating ACHN or A498 (4 × 106 cells) suspended in 200 μL of a mixture (1:1 (v/v)) of phosphate-buffered saline (PBS) and Matrigel (Corning, USA) subcutaneously in the right flank. On day 14 (ACHN) or day 21 (A498) after tumor inoculation, the mice were randomly allocated for treatments (35 days) of saline (control, i.v., twice/week), SU (5 mg/kg, i.v., twice/week), SU (40 mg/kg, p.o., daily), SU-MNC (5 mg/kg of equivalent SU, i.v., twice/week), and a combination (i.v., twice/week) of SU (5 mg/kg) and empty MNC (equivalent PEG-EGCG) (n = 10−12). The SU solution was prepared by dissolving SU malate in saline and citrate buffer (pH 3.5) for i.v. and p.o., respectively. Tumors were measured twice/week with a digital caliper, and the tumor volume (mm3) was calculated from the following formula: volume (V) = (length × width2)/2. Tumor growth inhibition at the day 56 end point was calculated according to the following formula:

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.9b00467. Supplementary experimental data; Tables S1−S5; Figures S1−S20 (PDF)

AUTHOR INFORMATION Corresponding Authors

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

ij Vtreatment,day 56 − Vtreatment,day 0 yzz zz × 100 = jjjj1 − j Vcontrol,day 56 − Vcontrol,day 0 zz k {

Tumor growth inhibition(%)

ORCID

Ki Hyun Bae: 0000-0001-8987-879X Jackie Y. Ying: 0000-0001-6938-2113 Motoichi Kurisawa: 0000-0003-2413-6933

where Vtreatment,day 0 and Vtreatment,day 56 are the tumor volumes of the treatment group on day 0 and 56, respectively, and Vcontrol,day 0 and Vcontrol,day 56 are the tumor volumes of the control group on day 0 and 56, respectively. The care and use of laboratory animals were regulated according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the Biological Resource Centre (BRC) in Biopolis, Singapore. Tumor Microvascular Density and Tumor Cell Proliferation and Apoptosis Induction. The tumors harvested at the day 56 end point were fixed with 4% paraformaldehyde in PBS, embedded in paraffin blocks, and then processed for histological analysis. The tumor microvascular density and tumor cell proliferation were analyzed by immunostaining using a rat monoclonal antimouse CD34 antibody (Clone MEC14.7, Biolegend, USA) and a rabbit antiKi67 polyclonal antibody (AB15580, Abcam, UK), respectively. For tumor apoptosis induction analysis, the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was conducted with ApopTag Plus peroxidase in situ apoptosis kit (S7101, Merck Millipore, Germany). The sections were counterstained with hematoxylin. Five representative hotspot areas were taken per mouse (n = 3) for analysis using ImageJ software (NIH, USA). Therapeutic Window Assessment. ACHN- and A498-xenografted mice were treated (i.v., twice/week) by SU (5−100 mg/kg), SU-MNC (2−100 mg/kg), or SU-PM (2−100 mg/kg) for 35 days (n = 10−12). The tumor volume, body weight, and survival rate were monitored until day 56. The MED was defined as the lowest dose that exhibited a statistically significant tumor growth inhibition (p < 0.05 versus control). The MTD was defined as the highest dose that did not cause statistically significant body weight loss (p < 0.05 versus control). Biodistribution, C max , and AUC Analysis. To analyze biodistribution of SU and SU-MNC, tumors and organs were collected at 8 h after a single injection of SU or SU-MNC (5 mg/kg of equivalent SU, i.v.) on ACHN- and A498-xenografted mice (n = 6). For kinetic distribution analysis, tumors, livers, and kidneys were collected at 0.5, 1, 2, 4, 8, 12, and 24 h after a single injection of SU, SU-MNC, or SU-PM (5 mg/kg of equivalent SU, i.v.) on ACHNxenografted mice (n = 6). The amount of SU in tumors and organs was measured by reverse-phase HPLC (Supporting Information section 10). PEG-EGCG amounts in tumors were measured using the PEGylated protein ELISA kit (Enzo Life Sciences, USA). Cmax and

Present Address †

NanoBio Lab, 31 Biopolis Way, The Nanos, #09-01, Singapore 138669. Funding

This work was funded by the Institute of Bioengineering and Nanotechnology (Biomedical Research Council, Agency for Science, Technology and Research (A*STAR), Singapore). Notes

The authors declare the following competing financial interest(s): The authors have filed a patent related to the design of sunitinib-loaded PEG-EGCG micellar nanocomplex and licensed this technology to a startup company, GreenT Biomed Pte. Ltd.

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Carrier-Enhanced Anticancer Efficacy of Sunitinib-Loaded Green Tea

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