Co-Delivery of Natural Compounds with a Dual-Targeted Nanoparticle

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Cite This: ACS Appl. Mater. Interfaces 2019, 11, 23880−23892

Co-Delivery of Natural Compounds with a Dual-Targeted Nanoparticle Delivery System for Improving Synergistic Therapy in an Orthotopic Tumor Model Pei-Yi Chu,† Shih-Chang Tsai,§ Han-Yu Ko,§ Chia-Che Wu,§ and Yu-Hsin Lin*,†,‡,∥

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Faculty of Pharmacy and ‡Institute of Biopharmaceutical Sciences, Department and Institute of Pharmacology, Center for Advanced Pharmaceutics and Drug Delivery Research, National Yang-Ming University, Taipei 11221, Taiwan § Department of Biological Science and Technology and ∥Department of Medical Research, China Medical University Hospital, China Medical University, Taichung 40402, Taiwan ABSTRACT: Various natural compounds including epigallocatechin gallate (EGCG) and curcumin (CU) have potential in developing anticancer therapy. However, their clinical use is commonly limited by instability and low tissue distribution. EGCG and CU combined treatment can improve the efficacy with synergistic effects. To improve the synergistic effect and overcome the limitations of low tissue distribution, we applied a dual cancer-targeted nanoparticle system to co-deliver EGCG and CU. Nanoparticles were composed of hyaluronic acid, fucoidan, and poly(ethylene glycol)-gelatin to encapsulate EGCG and CU. Furthermore, a dual targeting system was established with hyaluronic acid and fucoidan, which were used as agents for targeting CD44 on prostate cancer cells and P-selectin in tumor vasculature, respectively. Their effect and efficacy were investigated in prostate cancer cells and a orthotopic prostate tumor model. The EGCG/CU-loaded nanoparticles bound to prostate cancer cells, which were uptaken more into cells, leading to a better anticancer efficiency compared to the EGCG/CU combination solution. In addition, the releases of EGCG and CU were regulated by their pH value that avoided the premature release. In mice, treatment of the cancer-targeted EGCG/CU-loaded nanoparticles significantly attenuated the orthotopic tumor growth without inducing organ injuries. Overall, the dual-targeted nanoparticle system for the co-delivery of EGCG and CU greatly improved its synergistic effect in cancer therapy, indicating its great potential in developing treatments for prostate cancer therapy. KEYWORDS: cancer-targeted nanoparticle, hyaluronic acid, fucoidan, CD44, P-selectin, synergistic effect

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INTRODUCTION

To improve the efficacy of combined natural compound treatment in cancer therapy, applying nanoparticle (NP) systems with targeted delivery and drug release control properties is a promising strategy.16 Hyaluronic acid (HA), a linear glycosaminoglycan comprising alternating disaccharide units of D-glucuronic acid and N-acetylglucosamine, is a primary binding agent for cell surface molecule CD44,17 which is a surface protein widely overexpressed in solid tumors, including prostate cancer.18 Moreover, CD44 is highly present in cancer stem cells, which is involved in cancer initiation, maintenance, metastasis, and recurrence. Targeting cancer stem cells is important in cancer therapy.19 In addition, HA improves the tumor accumulation and increases the cellular uptake of NPs.20 Therefore, HA is a targeting molecule toward cancer cells in NP delivery systems.21 Fucoidan (FU), a natural component

Bioactive natural compounds have been regarded as a good source for developing anticancer drugs.1 Especially, various phenolic compounds extracted from plants are well known as a bioactive agent for preventing and curing many diseases.2 Epigallocatechin gallate (EGCG) found in green tea and curcumin (CU) extracted from turmeric are well-known polyphenols with potent anticancer properties.3−6 In recent years, the synergistic effect of EGCG and CU has been revealed. Combined treatment with EGCG and CU shows great benefits, as it inhibits angiogenesis, attenuates the cancer stem cell phenotype, and improves the antiproliferative effect.7−9 However, as the pharmacokinetic profiles of EGCG and CU are different, the synergistic effects of combined therapies are commonly reduced in vivo.10 In addition, the use of natural compounds is limited due to the rapid metabolism, low tissue distribution, and instability in vivo.11−14 Until now, EGCG and CU have not been approved as cancer therapeutic agents in clinical practices.15 © 2019 American Chemical Society

Received: April 8, 2019 Accepted: June 13, 2019 Published: June 13, 2019 23880

DOI: 10.1021/acsami.9b06155 ACS Appl. Mater. Interfaces 2019, 11, 23880−23892

Research Article

ACS Applied Materials & Interfaces

Figure 1. Illustration diagram of the dual-targeting NP delivery system encapsulated with EGCG and CU. NP, nanoparticle; HA, hyaluronic acid; FU, fucoidan; PEG, poly(ethylene glycol); EGCG, epigallocatechin-3-gallate; CU, curcumin; SEM, scanning electron microscopy; IHC, immunohistochemistry.

Table 1. Effect of Different FU/HA/EGCG Proportions on Particle Sizes, Zeta Potential Values, Polydispersity Index, and Loading Efficiency of the Prepared EGCG-Loaded FU/HA/PEG-Gelatin NPs (n = 5)a PEG-gelatin at 3.75 mg/mL FU/HA/EGCG (mg/mL) 0.30:0.30:1.00 0.45:0.45:1.00 0.60:0.60:1.00 0.75:0.75:1.00

mean particle size (nm) 315.78 198.65 181.78 213.10

± ± ± ±

15.87 9.89 7.30 5.52

polydispersity index 0.49 0.31 0.23 0.15

± ± ± ±

0.15 0.08 0.12 0.01

zeta potential value (mV) −24.85 −26.49 −29.77 −33.60

± ± ± ±

5.26 2.18 3.36 1.57

loading efficiency (%) 45.69 50.87 54.19 59.61

± ± ± ±

11.32 7.85 1.19 0.04

a

FU, fucoidan; HA, hyaluronic acid; PEG, poly(ethylene glycol); EGCG, epigallocatechin gallate.

EGCG and CU have the potential to become anticancer agents. The expression of P-selectin in prostate tumor vasculature was confirmed with P-selectin staining in tissues. Results showed that P-selectin was significantly expressed in tumor vasculature both in the edge part and the center part (Figure 1). In this study, we developed a dual-targeted NP delivery system to apply to the combination therapy of EGCG and CU. Moreover, the CD44 targeting agent (HA), P-selectin targeting agent (FU), and poly(ethylene glycol) (PEG)-gelatin were used to load anticancer agents EGCG and CU. HA and FU could help in dual-targeting and specifically delivering NPs to prostate tumors. Then, the co-delivery of EGCG and CU could provide an optimal synergistic treatment and superior anticancer abilities. An illustrative diagram is presented in Figure 1.

extracted from various kinds of brown algae and brown seaweeds, is a kind of polysaccharide composed of Lfucopyranose units and sulfated ester groups.22 It is a wellestablished targeting ligand for P-selectin.23 P-selectin is an inflammatory cell adhesion molecule expressed on endothelial cells and is responsible for leukocyte recruitment.24 The expression of P-selectin is upregulated in metastatic cancer cells and on active blood vessels, including the vasculature in tumors.25−27 Targeting P-selectin helps NP extravasation through the vascular barrier at the tumor site.28 Prostate cancer is one of the most common cancers among males, especially elderly individuals, and it represents 9.5% of all new cancer cases in the United States. With the highest incidence among all cancers, prostate cancer is diagnosed in more than 1 million men and causes more than 300,000 deaths in a year worldwide, accounting for 6.6% of total male cancer mortality.29,30 For inoperable prostate cancer, 40% of patients developed castration-resistant prostate cancer few years after hormone deprivation treatment.31 Under this condition, chemotherapy (docetaxel) is the primary treatment. However, the efficacy is still unsatisfied. The cytotoxicity on normal cells and drug resistance are still the problems in clinical treatment.3,32 It has been reported that EGCG and CU show anticancer effects against prostate cancer and castrationresistant prostate cancer in vitro and in vivo.33−40 Hence,

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EXPERIMENTAL SECTION

Materials. HA (molecular weight (MW), 200 kDa) and methoxyl PEG succinimidyl ester (molecular weight, 5000 Da) (mPEG5000NHS) were purchased from Lifecore Biomedical, LLC (Minnesota, United States) and Nanocs Inc. (New York, United States). Gelatin, 3-(4,5-dimethylthiazol-yl)-2,5-diphenyltetrazolium bromide (MTT), rhodamine 6G (Rh6G), 4,6-diamidino-2-phenylindole (DAPI), pluronic F-127 (PLF), CU, and EGCG were purchased from Sigma-Aldrich (St. Louis, United States). FU was purchased from 23881


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Table 2. Effect of Different CU/PLF Proportions on Particle Sizes, Zeta Potential Values, Polydispersity Index and Loading Efficiency of the Prepared EGCG/CU-Loaded FU/HA/PEG-Gelatin NPs (n = 5)a FU/HA/PEG-gelatin/EGCG at 0.600/0.600/3.750/1.000 by mg/mL Loading efficiency (%) CU/PLF (mg/mL) 0.025:0.000 0.025:0.250 0.025:0.500 0.025:0.750 0.025:1.000

mean particle size (nm) 950.80 382.30 197.73 223.70 270.20

± ± ± ± ±

284.89 197.15 18.53 8.14 81.69

polydispersity index 1.00 0.61 0.29 0.38 0.78

± ± ± ± ±

zeta potential value (mV) −26.78 −27.43 −33.30 −33.07 −35.23

0.32 0.18 0.07 0.15 0.09

± ± ± ± ±

7.98 4.58 2.19 1.53 1.45

EGCG 45.21 45.21 46.01 42.95 42.95

± ± ± ± ±

9.96 9.96 1.96 1.27 1.27

CU 48.98 59.62 67.76 72.66 72.66

± ± ± ± ±

19.59 8.97 6.67 3.18 3.18

a

FU, fucoidan; HA, hyaluronic acid; PEG, poly(ethylene glycol); EGCG, epigallocatechin gallate; CU, curcumin.

ChamBio (Taichung, Taiwan). All chemicals and reagents were of analytical grade. Preparation and Characterization of PEG-Gelatin and EGCG/CU-Loaded FU/HA/PEG-Gelatin NPs. PEG-gelatin was produced by adding 0.6 g of mPEG5000-NHS to the gelatin solution in dimethyl sulfoxide (DMSO, 2.0 g/20.0 mL). Then, the solution was stirred for 4.0 h. The PEG-gelatin was purified by dialyzing against deionized water in the dark. The water was replaced once a day for 5 days to remove the unconjugated materials. After freezedrying, the PEG-gelatin powder was collected. Fourier transform infrared (FTIR) spectroscopy analysis was used to identify the chemical structure in PEG-gelatin. EGCG-loaded FU/HA/PEGgelatin NPs were established first. To determine the optimal preparation conditions, different proportions of FU/HA were examined (Table 1). The NPs were prepared by dropping an aqueous EGCG solution into an aqueous FU/HA/PEG-gelatin mixed solution. Using a pipette, FU/HA aqueous mixed solutions (1.20/ 1.20, 1.80/1.80, 2.40/2.40, 3.00/3.00, 1.00 mL) were added to the PEG-gelatin aqueous solution (15.00 mg/mL; 1.00 mL), and then the solutions were gently shaken for 30 min at 37 °C. Afterward, the EGCG aqueous solution (2.00 mg/mL; 2.00 mL) was separately added to the FU/HA/PEG-gelatin mixed solutions with different FU/ HA concentrations under stirring at 37 °C. After centrifugation, FU/ HA/PEG-gelatin/EGCG NPs were formed at the following ratios: 0.30:0.30:3.75:1.00, 0.45:0.45:3.75:1.00, 0.60:0.60:3.75:1.00, and 0.75:0.75:3.75:1.00 mg/mL. The particle size, polydispersity index, and zeta potentials were measured with a Zetasizer instrument. The amount of free EGCG in the supernatant was detected using a highperformance liquid chromatography (HPLC) system with a reversedphase C18 column. Compounds were eluted with 5% acetic acid/ acetonitrile (50:50, v/v) at a flow rate of 1.00 mL/min. Subsequently, the condition of EGCG-loaded FU/HA/PEG-gelatin NPs was decided. Water-insoluble CU was encapsulated in these NPs using nonionic surfactant PLF. To determine the optimal condition for preparing EGCG/CU-loaded FU/HA/PEG-gelatin NPs, different proportions of PLF were examined (Table 2). In brief, EGCG/CUloaded FU/HA/PEG-gelatin NPs were prepared by dropping an aqueous EGCG/CU/PLF solution into an aqueous FU/HA/PEGgelatin mixed solution. CU dissolved in 99% ethanol (0.4 mg/mL; 0.25 mL) was mixed with PLF (1.33/2.67/4.00/5.33 mg/mL; 0.75 mL) and then added to the EGCG aqueous solution (4.00 mg/mL; 1.00 mL). The EGCG/CU/PLF aqueous solutions (2.00 mL), with different PLF concentrations, were added to the FU/HA/PEG-gelatin mixed solution under stirring at 37 °C. Then, EGCG/CU-loaded FU/ HA/PEG-gelatin NPs were formed, and their compositions were confirmed by FTIR analysis. Assessing the Release of EGCG and CU in NPs. The stabilities of the NPs in the pH 7.4, pH 6.5, and pH 5.0 environments were evaluated with the morphology changes by using transmission electron microscopy (TEM). The buffers with pH values of 7.4, 6.5, and 5.0 were chosen to simulate the environments of the circulatory system, tumor tissues, and endosomal compartments, respectively.41 The releases of EGCG and CU from NPs were assessed. In brief, the NP solutions (2.0 mg/mL; 1.0 mL of deionized water) were dialyzed against 10.0 mL of phosphate-buffered saline

(PBS) with different pH values (MW, 3500 Da dialysis bag), and the percentages of cumulative drug release were detected by using an HPLC system. Cell Culture and Evaluating the Anticancer Effect in Vitro. The luciferase-expressing stable human prostate cancer cells (Luc PC3) used in this study were given by Prof. Jer-Tsong Hsieh (Southwestern Medical Center at Dallas). Luc PC3 is a type of human prostate cancer cells with stable luciferase expression, which can be easily detected in vivo with an IVIS system. Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum in a humidified atmosphere of 5% CO2/95% air at 37 °C. Puromycin (1 μg/mL) and geneticin 418 (400 μg/mL) were added to sustain the expression of luciferase in the cell line. In a cell viability study, Luc PC3 cells (1.0 × 104 cells/well) were seeded into 96-well plates. Different concentrations of EGCG solution (ranging from 0 to 480 mg/L), CU solution (ranging from 0 to 18 mg/L), EGCG/CU combination solution (ranging from 0/0 to 480/18 mg/L), or EGCG/CU-loaded NPs (EGCG/CU ranging from 0/0 to 480/18 mg/L) were given to Luc PC3 cells for 2 h and then replaced with a drug-free cell medium. Cell viability was assessed for 24 h by MTT assay. The drug concentration decreasing 50% of cell viability (IC50) was calculated. We evaluated the combined effect of EGCG and CU on cancer cell viability by using the Chou−Talalay method.42 The combination index (CI) of test agents was calculated by the formula CI = DE/DxE + DC/DxC.43 DE and DC were the concentrations of EGCG and CU used in the combined treatment to achieve x% drug effect, respectively. DxE and DxC were the concentrations for single agents to achieve x% drug effect, respectively. CI values higher than 1.0 indicate antagonistic effects, those equal to 1.0 indicate additive effects, and those lower than 1.0 indicate synergistic effects of the combined agent action. In our experiments, the IC40 (fractional effect 0.4), IC30 (fractional effect 0.3), IC20 (fractional effect 0.2), and IC10 (fractional effect 0.1) values were calculated. Isobologram and CI−Fa plot were presented. In Vitro Cellular Uptake and Drug Distribution Evaluation. Cellular uptakes of NPs were detected by using fluorescent-dye conjugated HA and FU in NPs. Fluorescent polymer cyanine5 hydrazide-labeled FU (Cy5-FU) and Rh6G hydrazide-labeled HA (Rh6G-HA) were synthesized. Cy5 hydrazide and Rh6G hydrazide solutions (1.0 mg/0.1 mL in DMSO) were added gradually to aqueous FU solution and HA solutions (0.2 g/20.0 mL), respectively. The mixed solutions were then stirred for 12 h at 4 °C. To remove the unconjugated fluorescent dye, Cy5-FU and Rh6G-HA were dialyzed against deionized water in the dark. The remaining Cy5-FU and Rh6G-HA solutions were then lyophilized in a freeze dryer. The fluorescent EGCG/CU-loaded Cy5-FU/Rh6G-HA/PEG-gelatin NPs were prepared by the same procedure described before. To determine the cellular uptake ratio, the PC3 cells carrying fluorescent NPs were detected by flow cytometry. In brief, Luc PC3 cells were incubated with fluorescent NPs for 0, 0.25, 0.50, 1.00, and 2.00 h, respectively. Cells were collected and washed with PBS three times. After centrifugation, cells were suspended in 0.5 mL of PBS. The Cy5-FU and Rh6G-HA contents in 1.0 × 104 cells were detected with a Becton Dickinson FACSCalibur flow cytometer (Becton Dickinson, United 23882


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Figure 2. Preparation and Fourier transform infrared (FTIR) spectroscopy analysis of EGCG/CU-loaded FU/HA/PEG-gelatin NPs. (a) Size distribution of NPs produced under different CU/PLF concentrations. (b) FTIR analysis of the materials and the NPs. NP, nanoparticle; FU, fucoidan; HA, hyaluronic acid; PEG, poly(ethylene glycol); EGCG, epigallocatechin gallate; CU, curcumin. States) and analyzed with Cell Quest software WinMDI (Verity Software House, Inc., United States). In a drug distribution study, EGCG distribution was evaluated by detecting Rh6G-conjugated EGCG. CU distribution was evaluated by detecting the fluorescence of CU itself (EX455/EM540). Rh6G hydrazide-labeled EGCG (Rh6G-EGCG) was synthesized by adding Rh6G hydrazide solutions (1.0 mg/0.1 mL in DMSO) into the aqueous EGCG solution (0.2 g/20.0 mL). After stirring for 12 h, the Rh6G-EGCG was freeze-dried. To precipitate and remove the unconjugated Rh6G dye, deionized water (20 mL) was added to the lyophilized Rh6G-EGCG sample, and the precipitated Rh6G dye was separated by centrifugation (6000 rpm for 20 min). The precipitation procedure was repeated a few times until no precipitation of Rh6G-dye was found. The Rh6G-EGCG solution was lyophilized with a freeze dryer. The Rh6G-EGCG/CU-loaded FU/HA/PEG-gelatin NPs were prepared by the same procedure described before. The EGCG and CU cellular distributions were further observed by confocal microscopy. Briefly, cells (3 × 105 cells/cm2) were seeded on glass coverslips and precultured for 24 h. Then, cells were incubated with fluorescent Rh6G-EGCG/CU solutions and Rh6G-EGCG/CU-loaded FU/HA/ PEG-gelatin NPs for 2 h and then washed with PBS three times. After fixing with 3.7% paraformaldehyde, the nucleus was stained with DAPI. Stained cells were observed by confocal laser scanning microscopy (CLSM, Leica TSC SP2) with excitation wavelengths of 340, 488, and 525 nm. The binding of prepared NPs on prostate cancer cells was observed by using field emission scanning electron microscopy (FE-SEM). Luc PC3 cells were cultured on Costar Transwell plates (5 × 105 cells/insert) with RPMI medium added to the acceptor and donor compartments. The NPs were given into the donor compartment for 2 h. After washing with PBS and fixing in 3.7% paraformaldehyde, the cells were than dehydrated in an ethanol series. Using a mini-gold sputter, cell samples were coated with gold− palladium for visual inspection by SEM. Mice and Orthotopic Prostate Tumor Model. Six-week-old male severe combined immunodeficient mice (SCID) weighing 25 g were used to establish the xenograft tumor model. They were obtained from the National Laboratory Animal Center and housed in the Laboratory Animal Center at Yang-Ming University. Mice were housed in a room with central air conditioning (25 °C, 70% humidity) under a 12 h:12 h dark−light cycle. The guidelines of animal care and all experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC). The operative procedure of establishing the orthotopic prostate tumor model was according to a previous research published by Dr. JerTsong Hsieh’s research team with some modifications.44 During the operation, mice were anesthetized with 1−2% isoflurane mixed with 100% O2. After sterilizing with alcohol, a transverse incision was made on the lower abdomen. Seminal vesicles and the bladder were

carefully lifted, and the prostate were pulled out to expose the anterior prostate. A total of 3 × 106 Luc PC3 cells were diluted in matrigel to a final volume of 20 μL. The cell suspension was slowly injected into the prostate gland with an insulin syringe. After injection, the abdominal muscle layer and the skin were closed with a 3-0 absorbable suture and with a 3-0 silk suture, respectively. Evaluating the Anticancer Effects in Vivo. After the orthotopic prostate tumor was stably detected, drug treatment courses were started, and the tumor growths were detected by the IVIS spectrum imaging system through capturing the bioluminescence signals from Luc PC3 cells. The SCID mice were then randomly divided into three groups of six mice each for tail vein injection of the FU/HA/PEGgelatin solution (control) or 15.0 mg/kg EGCG/0.6 mg/kg CU in the EGCG/CU solution or EGCG/CU-loaded FU/HA/PEG-gelatin NPs every three days. To initiate the bioluminescence expression, luciferin was intraperitoneally injected to mice 10 min before imaging. IVIS acquired a photographic image and quantified the bioluminescence signals for assessing tumor growths. After mice were sacrificed at day 18, tumors and major organs were collected. Histological examinations for evaluating organ injury were performed by hematoxylin− eosin (H&E) staining. The effect of EGCG/CU-loaded NP treatments on tumor proliferation was evaluated by immunohistochemistry staining of Ki-67 (proliferation marker). In brief, paraffinembedded tumor sections were dewaxed and rehydrated by immersing the tissue in series concentrations of ethanol and xylene. After blocking for 30 min, sections were incubated with Ki-67 primary antibodies overnight and with secondary peroxidase antibodies for 1 h. Ki-67 expressions were expressed by adding a peroxidase substrate solution. Images of the pathological changes and Ki-67 expression were taken using a light microscope (×40, ×400). In an NP distribution study in vivo, the near-infrared fluorescent Vivo-Tag 750labeled NPs were intravenously injected to assess the NP distribution during cancer therapy. This fluorescence was detected with a filter set (excitation, 710−760 nm; emission, 810−875 nm) in the IVIS imaging system. The fluorescence images of mice were captured at 0, 3, 6, and 12 h. After sacrificing at 12 h, the fluorescence images of heart, lung, liver, spleen, kidney, and prostate were captured. Statistical Analysis. Data are expressed as the means ± standard deviation (SD). The significant differences were analyzed by using the statistical package Statistical Analysis System (SAS Institute lnc., United States; 6.08 version). One-way analysis of variance (ANOVA) followed by Tukey analysis was used to make pairwise comparisons between the groups. Statistical significance was set at P < 0.05.

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RESULTS Synthesis and Characterization of EGCG/CU-Loaded FU/HA/PEG-Gelatin NPs. To establish the NPs loaded with EGCG and CU, the optimum material composition ratio for 23883


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Figure 3. Release profile of EGCG/CU-loaded FU/HA/PEG-gelatin NPs. (a) Transmission electron micrographs of NPs in buffer solutions with different pH values. (b) Release of EGCG and CU in different pH environments at 37 °C. Data are expressed as mean ± SD (n = 5). NP, nanoparticle; FU, fucoidan; HA, hyaluronic acid; PEG, poly(ethylene glycol); EGCG, epigallocatechin gallate; CU, curcumin.

CO asymmetric stretching vibration at 1624 cm−1 and C−O symmetric stretching vibration at 1406 cm−1. Vibrations of N− H at 1539 cm−1 and C−O−C at 1103 cm−1 indicated the characteristic peaks of gelatin and PEG, respectively. The EGCG spectrum presented characteristic peaks of aromatic ring CC stretching vibrations at 1534 cm−1 and C−OH alcohol stretching vibrations at 1096 cm−1. The CU spectrum presented characteristic peaks of C−OH alcohol stretching vibrations at 3494 cm−1 and C−H stretching vibrations at 1425 cm−1. By virtue of its phenyl ring structure, CU also presented a peak at 856 cm−1, reflecting the vibration of C−O in −C− OCH3. The spectra of EGCG/CU-loaded FU/HA/PEGgelatin NPs revealed prominent peaks at 1628 cm−1, attributed to the stretching vibration of the CO group of HA; at 1531 cm−1, attributed to the stretching vibration of the N−H group of PEG-gelatin; and at 1252 cm−1, attributed to the stretching vibration of the OSO group of FU. Furthermore, the C− OH alcohol stretching vibrations of CU and EGCG at 3494 and 1096 cm−1 shifted to 3504 and 1107 cm−1, respectively, in EGCG/CU-loaded FU/HA/PEG-gelatin NPs. This reflects the hydrogen bonding (N−H···HO−C) between PEG-gelatin and EGCG/CU. NP Stability and the EGCG/CU Releasing Profile. To observe the NP stability and the drug releasing profile, the morphology of EGCG/CU-loaded NPs were observed in buffer solutions with different pH values, and then the releases of EGCG and CU were detected by using an HPLC system. As shown in Figure 3a, the morphologies of NPs were similar in deionized water and pH 7.4 buffer (simulating the circulatory system or normal tissues), which appeared as a stable spherical shape with matrix structure. In pH 6.5 buffer (simulating the tumor tissues), few NPs were slightly collapsed and some NP fragments were observed. In pH 5.0 buffer (simulating the endosomal compartment of tumor cells), the −COO− groups in HA were partially protonated, which led to conformational destabilization and affecting the electrostatic repulsion between NPs. NPs partially collapsed and aggregated under the condition, and the drug were released with the conformational change (Figure 3a). Figure 3b shows that the EGCG release proportions were 19.68 ± 1.64% in pH 7.4 buffer, 25.66 ± 1.20% in pH 6.5 buffer, and 50.03 ± 2.76% in pH 5.0 buffer at 3 h. At 24 h, the EGCG release proportion was significantly increased to 83.50 ± 1.92% in pH 5.0 buffer. Meanwhile, the CU release proportions were 36.20 ± 3.97% in pH 5.0 buffer at

synthesizing EGCG-loaded NPs was first found, and then the composition ratio for CU incorporation was adjusted. Four formulations fabricated by varying the weight proportion of FU and HA were tested. The mean size, polydispersity index, zeta potential value, and EGCG loading efficacy were determined. Table 1 shows that the proportions of FU and HA affected all these parameters. The NP composed of the FU/HA/PEGgelatin/EGCG proportion of 0.60:0.60:3.75:1.00 resulted in the lowest mean particle size (181.78 ± 7.30 nm), an acceptable polydispersity index (0.23 ± 0.12), an acceptable zeta potential value (−29.77 ± 3.36 mV), and an EGCG loading efficiency of 54.19 ± 1.19%. Although the formulation with the FU/HA/PEG-gelatin/EGCG proportion of 0.75:0.75:3.75:1.00 exhibited a higher EGCG loading efficacy (59.61 ± 0.04%), the mean particle size was bigger (213.10 ± 5.52 nm) than that of the NPs prepared with 0.60:0.60:3.75:1.00 formulation (181.78 ± 7.30 nm). To further load the insoluble CU into the NPs, the surfactant PLF was used to improve the loading efficiency of CU. Table 2 shows that without using PLF, the mean particle size was large (950.80 ± 284.89 nm), and the polydispersity index became unacceptably high (1.00 ± 0.32). The addition of a low concentration of PLF (0.250 and 0.500 mg/mL) decreased the particle size and polydispersity index. The CU loading efficiencies were significantly increased with PLF concentration. However, the high concentration (0.75 and 1.00 mg/mL) of PLF increased the particle size and polydispersity index. The NP prepared with 0.5 mg/mL PLF concentration presented the smallest size (197.73 ± 18.53 nm) and lowest polydispersity index (0.29 ± 0.07) compared to those prepared with other PLF concentration groups. These results were consistent with the size distributions shown in Figure 2a. Therefore, 0.500 mg/mL was the optimal concentration of PLF in the formulation FU/HA/PEG-gelatin/EGCG:CU of 0.600:0.600:3.750:1.000:0.025. FTIR analysis confirmed the composition of the EGCG/ CU-loaded FU/HA/PEG-gelatin NPs (Figure 2b). The spectra of FU, HA, PEG-gelatin, EGCG, and CU exhibited different peaks representing the different characteristic stretches of bonds. The spectrum of FU presented a stretching vibration of sulfate esters at broad band OSO at 1249 cm−1 and scissoring vibration of CH2 (galactose, xylose) or asymmetric bending vibration of CH3 (fucose, O-acetyls) at 1425 cm−1. The HA, by virtue of its carboxyl group, exhibited 23884


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Figure 4. Effects of EGCG, CU, and EGCG/CU-loaded FU/HA/PEG-gelatin NPs on PC3 cell viability. (a) Dose−response treatment of EGCG and CU for 2 h and then cell viabilities were detected at 24 h. (b) Dose−response treatments of EGCG/CU combination solution and EGCG/CUloaded NPs for 2 h and then cell viabilities were detected at 24 h. Data are expressed as mean ± SD (n = 8). (c) CI−Fa plot. CI < 1.0 indicated synergistic effects. (d) Isobologram. DE and DC were the concentrations of EGCG and CU used in the combined treatment to achieve x% drug effect. DxE and DxC were the concentrations for single agents to achieve x% drug effect. Dots presented below the slanted line indicated synergistic effects. CI, combination index; FA, fraction affected.

3 h, accumulating to 62.47 ± 6.78% within 24 h. On the contrary, the releases at pH 6.5 and pH 7.4 were significantly lower (35.47 ± 4.67% in pH 6.5 buffer; 22.76 ± 5.25% in pH 7.4 buffer). Synergistic Effect of EGCG and CU and the Anticancer Effect of EGCG/CU-FU/HA/PEG-Gelatin NPs. To confirm the synergistic effect of EGCG and CU, the effects of EGCG alone, CU alone, and EGCG/CU combination treatments on prostate cancer viability were assessed. Additionally, the anticancer effects of EGCG/CU-loaded NPs and EGCG/CU combination treatments were compared. The dose−response effects of EGCG and CU on prostate cancer cell viabilities were assessed. Both EGCG and CU significantly decreased the cell viability in a dose−response manner. At doses of 480 mg/L EGCG and 18 mg/L CU, the cell viabilities decreased to 55.41 ± 2.91% and 60.53 ± 2.43%, respectively (Figure 4a), whereas the EGCG/CU treatment (480/18 mg/ L) decreased the viability down to 20.11 ± 3.74%. In addition, the EGCG/CU treatment presented the potent synergistic effects on viabilities with the CI values of 0.68, 0.50, 0.62, and 0.68 for IC10, IC20, IC30, and IC40, respectively. The CI values versus fraction affected (Fa) plot and the isobologram were presented in Figure 4c,d, respectively. We also compared the efficacies of EGCG/CU combination solution and EGCG/ CU-loaded NP treatments. The cell viability in EGCG/CUloaded NP (480/18 mg/L for EGCG/CU) group decreased to 13.87 ± 4.08%, which was significantly lower than that of the EGCG/CU combination solution group. In addition, the treatment IC50 for EGCG/CU-loaded NPs was 80/3 mg/L, which was half of the concentration for EGCG/CU combination solution (160/6 mg/L). This reveals that the cancer-targeted EGCG/CU-loaded FU/HA/PEG-gelatin NP treatment had greater benefits than the combination drug treatment (Figure 4b). Cellular Uptake of NPs and the Internalization of EGCG and CU. To observe the targeting ability and the cellular uptake, FACS analysis was performed with cells after time course treatments of NPs with Cy5-FU and Rh6G-HA. Data show that the uptake rates and total fluorescence intensities were increased with treating time. According to the results of Cy5-FU detection, the uptake rates were 43.5 ± 1.77% at 0.25 h and 84.5 ± 2.40% at 2 h, and from the results of Rh6G-HA detection, the uptake rates were 52.8 ± 5.21% at 0.25 h and 91.3 ± 4.44% at 2 h, indicating that almost all cells

received the NPs (Figure 5a, top). The confocal laser scanning microscopy detection was assessed at 2 h. DAPI with blue fluorescence was used to label the nucleus. Figure 5a (bottom) shows that the fluorescence of Cy5-FU and Rh6G-HA were colocalized in cells. Moreover, EGCG and CU internalizations were assessed by detecting Rh6G-EGCG and CU fluorescence signals. The delivery efficacies of Rh6G-EGCG/CU solution and Rh6G-EGCG/CU-loaded FU/HA/PEG-gelatin NPs were compared after 2 h treatments. Figure 5b shows that there were more Rh6G-EGCG and CU presented in prostate cancer cells in the Rh6G-EGCG/CU-loaded NP group than in the Rh6G-EGCG/CU solution group. It is suggested that the NP system improve the delivery efficacy of EGCG and CU. To detect the binding of EGCG/CU-loaded NPs on prostate cancer cells, FE-SEM was used to observe the morphology of the NP-treated prostate cancer cell monolayer in Costar Transwell plates. Figure 5c shows the attachment of NPs on PC3 cells (white arrows), suggesting that the NPs actively contacted the prostate cancer cells. Anticancer Efficacy of EGCG/CU-Loaded FU/HA/PEGGelatin NPs in an Orthotopic Prostate Tumor Mice Model. To evaluate the therapeutic efficacy of EGCG/CUloaded FU/HA/PEG-gelatin NPs in vivo, an orthotopic prostate tumor model was established in SCID mice. Luciferase-expressing stable PC3 cells was used. After the tumors were stably detectable in IVIS system, treatments were started with the i.v. injection of FU/HA/PEG-gelatin solution, EGCG/CU combination solution, or EGCG/CU-loaded NPs every three days for 5 injections. The tumor growths were slower in the EGCG/CU combination group (2.39 ± 0.18 units) compared with the FU/HA/PEG-gelatin solution control group (3.16 ± 0.29 units). Furthermore, EGCG/CUloaded NP treatment (1.60 ± 0.27 units) showed better inhibitory effect on tumor growth compared to EGCG/CU combination solution treatment (Figure 6a,b), indicating significant antitumor activity. There was no difference on the body weight loss between mice treated with EGCG/CUloaded NPs and mice-treated with FU/HA/PEG-gelatin control solution (Figure 6c). After sacrificing at day 18, tumor and organs were collected. Orthotopic prostate tumors were observed inside the prostate tissue with histological examination (Figure 7a). To further evaluate the effect of EGCG/CU-loaded NPs on tumor proliferation, the expressions of proliferation marker Ki-67 were detected. Data show 23885


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Figure 5. In vitro cellular uptake of EGCG/CU-loaded NPs. PC3 cells were incubated with various fluorescent-conjugated NPs. (a) To confirm the targeting of prostate cancer cells by FU and HA in nanoparticles, Cy5-FU and Rh6G-HA were detected by flow cytometry after treating NPs. Confocal images of Cy5-FU and Rh6G-HA fluorescence signals in the cells were observed after 2 h incubation of NPs. (b) Intakes of Rh6G-EGCG and CU were observed after Rh6G-EGCG/CU-loaded NPs or Rh6G-EGCG/CU solution treatment by using confocal laser scanning microscopy. (c) Binding of NPs on cells was observed by using field emission scanning electron microscopy. The right panels presented a higher magnification image of the framed area in the middle panels. The white arrows indicate NPs.

the tumor appeared at 6 h. The distribution of NPs in the lower part of the body was markedly higher in mice implanted with orthotopic prostate tumors compared to control mice without the tumor (Figure 8a), indicating the targeting capability for enhancing NP accumulation in the prostate tumor. To further clarify the NP distribution in organs, fluorescence signals were detected in the prostate tumor and other major organs at 12 h. The prostate tumor was presented by detecting the bioluminescence signals produced from luciferase-expressing stable PC3 cancer cells. Data show that the orthotopic prostate tumor expressed a high level of NP signal (11.03 ± 3.28 relative unit). In contrast, the signal was low in normal prostate (3.19 ± 1.88 relative unit). The increase in NP distribution in the prostate tumor led to the decrease in the distribution in other organs including liver, spleen, and kidney (Figure 8b). It seems that the tumor

that Ki-67 (brown color stain) was highly expressed in tumor edge (block arrow) in FU/HA/PEG-gelatin control group which was markedly reduced in EGCG/CU-loaded NPstreated tumor (Figure 7a). Safety is the basic evaluation item for drug development. According to the histological examination, EGCG/CU-loaded NPs did not cause any damage in the major organs including heart, lung, liver, spleen, and kidney (Figure 7b). It is suggested that the prepared EGCG/CUloaded NPs inhibit tumor growth through attenuating the cancer cell proliferation without inducing major organ injuries. EGCG/CU-Loaded FU/HA/PEG-Gelatin NP Targeting and Distribution In Vivo. The NP local accumulation was further evaluated by the IVIS fluorescence imaging system. After fluorescent NPs were injected into the tail vein, the fluorescence signal was detected by the IVIS technique at different time points. The maximum accumulation of NPs in 23886


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Figure 6. Therapeutic efficacy of EGCG/CU-loaded FU/HA/PEG-gelatin NPs in the orthotopic model. Mice were treated with FU/HA/PEGgelatin control solution, EGCG/CU combination solution, and EGCG/CU-loaded FU/HA/PEG-gelatin NPs by intravenous injection every 3 days for 18 days. (a) Orthotopic prostate tumor were detected by the IVIS imaging. (b) Tumor growths were evaluated with relative luminescence intensity. (c) Body weight change. Data are expressed as mean ± SD (n = 6).

show that the anticancer synergistic efficacy of EGCG/CUloaded FU/HA/PEG-gelatin NPs was better than that of EGCG/CU combination solution in decreasing cell viability in vitro and inhibiting tumor growth and proliferation in vivo. Previous studies have reported that the combined treatment of EGCG and CU synergistically enhanced the p21-induced growth arrest, which led the attenuation of prostate cancer proliferation and inhibited angiogenesis by JAK/STAT3/IL-8 pathway in colorectal cancer.7,9 The phenomenon of tumor growth and proliferation attenuations in prostate cancer cells is consistent with our results. No significant cell injury in major organs, including heart, lung, liver, spleen, and kidney, was found after NP treatment. This suggests that the anticancer EGCG/CU-loaded FU/HA/PEG-gelatin NPs with low toxicity could be a potential adjuvant treatment for patients experiencing severe side effects during chemotherapy. Nanoparticles are popular tools for developing disease therapies. According to our purpose, we designed NPs with multiple functions by synthesizing them with different materials. To form stable NPs, PEG-gelatin, HA, and FU were used as a carrier for EGCG and CU. Moreover, PEG is a steric stabilizer with the ability to prevent the interactions of the NPs with the bloodstream components. The role of PEG is to improve the drug loading efficiency, reduce the immunogenicity, and increase the blood circulation half-life of systemic NP administration.47,48 Gelatin is a polymer produced from the hydrolysis of collagen. It contains repeating amino acid sequences of glycine-A-B, where A and B are mainly proline and 4-hydroxyproline, respectively. Because of the nontoxic and gelling properties, gelatin is commonly used in food and medication. Gelatin can interact with polyphenol through hydrogen bonding of the hydrophobic amino acids to the phenol rings.49−51 It has been used to protect EGCG in microcapsules produced by coacervating with various poly-

targeting ability of EGCG/CU-loaded FU/HA/PEG-gelatin NPs was working in the orthotopic prostate tumor model in mice.

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DISCUSSION

The EGCG/CU combination treatment is beneficial for treating cancer. Chemotherapies are commonly accompanied by high toxicity in normal cells, and thus, the dosage used in the course is limited. Low-toxicity adjuvant therapy to maintain the efficacy and reduce the damage to organs is being developed to improve cancer treatments. EGCG and CU are natural compounds with potent anticancer properties and very low toxicity to normal cells. No common toxicity has been reported. In mice, the LD50 values of EGCG and CU are 1.9 and 2.5 g/kg, respectively.14 In addition, the combined treatment of EGCG and CU has shown great combinative and synergistic effects in treating colorectal and prostate cancers.7,9 Furthermore, both EGCG and CU have shown protective effects in various organ injury models through their antiinflammatory and antioxidative properties.45 Treatments for castration-resistant prostate cancer are very few, with docetaxel being the main medication option. Moreover, nearly 60% of castration-resistant prostate cancer develops into metastatic prostate cancer within 5 years.46 Although the application of EGCG and CU could be a promising strategy, treatment with natural compounds still has many limitations. In this study, the limitations of rapid metabolism, low tissue distribution, and instability were overcome by the use of the EGCG/CU-loaded FU/HA/PEG-gelatin NP delivery system, which can target cancer and avoid premature releasing. Furthermore, this system improved the co-delivery of EGCG and CU and the simultaneous EGCG/CU stimulations to the targeted site, which enhanced the synergistic effects. The data 23887


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Figure 7. Histological examination. Eighteen days after initial treatment, mice were sacrificed. The tumor and major organs tissues were collected. (a) Tumor morphology and cell proliferation were evaluated by H&E staining and Ki-67 protein detection, respectively, in prostate tumor tissues. (b, c) Organ injuries were evaluated with hematoxylin and eosin staining.

saccharides.52 EGCG features abundant hydroxyl groups in its structure, which forms hydrogen bonds with polysaccharides HA and FU and gelatin. Therefore, FU/HA/PEG-gelatin complexes were produced when the EGCG solution is mixed with the FU/HA/PEG-gelatin solution. CU is an insoluble agent. To incorporate CU into polymer networks, a hydrophilic nontoxic copolymer PLF can be used.53 PLF surfactant is an amphiphilic copolymer comprising (i) polyoxyethylene units and (ii) polyoxypropylene units (a-b-a-type triblock

copolymer), which can carry the insoluble agent and increase its solubility. When the FU/HA/PEG-gelatin solution was mixed with the EGCG/CU/PLF solution, CU/PLF was trapped into the FU/HA/PEG-gelatin/EGCG complex. In this study, we successfully established the NPs with PEGgelatin/FU/HA/EGCG/CU/PLF at a 3.75:0.60:0.60:1.00:0.025:0.50 mg/mL ratio. The mean size of the NP was 197.73 ± 18.53 nm. The polydispersity index was 0.29 ± 0.07. The EGCG and CU loading efficacies were 23888


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Figure 8. Distributions of EGCG/CU-loaded FU/HA/PEG-gelatin NPs in mice and organs after treatments. Near-infrared fluorescent NPs were intravenously injected into mice with or without the orthotopic prostate tumor. (a) Prostate tumors were detected with IVIS bioluminescence. Near-infrared fluorescent NPs were detected with IVIS fluorescence. (b) Left: IVIS images of images of bioluminescence expression presenting prostate tumor. Right: IVIS images of fluorescence expression presenting NPs distribution.

46.01 ± 1.96 and 67.76 ± 6.67%, respectively. Furthermore, because of the NPs with HA and FU, the zeta potential of the NPs was −33.30 ± 2.19 mV. The negatively charged surface of the NPs is beneficial for tolerating the adsorption of blood components, delivering drugs into deep tissues, and decreasing nontarget cell uptake, which promotes the targeting efficiency to tumor cells.54,55 To increase the therapeutic efficacy and lower the effect on normal cells, targeting the drug delivery site and avoiding premature release are the keys. In this study, HA and FU were used to target cancer cells. HA, an amphiphilic polysaccharide, is a well-known CD44 ligand, and CD44 is a cell surface adhesion receptor highly expressed in prostate cancer and cancer stem cells. CD44 also plays an important role in

prostate cancer metastasis. The development of drug-resistant subpopulations possessing a cancer stem cell morphology is an emerging mechanism of docetaxel resistance. Therefore, targeting CD44 is of great benefit in prostate cancer therapy. In our previous study, we proved that the drug-loaded HA/ PEG-gelatin NP delivery system can successfully target and deliver drugs to prostate cancer cells in a subcutaneous tumor model.56 In the present study, we improved our delivery system by introducing FU as a component of this NP and then examined the system in an orthotopic tumor model. FU is a promising polysaccharide-based material to be used as a carrier. It is a heparin-like molecule containing abundant fucose, sulfated ester groups, and small amounts of xylose, galactose, mannose, and glucuronic acid.57 It is a binding agent 23889


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ACS Applied Materials & Interfaces for P-selectin,23 which is upregulated on active blood vessels in tumors. It has been reported that targeting P-selectin by FUbased NPs helps particle extravasation through the vascular barrier to the tumor site.28 Therefore, in this study, the dualtargeted NPs will first target the active tumor vasculature by binding to P-selectin, which helps the NPs pass through the blood vessel and then target the tumor cancer cell by binding to CD44, and this enhances the intake of the NPs. Furthermore, to prevent the premature drug release, the NP system adopts pH-sensitive regulation strategy. The pH value is different in blood and healthy tissues (pH 7.4), tumor (pH 6.0 to 7.2), and endosomes (pH 5.0 to 6.0).58,59 In cancer therapy, ideal NPs remain stable in pH 7.4 environments and collapse in pH 5.0 environments. This prevents the premature drug release during circulation so that the release occurs after the uptake of NPs by targeted cancer cells. 60 The incorporation of charged residues within NPs disrupted through protonation/deprotonation and electrostatic repulsion results in developing a pH-sensitive response of NP assemblies.61−63 The decrease in the ionization of carboxyl groups between pH 7.4 and pH 5.0 leads to conformational changes of HA, which results in the release of the encapsulated agents.64−68 In the present study, EGCG/CU-loaded FU/HA/ PEG-gelatin NPs were stable in pH 7.4 and pH 6.5 buffer solutions and collapsed in pH 5.0 buffer solution. This means that an acidic environment is required for the releases of EGCG and CU. This was consistent with the pH-sensitive regulation strategy. Under the treatment using the developed cancer-targeted NPs, the drug distribution was significantly increased in the prostate tumor and decreased in other organs in mice with orthotopic tumor, compared to mice without the prostate tumor. Moreover, EGCG/CU-loaded FU/HA/PEGgelatin NP treatment inhibited 60% of prostate tumor growths compared to the control group, which was much better than the inhibitory efficacy of EGCG/CU combination solution treatment (25% inhibition). Hence, EGCG/CU-loaded FU/ HA/PEG-gelatin NPs successfully targeted the prostate tumor in mice and enhanced the synergistic efficacy of EGCG/CU cancer therapy. The system may also avoid the possible damage caused by the high-dosage treatment of the NPs in the future.

ORCID

Yu-Hsin Lin: 0000-0003-1947-1809 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS EGCG, epigallocatechin gallate CU, curcumin NP, nanoparticle HA, hyaluronic acid PEG, poly(ethylene glycol) FU, fucoidan MTT, 3-(4,5-dimethylthiazol-yl)-2,5-diphenyltetrazolium bromide Rh6G, rhodamine 6G DAPI, 4,6-diamidino-2-phenylindole PLF, pluronic F-127 FTIR, Fourier transform infrared TEM, transmission electron microscopy CI, combination index H&E, hematoxylin−eosin IACUC, Institutional Animal Care and Use Committee SD, standard deviation ANOVA, one-way analysis of variance

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REFERENCES

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CONCLUSIONS In summary, we first applied a dual cancer-targeted NP delivery system to co-deliver natural compounds EGCG and CU to prostate cancer cells. The delivery efficiency was enhanced using CD44 targeting material HA and P-selectin targeting material FU. The drug releases were regulated by environmental pH. In addition, the synergistic anticancer effects of EGCG and CU were significantly improved with the co-delivery system, without inducing injuries in major organs. The targeting led to the enhancement of NP distributions in cancer cells and tumors and their decrease in major organs in orthotopic prostate cancer mice. The results suggest that the EGCG/CU co-delivery therapeutic strategy based on the cancer-targeted HA/FU NP system shows great promise for developing prostate cancer therapy.

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ACKNOWLEDGMENTS

This work was supported by grants from the Ministry of Science and Technology (MOST 106-2314-B-010-051-MY3, 107-2221-E-010-004-MY3, and 108-2811-B-010-501).

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: 886-2-2826-7000 Ext. 6493. Fax: 886-2-2823-2940. 23890


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