Nanoparticle Targeting CD44-Positive Cancer Cells for Site-Specific

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Nanoparticle Targeting CD44-Positive Cancer Cells for Site-Specific Drug Delivery in Prostate Cancer Therapy Wen-Ying Huang, Jia-Ni Lin, Jer-Tsong Hsieh, Shen-Chieh Chou, Chih-Ho Lai, Eun-Jin Yun, U-Ging Lo, Rey-Chen Pong, Jui-Hsiang Lin, and Yu-Hsin Lin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b10029 • Publication Date (Web): 27 Oct 2016 Downloaded from http://pubs.acs.org on October 28, 2016

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ACS Applied Materials & Interfaces

Nanoparticle Targeting CD44-Positive Cancer Cells for Site-Specific Drug Delivery in Prostate Cancer Therapy

Wen-Ying Huang1†, Jia-Ni Lin2†, Jer-Tsong Hsieh3, Shen-Chieh Chou2, Chih-Ho Lai4, Eun-Jin Yun3, U-Ging Lo3, Rey-Chen Pong3, Jui-Hsiang Lin5*, Yu-Hsin Lin2,3*

1

Department of Applied Cosmetology, Master Program of Cosmetic Science, Hung-Kuang University, Taichung, Taiwan

2

Department of Biological Science and Technology, China Medical University, Taichung, Taiwan

3

Department of Urology, University of Texas Southwestern Medical Center, Dallas, Texas, USA

4

Departmentof Microbiology and Immunology, Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan,

5

Bio-medical Carbon Technology Co., Ltd, Taichung, Taiwan

*

Correspondence to:

Yu-Hsin Lin, PhD Department of Biological Science and Technology, China Medical University, Taichung, Taiwan Department of Urology, University of Texas Southwestern Medical Center, Dallas, Texas, USA Fax: 886-4-2207-1507 E-mail: [email protected]

†

The first two authors (Wen-Ying Huang and Jia-Ni Lin) contributed equally to this

work. To whom correspondence should be addressed: [email protected] (Yu-Hsin Lin) and [email protected] (Jui-Hsiang Lin).

*

1

ACS Paragon Plus Environment


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Abstract Prostate cancer is one of the leading causes of cancer death in adult men and is a multi-stage disease with therapeutic challenges of local recurrent advanced tumors and distant metastatic disease. CD44 is a multifunctional and multistructural cell surface glycoprotein that is involved in cell-cell interactions, cell proliferation, and cell migration. In the study, we produced negatively charged and biocompatible hyaluronic acid-based nanoparticles as a therapeutic system for targeting CD44-positive cancer cells. Subsequently, we confirmed the delivery of bioactive epigallocatechin-3-gallate and site-specific inhibition of prostate tumor growth. In this study,

hyaluronic

acid-based

nanoparticles

successfully

encapsulated

epigallocatechin-3-gallate and were efficiently internalized into cancer cells via CD44 ligand receptor recognition, induced cell cycle arrest at G2/M phase, and inhibited prostate cancer cell growth. Furthermore, in vivo assays indicated that these nanoparticles specifically bind CD44 receptors and increase apoptosis of cancer cells, leading to significant decreases in prostate tumor activity and tumor tissue inflammation.

Keywords:

prostate

cancer;

CD44;

hyaluronic

epigallocatechin-3-gallate

2


acid;

nanoparticles;

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1. Introduction Prostate cancer is the most frequently diagnosed cancer in men and presents therapeutic challenges of local recurrent advanced tumors and distant metastases.1–3 Cancer stem cells have been shown to be resistant to chemotherapy and radiotherapy and to form metastatic tumors in different organs.4,5 CD44 is a well-known marker of cancer stem cells and mediates intercellular adhesion, cell orientation, migration, and matrix-cell signaling processes.6–8 Some studies have shown that CD44+ prostate cancer cells have stem-like properties, such as improved tumorigenic/clonogenic characteristics and enhanced tumor formation ability in animal models.4,9,10 To improve the efficacy of chemotherapies and decrease drug-induced toxicity, many bioactive agents have been used alone or as adjuncts to standard chemotherapies.11 Among these, green tea has been widely considered for its diverse biological and pharmacological activities. In particular, green tea catechins have various health promoting properties that reflect antioxidant, anti-inflammatory, anti-carcinogenic, and antibacterial effects.12 Among tea catechins, (−)-epigallocatechin-3-gallate (EGCG) is a major constituent of green tea polyphenol extracts and has been found to inhibit matrix metalloproteinases that are tightly associated with tumor metastasis and invasion, leading to apoptosis-inducing activities against prostate cancer cells and prostate cancer stem cells.13–15 Previous studies have shown that EGCG is unstable under physiological conditions and is metabolized or degraded via interactions with hydroxyl groups on its phenol rings.16,17 Thus, to improve the therapeutic effects of specific localized cancer treatments, nanoparticles have been generated to target tumors (actively and/or passively) and increase the bioavailability of novel cancer drugs.18–20 Hyaluronic acid (HA) is a linear glycosaminoglycan comprising alternating disaccharide units of N-acetyl-D-glucosamine and D-glucuronic acid with β(1→4) interglycosidic linkages.21 HA has crucial roles in embryonic development, cell growth, and tumor formation.22 Additionally, HA plays many roles in the regulation of cell migration, 3



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cell adhesion, and cell differentiation.23 HA also participates in hydrogen bonding and van der Waals binding interactions with migrating cells through CD44 receptor ligand, which is a cell surface molecule with reported roles in cell migration, differentiation, and proliferation.24–26 We developed nanoparticles of HA and polyethylene glycol-gelatin (PEG-gelatin) to encapsulate the green tea polyphenol EGCG. PEG is soluble in water and in organic solvents and has no toxic, antigenic, or immunogenic properties. Furthermore, PEG is biocompatible and is widely used in graft-forming polymers as a crosslinker that constitutes interconnected channels to control drug release.27–29 Herein, we examined physicochemical characteristics of the nanoparticles using nuclear magnetic resonance (NMR), fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), and dynamic light scattering. Subsequently, drug effects and nanoparticle interplay mechanisms with the CD44 protein receptor have been investigated in human prostate cancer cells using field emission scanning electron microscopy (FE-SEM) and confocal laser scanning microscopy (CLSM), and apoptotic protein expression was determined via Western blot analysis. We also performed in vivo experiments to confirm inhibition of prostate tumor growth following

intravenous

injections

of

nanoparticles,

and

conducted

immunohistochemical analyses of the tumor cell proliferation marker Ki-67 and the apoptosis marker poly(ADP)ribose polymerase (PARP).

2. Experimental Section 2.1.Materials Acetic acid, dimethyl sulfoxide, fluoresceinamine (FA), EGCG, gelatin (type A; molecular weight 25 kDa), puromycin, rhodamine 6G (Rh6G), Triton X-100, and 4′,6-diamidino-2-phenylindole (DAPI) were acquired from Sigma-Aldrich (St Louis, United States). HA with average molecular weight of 60, 100 and 200 kDa were purchased from Lifecore Biomedical, LLC (Minnesota, United States). Methoxyl 4


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PEG succinimidyl ester, molecular weight 5000 Da (mPEG5000-NHS) and RNase A were purchased from Nanocs Inc (New York, United States) and Zymo Research Corporation (California, United States). Roswell park memorial institute (RPMI) 1640 medium and penicillin-streptomycin were obtained from Gibco (New York, United States). The rabbit polyclonal anti-caspase-9, rabbit polyclonal anti-poly ADP ribose polymerase (anti-PARP) were purchased from Cell Signaling Technology (Massachusetts, United States) and mouse monoclonal anti-caspase-3, mouse monoclonal anti-β-actin were from Novus Biologicals (California, United States). All reagents and chemicals used were of analytical grade. 2.2.Preparation and characterization of PEG-gelatin and HA/PEG-gelatin/EGCG nanoparticles PEG-gelatin was synthesized according to a previously reported procedure, with few modifications.30 Briefly, 2.0 g of gelatin was dissolved in 20.0 mL of dimethyl sulfoxide (DMSO) containing 0.6 g of mPEG-NHS with stirring at room temperature for 4.0 h. To remove unreacted mPEG-NHS, samples were purified by dialysis against 5 L distilled water, which was substituted five times per day. The resulting PEG-gelatin was freeze-dried by lyophilization technology, and its structure was defined using NMR and FTIR analyses. Finally, the grafting densities on PEG-gelatin were defined using a potassium polyvinyl sulfate titration method. Nanoparticles were produced by dropping of an EGCG aqueous solution into an HA/PEG-gelatin aqueous solution. In these procedures, aqueous PEG-gelatin solution (15.00 mg/mL; 1.0 mL) was added to various concentrations of aqueous HA (2.50, 5.00, 7.50, and 10.00 mg/mL; 1.0 mL) to form HA/PEG-gelatin mixed solutions (1.25:7.50, 2.50:7.50, 3.75:7.50, and 5.00:7.50 mg/mL, respectively; 2.0 mL). Subsequently, EGCG aqueous solution (4.00 mg/mL, 2.0 mL) was added into 2.0 mL of the HA/PEG-gelatin mixed solution under stirring for 0.5 h to produce HA/PEG-gelatin/EGCG nanoparticles. Nanoparticles were then collected by centrifugation at 60000 × g for 50 min at 37°C and were resuspended in distilled 5



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water. The particle size and zeta potential values of nanoparticles were estimated using a Zetasizer Nano apparatus (Malvern Instruments Ltd., United Kingdom) and characterization of the nanoparticles was performed using an FTIR spectrometer (Shimadzu Scientific Instruments, United States). Free EGCG concentrations in supernatants were identified using high-performance liquid chromatography (HPLC) with a UV detector and a C18 reversed-phase column, which was eluted with the mobile phase, consisting of 0.005 M citric acid-acetonitrile (86:14; v/v) at a flow rate of 1.0 mL/min. EGCG loading efficiencies and contents in nanoparticles were calculated using the equation: Loading efficiency =

total amount of EGCG – amount of free EGCG in supernatant × 100% total amount of EGCG

Loading content =

total amount of EGCG – amount of free EGCG in supernatant × 100% Weight of nanoparticles

2.3.Characterization and EGCG release profiles of prepared nanoparticles To investigate the stability of test nanoparticles, size distributions and nanoparticle morphology in phosphate buffered saline (PBS) with and without fetal bovine serum (FBS) were measured and observed using a zetasizer and TEM. Briefly, nanoparticle suspensions were placed onto 400 mesh copper grids coated with carbon, and deposition was allowed to proceed for about 10 min. Subsequently, the surface water on the grids was removed by tapping with filter paper, and samples were then stained with osmium tetroxide for TEM observations. To determine EGCG release, 1.0 mL of test nanoparticle solution (2.0 mg/mL) was introduced into a dialysis bag (molecular weight 3.5 kDa) and was then placed into a tube containing 10.0 mL of release media. Release tests were performed at 37°C with gentle shaking at 100 rpm. At specific time intervals, release media were removed and replaced with fresh media to prevent drug saturation. EGCG release was then determined using a HPLC system and release percentages of cumulative EGCG were calculated from a standard calibration curve. 6


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2.4.Viability of prostate cancer cells after treatments with EGCG solution or HA/PEG-gelatin/EGCG nanoparticles PC3 human prostate cancer cells were transfected with CD44 knock down (PC3 shCD44) and control (empty vector, PC3 shVec) vectors from Prof. Jer-Tsong Hsieh (Southwestern Medical Center at Dallas). Cells were then grown in RPMI 1640 medium supplemented with 10% FBS, puromycin (1 µg/mL), penicillin (100 U/mL), and streptomycin (100 µg/mL) in an atmosphere of 95% air/5% CO2 at 37°C, and were then harvested for cytotoxicity experiments. After seeding in 96 well plates at 1.0 × 104 cells/well, cancer cells were allowed to adhere overnight, and were then treated with test samples containing various EGCG concentrations (EGCG solution or HA/PEG-gelatin/EGCG nanoparticles) for 2 h. At the designated time points, test samples were then gently aspirated from cancer cells. The cancer cells were washed with PBS and refreshed with culture medium for an additional 22 h before determining cytotoxicity using 3-(4,5-dimethyl-thiazol-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. 2.5.In vitro cellular uptake and CLSM visualization of fluorescent nanoparticles on PC3 cancer cells Fluorescent FA-HA/PEG-gelatin/Rh6G-EGCG nanoparticles were produced according to the described above procedure, and Rh6G-labeled EGCG was synthesized as described elsewhere.31 Fluorescent FA-labeled HA (FA-HA) was synthesized in reactions between the carboxylic acid groups of HA and the amine groups of FA. Briefly, HA (50 mg) was fully dissolved in 10 mL distilled water, and FA (1 mg) was completely dissolved in 1 mL acetonitrile. Subsequently, FA solutions were

added

to

HA

solutions,

and

1

mg

of

1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added under stirring at 4°C. To remove unconjugated fluorescent dye, FA-HA was dialyzed against distilled water in the dark until no fluorescence was detected in the supernatant, and the resulting FA-HA was obtained as a powder by freeze-drying. 7



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To quantify cellular HA molecules of varying molecular weights, PC3 shVec cells were seeded into 24 well plates and were treated with test samples for specific times. The cells were then washed twice with PBS and solubilized in a solution containing Triton X-100 (5.0 mg/mL) and sodium hydroxide (1.6 mg/mL). The cell lysates of cell-associated

test

spectrofluorometer.

samples To

track

were

then

fluorescent

quantified

using

a

microplate

FA-HA/PEG-gelatin/Rh6G-EGCG

nanoparticle internalization in three-dimensional prostate cancer cells, cells (2 × 105) were grown in matrigel coated global eukaryotic microcarrier (GEM™, Global Cell Solution, United States) at 37°C in 95% air/5% CO2 for 7 days. Fluorescent nanoparticles were added to cells for 2 h and test samples were aspirated. Next, cells were washed with PBS, and fixed and permeabilized by incubation with 3.7% formaldehyde and 0.2% Triton X-100 for 15 min. Cells were then washed again and stained with the nuclear stain DAPI, and were observed using CLSM after excitation at 340, 488, and 543 nm, and then images were superimposed using LCS Lite software. Following treatments with nanoparticle samples, separate cells were washed with PBS, fixed in 4.0% glutaraldehyde, dehydrated with a series of graded ethanol solutions (35%, 50%, 75%, 95%, 100%), and soaked in 100% ethanol. Cell samples were then subjected to supercritical drying and were sputter coated with gold-palladium for visual inspection using FE-SEM. 2.6.Flow cytometry analysis of cell cycle and Western blotting of apoptosis-related proteins For analysis of the effects of nanoparticles on cell cycle progression, cells were incubated with EGCG-loaded HA/PEG-gelatin samples for 2 h, washed with prewarmed PBS, and incubated in culture medium for an additional 22 h. Subsequently, cells were washed twice in ice cold PBS to remove residual ethanol and resuspended in 1 mL of hypotonic buffer (PBS containing 1.0 mg/mL Triton X-100 and 0.1 mg/mL RNase A) and incubated at 37°C for 1 h. Subsequently, 0.01 mL aliquots of 1.00 mg/mL propidium iodide (PI; Calbiochem-Behring Co., California, 8


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United States) solution were added, mixed, and incubated for 30 min at 4°C in the dark. Analyses of cell cycle phases were then performed using PI staining and a BD FACSCanto system (BD Biosciences, New Jersey, United States). The expression of apoptotic proteins was determined in cultured PC3 shVec cells using Western blot analysis after treatments with EGCG-loaded HA/PEG-gelatin nanoparticles. In these experiments, cells were lysed by three freeze-thaw cycles in 0.1 mL of lysis buffer containing PBS, 1.0% sodium deoxycholate, 1.0% Triton X-100, 0.1% sodium dodecyl sulfate, 50 mM Tris-HCl, 150 mM sodium chloride, 1 mM ethylenediaminetetraacetic acid, 1 mM phenylmethanesulfonyl fluoride, 10 µM aprotinin, 0.01 mM leupeptin, 1 mM sodium orthovanadate, and 1 mM sodium fluoride, and were then scraped into eppendorf tubes. After incubating on ice for 30 min, cell lysates were centrifuged at 10000 × g for 30 min at 4°C. Equal amounts of cell lysates (30 µg/well) were then electrophoresed on 8%–15% sodium dodecyl sulfate-polyacrylamide gels and proteins were transferred to polyvinylidene difluoride membranes. Membranes were then incubated with 5% w/v nonfat dry milk in PBS containing Tween 20 for 1 h at room temperature. Membranes were probed using the following primary antibodies overnight at 4°C with gentle shaking: rabbit polyclonal anti-caspase-9, mouse monoclonal anti-caspase-3, rabbit polyclonal anti-PARP, and mouse monoclonal anti-β-actin. Bands were then visualized with horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Pennsylvania, United States) for 1 h and observed using enhanced chemiluminescence reagents (Amersham Life Science, Illinois, United States). 2.7.Cell apoptosis and immunofluorescence staining visualization by confocal microscopy image To

assess

the

effects

of

nanoparticles

on

apoptosis,

fluorescein

isothiocyanate-conjugated Annexin V (Annexin V-FITC) solution was used to detect apoptotic cells (Biovision lnc., Mountain View, Canada) according to the manufacturer’s instructions. Briefly, cells were plated onto glass coverslips at 3 × 105 9



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cells/cm2 density and were incubated for 24 h. EGCG-loaded nanoparticles were added to cells for 2 h and were grown in cultured media for an additional 22 h. Cells were then washed two times in ice cold PBS and were resuspended in 400 µL of binding buffer. Subsequently, 2 µL of Annexin V-FITC was added, mixed, and incubated at 4°C for 15 min in the dark. Stained cells were exposed to excitation at 488 nm with a laser line and emissions were recorded at 525 nm using CLSM. In further experiments, interactions of prepared EGCG-loaded nanoparticles with cell surface CD44 were examined using immunofluorescence staining analyses. Breifly, cells were treated with fluorescent FA-HA/PEG-gelatin/EGCG nanoparticles and were fixed in 3.7% paraformaldehyde. Subsequently, fixed cells were permeabilized by adding 5.0 mg/mL Triton X-100 and 0.1 mg/mL ribonuclease for 15 min, blocked in 5% normal goat serum in PBS for 1 h, and treated with mouse anti-CD44 antibody at a dilution of 1:100 for 1 h at 37°C. Following three washes in PBS, cells were treated with Cy5-conjugated goat anti-rabbit IgG at a dilution of 1:200 (Jackson ImmunoResearch Laboratories lnc., Pennsylvania, United States), uniformly mounted on slides, and then observed using CLSM. 2.8.Evaluation of antitumor activity and histology All animal procedures were performed in compliance with the Guide for the Care and Use of Laboratory Animals prepared by the National Research Council Institute for Laboratory Animal Resources and published by the National Academy Press, revised in 1996. Anti-tumor efficacies of EGCG formulations were evaluated in nude mice with subcutaneously implanted solid PC3 shVec cell (5 × 106/100 µL) tumors. Treatments were initiated on day 0 when tumors reached a tumor volume of 50 mm3.32 Mice were divided into four groups of six for treatments with normal saline solution

(control),

EGCG

solution,

HA/gelatin/EGCG

nanoparticles,

or

HA/PEG-gelatin/EGCG nanoparticles. EGCG formulations were then injected via tail veins as single doses of 2.0 mg EGCG/kg every second day, and prostate tumor volumes and changes in body weights of mice were recorded. One day after the final 10


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observation, mice were sacrificed and tumors were removed for histological examinations. Tumor biopsies (approximately 5 µm thick) were sectioned, placed on tissue slides, and stained with hematoxylin-eosin for histology or with antibodies against cleaved PARP (Cell Signaling Technology, United States) and Ki-67 (Thermo Fisher

Scientific,

United

States)

for

immunohistochemical

analyses.

Microdistributions and tissue inflammatory reactions of stained sections were observed under a light microscope at various magnifications. 2.9.Statistical analysis Differences between treatment groups were identified using one-way analysis of variance with determination of confidence intervals using the Statistical Analysis System (SAS), version 6.08 (SAS Institute, NC, United States). All data are presented as means ± standard deviations. Differences with p values of less than 0.05 were considered statistically significant.

3. Results 3.1.Preparation and characterization of PEG-gelatin Active ester terminal groups of methoxyl PEG succinimidyl ester were coupled with amino groups of gelatin to prepare PEG-grafted gelatin (PEG-gelatin). The 1

H-NMR spectrum of PEG-gelatin showed a signal at δ 3.29 ppm corresponding to a

methyl group, and signals at δ 3.52 and 3.78 ppm corresponding to H-2 and H-4 of PEG, respectively. Signal peaks in gelatin were assigned as follows: peak 1 (0.82 ppm), methyl residues of amino acids such as leucine, valine, and isoleucine; peaks 2, 3, 4, and 5 (1.21, 1.36, 1.60, and 1.71 ppm, respectively) methyl residues of threonine, alanine, lysine, and arginine, respectively; peaks 6, 7, 8, and 9 (2.62, 2.90, 3.12, and 3.56 ppm, respectively), methylene residues of aspartic acid, lysine, arginine, and proline, respectively (Figure 1a). In further experiments, the chemical structure of PEG-gelatin was characterized using FTIR (Figure 1b), which showed characteristic peaks at 839, 1243, and 953 cm−1 corresponding with C-C and C-O stretching on PEG, 11



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and a peak at 1541 cm−1 reflecting contributions of -NH bending vibrations on gelatin. These results indicate that PEG was bound to gelatin during preparation of the conjugate. Accordingly, a substitution value of 0.27 ± 0.07 was examined with potassium polyvinyl sulfate titration method.

Figure 1. (a) Nuclear magnetic resonance of PEG, gelatin, and PEG-gelatin; (b) Fourier transform infrared analyses of PEG-gelatin, HA, EGCG, and HA/PEG-gelatin/EGCG nanoparticles.

12


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3.2.Characterization of HA/PEG-gelatin/EGCG nanoparticles and EGCG release profiles Nanoparticles were prepared through gelation of EGCG aqueous solution in mixtures of varying aqueous HA/PEG–gelatin concentrations (Figure 2a). After centrifugation, aggregates of the HA/PEG-gelatin/EGCG nanoparticle solution (HA:PEG-gelatin:EGCG 0.625:3.750:2.000 mg/mL; Figure 2a; red arrow) were precipitated.

Subsequently,

particle

sizes

and

zeta

potentials

of

aqueous

HA:PEG-gelatin:EGCG nanoparticles of various ratios were determined using a dynamic light scattering analyzer (Table 1 and Figure 3a), and were 700.92 ± 96.83 nm and −18.41 ± 6.05 mV for 0.625:3.750:2.000 mg/mL, 198.37 ± 11.62 nm and −27.83 ± 2.98 mV for 1.250:3.750:2.000 mg/mL, 259.65 ± 26.79 nm and −33.83 ± 5.17 mV for 1.875:3.750:2.000 mg/mL, and 368.92 ± 51.51 nm and −41.51 ± 7.67 mV for 2.500:3.750:2.000 mg/mL mixtures, respectively. Furthermore, at a HA concentration of 1.250 mg/mL, nanoparticles formed opalescent suspensions after centrifugation (Figure 2a) and had appreciably narrower distributions (0.18 ± 0.04). Corresponding EGCG percentage loading efficiencies and contents were 66.37% ± 5.61% and 29.90% ± 2.53%, respectively. Therefore, HA:PEG-gelatin:EGCG at 1.250:3.750:2.000 mg/mL had optimal loading efficiencies, particle sizes, and distributions for subsequent experiments. In FTIR analyses of HA/PEG-gelatin/EGCG nanoparticles (Figure 1b), characteristic HA and EGCG peaks were detected at 1401 and 1620 cm−1 for symmetric (C-O) and asymmetric (C=O) stretching modes of planar carboxyl groups on HA, and at 1098 and 1534 cm−1 for the alcohol C-OH stretch and the C=C aromatic ring of EGCG. Spectra for the HA/PEG-gelatin/EGCG complex showed a characteristic band at 1543 cm−1 corresponding to -NH bending on PEG-gelatin. Moreover, the 1620 cm−1 peak for C=O stretching on HA was replaced by peaks at 1625 cm−1, and the characteristic C-OH peak of EGCG at 1098 cm−1 was shifted to 1092 cm−1. These observations reflect hydrogen bond interactions between H atoms 13



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Page 14 of 51

of EGCG and N atoms of PEG-gelatin (C–OH⋯N–C) or O atoms of HA (C– OH⋯O=C). Furthermore, PEG–gelatin, HA, and EGCG complexes were segregated into colloidal nanoparticles (Figure 2a). We also evaluated the stability of the prepared nanoparticles by suspending them in PBS with or without FBS. These experiments showed no significant changes in varieties of particle morphologies in the absence of FBS, demonstrating excellent colloidal stability in PBS (Figure 3b). However, in the presence of FBS, HA/gelatin/EGCG nanoparticles adsorbed FBS and fouling of proteins on the surface of nanoparticles lead to partial collapse (Figure 3b), gradual increases in particle size (359.78 ± 95.64 nm), and EGCG release of 44.57% ± 5.12% over 4 h, compared with 20.65% ± 2.98% from HA/PEG-gelatin/EGCG nanoparticles (Figure 3c). Moreover, interactions involving hydrogen bonds between N atoms on PEG-gelatin and H atoms in hydroxyl groups on EGCG caused slower release of EGCG from HA/PEG-gelatin/EGCG nanoparticles, and accumulated release was 48.87% ± 5.98% at 24 h and 74.85% ± 5.87% at 48 h. Table 1. Effect of varying concentrations of hyaluronic acid on particle size, polydispersity index, zeta potential value, and loading efficiencies of hyaluronic acid/PEG-gelatin/EGCG nanoparticles (n = 5).

Hyaluronic acid: PEG-gelatin:EGCG (mg/mL)

Mean particle size (nm)

Loading Zeta potential Polydispersity efficiency value (mV) index (%)

0.625:3.750:2.000

700.92 ± 96.83

-18.41 ± 6.05

0.68 ± 0.23

44.36 ± 9.72 19.98 ± 4.37

1.250:3.750:2.000

198.37 ± 11.62

-27.83 ± 2.98

0.18 ± 0.04

66.37 ± 5.61 29.90 ± 2.53

1.875:3.750:2.000

259.65 ± 26.79

-33.83 ± 5.17

0.26 ± 0.13

68.57 ± 6.34 28.04 ± 2.59

2.500:3.750:2.000

368.92 ± 51.51

-41.51 ± 7.67

0.49 ± 0.12

64.68 ± 9.93 29.14 ± 4.47

14


Loading content (%)

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Figure 2. (a) Schematic representation of the HA/PEG-gelatin/EGCG nanoparticle strategy and observation of effects in prostate carcinoma cells; (b) HA fluorescence intensity of PC3 shVec cells indicates uptake efficiency of HA molecules with varying molecular weights and confocal images of CD44 protein staining in PC3 shVec cells incubated with FA-HA/PEG-gelatin/EGCG nanoparticles.

15



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Figure 3. (a) Particle size distributions and zeta potentials of HA/PEG-gelatin/EGCG nanoparticles with varying

constituent

weight

ratios;

(b)

Transmission

electron

microscopy

micrographs

of

HA/PEG-gelatin/EGCG and HA/gelatin/EGCG nanoparticles in distinct buffers; (c) In vitro EGCG release profiles from HA/PEG-gelatin/EGCG and HA/gelatin/EGCG nanoparticles at 37°C (n = 5).

3.3.Viability of PC3 cancer cells after treatment with EGCG solution and HA/PEG-gelatin/EGCG nanoparticles To evaluate tumor cytotoxicity of test samples, PC3 shVec prostate cancer cell viability was determined using MTT assays after treatments with various concentrations of EGCG. In these experiments, EGCG-loaded nanoparticles and EGCG

solution

significantly

reduced

cancer

cell

survival

in

a

concentration-dependent manner, with half inhibitory concentrations (IC50) of 250.0 and 500.0 mg/L for EGCG-loaded nanoparticles and EGCG solution, respectively (Figure 4a). Mean Rh6G–EGCG fluorescence intensities in PC3 shVec cells were 723.93 ± 35.61 after 2 h treatment with HA/PEG-gelatin/Rh6G–EGCG nanoparticles and 405.35 ± 17.03 after 2 h treatment with Rh6G–EGCG solution. Moreover, maximum fluorescence of EGCG-loaded nanoparticles was 1.8 times greater than that 17



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of EGCG solution (p < 0.05). Subsequent CLSM analyses showed predominant localization of Rh6G–EGCG fluorescence (red; white arrows) signals from HA/PEG-gelatin/Rh6G–EGCG in intercellular spaces and cytoplasm, compared with that following treatment of Rh6G–EGCG solution alone (Figure 4b).

18


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Figure 4. (a) Cell viability was determined using 3-(4,5-dimethyl-thiazol-yl)-2,5-diphenyltetrazolium bromide assays after treatments with EGCG solution or HA/PEG-gelatin/EGCG nanoparticles for 2 h followed by 22 h in growth medium (n = 8); (b) Confocal images and EGCG fluorescence intensities of PC3 shVec cells indicate the uptake efficiency of Rh6G-EGCG-loaded HA/PEG-gelatin nanoparticles or Rh6G-EGCG solution at specific times. *Statistical significance at the p < 0.05 level.

3.4.Association of nanoparticles with PC3 shVec cells in FE-SEM and CLSM analyses Nanoparticle morphologies were examined in prostate cancer cells on matrigel-coated global eukaryotic microcarriers using FE-SEM (Figure 5a). These experiments showed three-dimensional cell growth morphology in matrigel-coated microcarriers. Moreover, HA/PEG-gelatin/EGCG nanoparticles (white arrows) were in active contact with PC3 shVec cells and released EGCG to inhibit prostate cancer cell growth. In further experiments, three-dimensional cancer cell cultures were incubated with fluorescent FA-HA/PEG-gelatin/Rh6G–EGCG nanoparticles (FA–HA: green spot and Rh6G–EGCG: red spot), and attachment and co-localization of nanoparticles and EGCG in intercellular spaces (white arrows) were observed using CLSM. These experiments indicated that the present nanoparticles deliver EGCG to cells, and were confirmed in CLSM analyses of tumor sections from mice that were injected with Rh6G–EGCG-loaded HA/PEG-gelatin nanoparticles or Rh6G–EGCG solution (Figure 5b; red spot; white arrows). In these observations, EGCG fluorescence of tumors in mice injected with Rh6G–EGCG-loaded-nanoparticles was significantly stronger than in mice injected with Rh6G–EGCG solution.

19



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Figure 5. (a) Field emission scanning electron microscope and confocal images of three-dimensional PC3 shVec cells on matrigel-coated microcarriers incubated with HA/PEG-gelatin/EGCG or fluorescent FA-HA/PEG-gelatin/Rh6G-EGCG nanoparticles; (b) Fluorescent images of nuclear staining in prostate tumor

cells

after

intravenous

injections

of

fluorescent

Rh6G-EGCG-loaded-HA/PEG-gelatin

nanoparticles or Rh6G-EGCG solution.

3.5.Evaluation of cell cycle arrest and apoptosis following HA/PEG-gelatin/EGCG nanoparticles treatments To determine whether HA/PEG-gelatin/EGCG nanoparticles induce cell-cycle arrest of prostate cancer cells, we performed PI staining and determined cell cycle distributions (Figure 6a). Exposure of PC3 shVec cells to EGCG-loaded nanoparticles

induced

G2/M

phase

cell

cycle

arrest

in

an

EGCG

concentration-dependent manner. In subsequent Western blot experiments, apoptotic mechanisms of HA/PEG-gelatin/EGCG nanoparticles were investigated according to expression levels of apoptosis-related proteins PARP, caspase-3, and caspase-9. In these experiments, cleaved PARP protein expression was markedly increased after treatments with HA/PEG-gelatin/EGCG nanoparticles carrying various EGCG concentrations in prostate cancer cells (Figure 6b). Accordingly, cleavage of caspases-9 and -3 was significantly increased at higher EGCG concentrations of HA/PEG-gelatin/EGCG nanoparticles (Figure 6b). In addition, Annexin V-FITC staining (Figure 6c) showed that treatment with various EGCG concentrations of HA/PEG-gelatin/EGCG nanoparticles led to increased green fluorescence expression of apoptotic cells in CLSM analyses.

21



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Figure 6. (a) Percentages of PC3 shVec cells in G0/G1, S, and G2/M phases after treatment with HA/PEG-gelatin/EGCG nanoparticles are indicated in flow cytometry histograms. The data are the means ± standard deviations of three independent experiments; (b) Western blot analysis of apoptosis-related proteins caspase-3, -9, and PARP in PC3 shVec cells after incubation with HA/PEG-gelatin/EGCG nanoparticles; β-actin was used as an internal control; (c) Fluorescence detection of apoptotic PC3 shVec cells after treatment with HA/PEG-gelatin/EGCG nanoparticles; Untreated controls were labeled with Annexin V-FITC and analyzed using confocal laser scanning microscopy.

3.6.Evaluation of antitumor activity and histology To investigate tumor-specific delivery of the present nanoparticles in prostate carcinoma xenografts in nude mice (Figure 7), we monitored tumor growth in nude mice for 20 days after injections of saline (control), EGCG solution, or various EGCG-loaded nanoparticle formulations. In these experiments, tumor sizes increased 22


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significantly over time in the control group, and slightly delayed tumor growth was observed in mice injected with EGCG aqueous solution and EGCG-loaded HA/gelatin nanoparticle, with 1.93 ± 0.13- and 1.62 ± 0.08-fold increases in prostate tumor volumes, respectively. Tumor growth was significantly inhibited in mice after treatment with HA/PEG-gelatin/EGCG nanoparticles, with smaller tumor sizes and weights than those from HA/gelatin/EGCG nanoparticle-treated mice (p < 0.05; Figure 7). These data confirm that large numbers of HA/PEG-gelatin/EGCG nanoparticles are actively targeted to tumor sites (Figure 5), and that subsequent release of encapsulated EGCG inhibits tumor growth. After sacrificing mice, prostate tumor tissue biopsies were subjected to histological examination with hematoxylin and eosin staining (Figure 8). Tumor sections from control (saline-treated) animals showed moderately abundant granular eosinophilic cells (red arrows) compared with those in mice treated with HA/PEG-gelatin/EGCG nanoparticles. Furthermore, immunohistochemical analyses showed increased cleaved PARP and diminished expression of the proliferation marker protein Ki-67 in tumors from mice treated with HA/PEG-gelatin/EGCG nanoparticles (coffee dots, Figure 8). These observations indicate that the targeted nanoparticles induce apoptosis in cancer cells, leading to significant increased antitumor activity and reduced inflammation.

23



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Figure 7. Antitumor effects of EGCG-loaded nanoparticles in vivo; Nude mice xenograft models were generated using tumor cells that were derived from human prostate cancers. PC3 shVec cells were injected into the flanks of nude mice to establish human prostate cancer xenografts. Mice were divided into four groups of six mice and were treated with normal saline solution (■), EGCG solution (●), HA/gelatin/EGCG nanoparticles (♦), or HA/PEG-gelatin/EGCG nanoparticles (▲) every second day for 20 days, as described in the materials and methods section. (a) Tumor volumes were calculated every second days. (b) Average masses and images of tumors; (c) Changes in relative body weights. *Statistical significance at the p < 0.05 level.

24


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Figure

8.

Prostate

tumor

biopsy

of

histological

staining

with

hematoxylin–eosin

and

immunohistochemical staining analyses of prostate tumor-bearing mice after treatment with normal saline

solution,

EGCG

solution,

HA/gelatin/EGCG

nanoparticles,

or

HA/PEG-gelatin/EGCG

nanoparticles.

4. Discussion Prostate cancer treatment has improved dramatically with increased awareness and earlier diagnosis, large numbers of cases progress to aggressive metastatic disease.33 Accordingly, prostate cancer cells were previously derived from a metastatic lesion in bone, adhered strongly to bone marrow endothelial cells, and had high CD44 expression on cell membranes.34-36 Herein, we investigated tumor formation in a mouse prostate cancer cell (PC3 cells) xenograft model with 25



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differential CD44 protein expression. Following subcutaneous injections of scramble or CD44 shRNA infected PC3 cells into the dorsal flanks of nude mice, CD44 knock down cells (PC3 shCD44 cells) produced smaller prostate tumors than control PC3 shVec cells (Figure 2a). HA is a straight chain glycosaminoglycan polymer that is a predominant component of extracellular matrixes in almost all vertebrate tissues.37,38 Previous studies demonstrate covalent attachment of HA to the surfaces of nanoparticles and polymersomes that efficiently target epithelial cancer cells by HA receptors such as CD168 and CD44.21 These proteins are highly expressed in many types of tumors, including prostate cancers and gastric cancers, and in various types of leukemias, lymphomas, and myelomas.36,39 Mizrahy et al. previously used high molecular weight HA-bound nanoparticles as vehicles for targeting to CD44 overexpressing tumors and aberrantly activated leukocytes under inflammatory conditions.21 Therefore, we examined the kinetics of HA internalization by PC3 shVec cells at various HA molecular weights using spectrofluorometry. In these analyses, HA molecules of 200 kDa showed maximal fluorescent intensity in cells until 2 h (Figure 2b). Moreover, after incubation with fluorescent nanoparticles (FA-HA/PEG-gelatin/EGCG; FA-HA, green spot), co-localization and interaction with cell surface CD44 (red spot) were observed in PC3 shVec cells (white arrows indicate superimposed red/green spots; Figure 2b). The development and design of nanoparticles that can simultaneously deliver therapeutic and diagnostic agents to target sites are being intensively studied by researchers in the nanomedical field.40,41 HA complexes with EGCG did not form complete structures, and broad size distributions were indicated by the polydispersity index (PDI) of 0.87 ± 0.21, and were associated with low EGCG loading efficiencies (14.68% ± 5.62%). Thus, mixtures of PEG-gelatin were blended to form appropriate structures of encapsulated EGCG. Gelatin is a natural polymer that has been used to bind polyphenols through hydrogen bonding interactions between hydrophobic proline residues and polyphenols on phenol rings.42 Furthermore, PEG is a 26


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biocompatible nontoxic polymer that has been used widely to modify other polymers, offering reduced immunogenicity and improved plasma drug half-lives.29,43 The present nanoparticle system comprising HA:PEG-gelatin:EGCG at a ratio of 1.25:3.75:2.00 mg/mL had a spherical matrix structure (Figure 3b), a mean particle size of 198.37 ± 11.62 nm and a mean negative zeta potential value of −27.83 ± 2.98 mV (Table 1). Moreover, HA/PEG-gelatin/EGCG nanoparticles formed stable structures in PBS, and achieved controlled EGCG release of 13.78% ± 3.12% over 2 h (Figure 3c). Numerous previous studies have identified naturally-occurring chemopreventive or chemotherapeutic botanicals that inhibit, retard, or reverse carcinogenesis, including that in prostate cancer patients. Polyphenols are the abundant and pharmacological component in green tea, and the ensuing food functions have been related to EGCG.44 Moreover, EGCG reportedly suppresses tumor growth by inhibiting cell proliferation and inducing cell apoptosis via various biological mechanisms.45 HA/PEG-gelatin/EGCG nanoparticles were used to target prostate cancer cells (Figure 4). Subsequently, fluorescent HA/PEG-gelatin/Rh6G– EGCG nanoparticles led to greater Rh6G–EGCG fluorescence in cancer cells than Rh6G–EGCG solution alone, demonstrating the potential for increased cancer cell growth inhibition. In addition, EGCG-loaded nanoparticles were attached to prostate cancer PC3 shVec cells in three-dimensional cultures (Figure 5). To determine mechanisms of PC3 shVec cell growth inhibition by EGCG-loaded nanoparticles, we investigated cell cycle regulation and showed induction of cell cycle arrest at the G2/M phase by EGCG-loaded nanoparticles (Figure 6a). Moreover, EGCG-induced cell growth inhibition was accompanied by accumulation of cancer cells in the G2/M phase.46,47 Apoptosis is a highly conserved process, and involves numerous signaling pathways that coordinate cellular changes throughout the cell death process.48,49 Among these, apoptosis is predominantly regulated by sequential activation of caspases.50 Polyphenols such as EGCG reportedly trigger intrinsic apoptotic pathways by regulating mitochondrial function, activating caspases-3 and -9, 27



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Page 28 of 51

and inducing cleavage of PARP.51 Western blot analyses showed that EGCG-loaded nanoparticles triggered apoptosis by activating caspases-9 and -3 and inducing proteolytic cleavage of PARP (Figure 6b). These data were confirmed by increased numbers of Annexin V bound apoptotic cells via fluorescence immunohistochemical analyses (Figure 6c). Additionally, in vivo studies confirmed antitumor activities against prostate tumors at doses of 2.0 mg/kg EGCG in EGCG-loaded samples. Specifically, significant increases in prostate tumor growth inhibition were reflected by

smaller

tumor

sizes

and

no

differences

in

body

weight

loss

in

EGCG-loaded-HA/PEG-gelatin-nanoparticle-treated animals compared with control and EGCG-treated animals (Figure 7). Finally, histological analyses demonstrated that HA/PEG-gelatin/EGCG nanoparticles deliver EGCG to tumors (Figure 5b) and reduce tissue inflammation (Figure 8). Taken together, the present data indicate that our targeted nanoparticles specifically interact with prostate tumor cells via CD44 receptors and warrant further studies to identify alternative anticancer treatments in future clinical trials.

5. Conclusion In conclusion, the present experiments indicate that HA/PEG-gelatin/EGCG nanoparticles can effectively target prostate cancer sites via CD44 receptor-mediated recognition, leading to significant reductions in prostate tumor activities and tumor tissue inflammation and increases in cancer cell death via apoptosis. These targeted nanoparticles have potential as a delivery system for anticancer drugs.

Acknowledgments This work was supported by Ministry of Science and Technology (MOST 103-2320-B-039-002-MY3),

China

Medical

University

(CMU104-S-19

and

CMU105-S-36). The PC3 shVec and PC3 shCD44 prostate cancer cells were provided from Prof. Jer-Tsong Hsieh in Department of Urology, Southwestern Medical Center

28


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at Dallas. Authors would like to thank Enago English editing for the English language review.

Disclosure Statement: all authors have no conflicts of interest to declare for the work.

29



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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table of Contents

36


Page 36 of 51

1

O

3

CH3 O

2

PEG

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

2,3,4 PEG

O n

O

O

4

1 PEG

NH2 COO-

Lysine

Lysine

NH3+

NH2

Gelatin

Figure 1


a.

3gelatin 1gelatin 9gelatin

8gelatin

7gelatin

4gelatin 5gelatin

6gelatin

2gelatin

NH2 NH3+ 2,3,4 PEG

Lysine

COO-

Lysine O

NH O

1 PEG

1

3

O

4

O

2

O

CH3

n

3gelatin 9gelatin

PEG–gelatin

Page 37 of 51

5

1gelatin

4gelatin 5gelatin

8gelatin

7gelatin

4

3

6gelatin

2

ppm ACS Paragon Plus Environment

2gelatin

1

0

PEG-gelatin

Page 38 of 51

953

839

1243

HA

1541

1401 1620

EGCG

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

1534

HA/PEG-gelatin/ EGCG nanoparticles

Figure 1


b.

1098

950 1550

1387 827

1539

1236

1092

1625

4000

3200

2400 2000 1600 ACS Paragon Plus Environment

Wavenumber (cm-1)

1200

800

400

Page 39 of 51


Figure 2

a. PC3 shVec

PC3 shCD44

Hyaluronic acid (HA) CD44 protein

HA/PEG-gelatin/EGCG nanoparticles

Polyethylene glycol-gelatin (PEG-gelatin)

SEM morphology

Epigallocatechin-3-gallate (EGCG) GAPDH protein

Prostate tumor model

Nanoparticle solution situation

PC3 shVec

2.500:3.750:2.000

CD44

1.875:3.750:2.000

PC3 shCD44

1.250:3.750:2.000

Tumor weight 0.278  0.015 g

0.625:3.750:2.000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

CD44

HA:PEG-gelatin:EGCG concentration (mg/mL)

Tumor weight 0.058  0.006 g



Page 40 of 51

Figure 2

b.

600

(n = 6)

HA molecules (molecular weight 60 KDa) HA molecules (molecular weight 100 KDa)

Mean fluorescence intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Prostate cancer cells incubated with FA-HA/PEG-gelatin/ EGCG nanoparticles and CD44 immunofluorescence staining

HA molecules (molecular weight 200 KDa)

500 400 300 FA-HA

Cy5-CD44

Nuclei

Superimposition

200 Cell fluorescence observed treatment with FA-HA molecules by CLSM images

100 0

molecular weight 60 kDa

0.0

0.5

1.0


1.5


2.0

2.5

3.0

Incubation time (h)


Page 41 of 51


Figure 3

1

10

100

1000

10000

-100

Size (nm)

0

100

800000 1

10

Zeta Potentials (mV)

0 1

10

100

Size (nm)

1000

10000

100

200

500000

400000 500000 0

0

Total counts

Intensity (%)

10

5

100000

5

-100


100000

10

10000

15

300000

Total counts

15

1000

HA:PEG-gelatin:EGCG = 2.500:3.750:2.000 mg/mL

200000

20

100

Size (nm)

HA:PEG-gelatin:EGCG = 1.250:3.750:2.000 mg/mL 25

600000 0

0

200

400000 200000

5

400000

0

0

10

300000

2

Total counts

400000 100000

4

15

Intensity (%)

6

300000

Total counts

8

20

200000

10

Intensity (%)



200000

a.

Intensity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

-100

0

100

200

0

0 1

Zeta Potentials (mV) ACS Paragon Plus Environment

10

100

Size (nm)

1000

10000

-100

0

100


200


Page 42 of 51

Figure 3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

b.

HA/PEG-gelatin/EGCG nanoparticles in distinct buffer

Deionized water

PBS buffer

PBS with FBS buffer

HA/gelatin/EGCG nanoparticles in distinct buffer

Deionized water

PBS buffer

PBS with FBS buffer 200 nm


Page 43 of 51


Figure 3 c.

100 Cumulative amount of EGCG release (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

(n = 5)


90

HA/gelatin/EGCG nanoparticles

80 70 60 50 40 30 20 10 0 0

6

12

18

24 Time (h)


30

36

42

48


Page 44 of 51

Figure 4 a.

110 (n = 8)

100

Cell viability (%)

90 80 70 60 50 40

*

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

30 20 EGCG solution

10


0 0

50 100 150 200 250 300 350 400 450 500 550 EGCG concentration (mg/L)


Page 45 of 51


Figure 4

b. 900 800

Mean fluorescence intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

(n = 6)

HA/PEG-gelatin/Rh6G-EGCG nanoparticles * Rh6G-EGCG solution

HA/PEG-gelatin/ Rh6G-EGCG nanoparticles

*

700 600

* 500 400 Rh6G-EGCG solution

300 200 100 0 0.0

0.5

1.0

2.0

3.0

Incubation time (hrs)



Page 46 of 51

Figure 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

a.

Control group

1000X

3000X

7000X

30000X


1000X

3000X

7000X

30000X

FA-HA/PEG-gelatin/Rh6G-EGCG nanoparticles

DIC Image

ACS Paragon Plus FA-HAEnvironment

RH6G-EGCG

Superimposition 40 μm

Page 47 of 51

Figure 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


b.

HA/PEG-gelatin/Rh6G-EGCG nanoparticles 6 hr

DIC Image

RH6G-EGCG

Superimposition

DIC Image

RH6G-EGCG

Superimposition

24 hr

Rh6G-EGCG solution 24 hr

DIC Image

ACS Paragon Plus Environment RH6G-EGCG

Superimposition

Control group

G0/G1 57.97 ± 1.59 S 12.64 ± 1.16 G2/M 29.39 ± 1.13

G2/M

S

0

G0/G1

G0/G1 51.62 ± 1.24 S 13.39 ± 0.85 G2/M 34.99 ± 0.73

150

G2/M

S

0

DNA content

c.

DNA content Control group

EGCG 250 mg/L 512

G0/G1 45.25 ± 1.33 S 14.85 ± 1.29 G2/M 39.90 ± 0.53 G0/G1 G2/M

S

0

DNA content HA/PEG-gelatin/EGCG nanoparticle (EGCG 50 mg/L)

Annexin V-FITC (green spot)

250

Caspase-9

50

G0/G1 47.28 ± 0.98 S 14.09 ± 1.29 G2/M 38.63 ± 0.72

G0/G1

S

HA/PEG-gelatin/EGCG nanoparticle (at EGCG con. mg/L)

－

EGCG 150 mg/L 512

G2/M

0

DNA content

Procaspase-9 Cleaved caspase-9 1.00

1.09

1.54

1.61

Caspase-3

Procaspase-3

Cleaved caspase-3 1.15

1.21

1.39

PARP

1.00

HA/PEG-gelatin/EGCG nanoparticle (EGCG 150 mg/L) Annexin V-FITC (green spot)

PARP Cleaved PARP

1.00

β-actin

b.

512

Cell number

G0/G1

Cell number

512

Page 48 of 51

HA/PEG-gelatin/EGCG nanoparticle groups EGCG 50 mg/L

Cell number

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


Cell number

Figure 6 1a.

1.05

1.12

1.24


HA/PEG-gelatin/EGCG nanoparticle (EGCG 250 mg/L) Annexin V-FITC (green spot)

2.8

(n = 6)

Normal saline solution EGCG solution HA/gelatin/EGCG nanoparticles

2.6 2.4

b. (n = 6) 0.35


2.2

0.30

2.0 1.8 1.6 1.4

*

Tumor weight (g)

*

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Tumor volume ratio

Figure 7


a.

0.25

0.20

0.15

1.2

0.10

1.0

0.05

0.8

0.00

0.6

Normal saline solution

EGCG solution

HA/gelatin/ EGCG nanoparticles


0.4 0

2

4

6

8

10

12

14

16

18

20

22

Days after treatment c.

(n = 6)

Normal saline solution EGCG solution

1.4

HA/gelatin/EGCG nanoparticles HA/PEG-gelatin/EGCG nanoparticles

Relative body weight

Page 49 of 51

1.2

1.0

0.8

0.6

0

2

4

6

8

10

12

14

Days after treatment

16

Normal saline ACS Paragon Plus Environment solution 18

20

22

EGCG solution

HA/gelatin/EGCG HA/PEG-gelatin/EGCG nanoparticles nanoparticles


Page 50 of 51

Figure 8

Cleaved-PARP

Ki-67

HA/gelatin/ EGCG nanoparticles

EGCG solution

Normal saline solution

Hematoxylin and eosin


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


400X

Page 51 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


Table of Contents

Intravenous injection of HA/PEGgelatin/EGCG nanoparticles

FA-HA (green spot)/PEG-gelatin/EGCG nanoparticle targeting CD44 (red spot)

CD44

Prostate tumor

EGCG

drug release

Rh6G-EGCG nucleus

Prostate tumor

nucleus


Nanoparticle Targeting CD44-Positive Cancer Cells for Site-Specific

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