Article pubs.acs.org/Biomac
Active Targeted Nanoparticles for Oral Administration of Gastric Cancer Therapy Yu-Hsin Lin,*,†,‡ Zih-Rou Chen,† Chih-Ho Lai,#,§ Chia-Hung Hsieh,∥ and Chun-Lung Feng⊥ †
Department of Biological Science and Technology, China Medical University, Taichung, Taiwan Department of Urology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, United States # Department of Microbiology and Immunology, Graduate Institute of Biomedical Sciences, Chang Gung University, Taoyuan, Taiwan § Graduate Institute of Basic Medical Science & School of Medicine, China Medical University, Taichung, Taiwan ∥ Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan ⊥ Division of Hepatogastroenterology, Department of Internal Medicine, China Medical University Hospital, Taichung, Taiwan Downloaded by EMORY UNIV on August 25, 2015 | http://pubs.acs.org Publication Date (Web): August 19, 2015 | doi: 10.1021/acs.biomac.5b00907
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ABSTRACT: Gastric carcinogenesis is a commonly diagnosed type of cancer and has a dismal prognosis because of the rate at which it aggressively spreads and because of the lack of effective therapies to stop its progression. This study evaluated a type of oral drug delivery system of a potential target-activated nanosizer comprising a fucoseconjugated chitosan and polyethylene glycol-conjugated chitosan complex with gelatin containing encapsulated green tea polyphenol extract epigallocatechin-3-gallate, allowing oral administration of the drug through a site-specific release in gastric cancer cells. The results demonstrated that the nanoparticles effectively reduced drug release within gastric acids and that a controlled epigallocatechin-3-gallate release inhibited gastric cancer cell growth, induced cell apoptosis, and reduced vascular endothelial growth factor protein expression. Furthermore, in vivo assay results indicated that the prepared epigallocatechin-3-gallate-loaded fucose-chitosan/ polyethylene glycol-chitosan/gelatin nanoparticles significantly affected gastric tumor activity and reduced gastric and liver tissue inflammatory reaction in an orthotopic gastric tumor mouse model.
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INTRODUCTION Gastric cancer is a commonly diagnosed cancer and results in deaths worldwide.1,2 Interest in combination therapy for gastric cancer began with the realization that single agents (e.g., fluorouracil, doxorubicin, mitomycin, or cisplatin) have only modest activity against this disease.3,4 Although the disease is relatively chemosensitive, chemotherapy delivered in an advanced setting is limited by a low complete response rate, short-lived response durations, and considerable toxicities, such as neutropenia or anemia.5,6 This has compelled researchers to seek alternative types of reagents for treating gastric cancer. Green tea has been widely used for a long period of time and has diverse biological and pharmacological activities. Among tea catechins, (−)-epigallocatechin-3-gallate (EGCG), a major constituent in the green tea polyphenol extract, has shown protection against inflammation or oxidative damage.7−9 The green tea constituent EGCG is the most abundant and active component of green tea and has been demonstrated to inhibit tumor growth by antiangiogenesis, by inhibiting proliferation, and by inducing apoptosis.10,11 However, a limiting factor of treatment with this compound is its poor bioavailability of tea catechins that result from instability under digestive conditions, poor transcellular transport, and rapid metabolism followed by excretion.12,13 © XXXX American Chemical Society
Development of a drug delivery carrier to cancer cells remains a major challenge. Several approaches, such as through liposomes, micelles, and nanoparticle carrying anticancer drugs, have been utilized for drug delivery to cancer cells.14−16 However, the challenge with this method of treatment is poor drug bioavailability, primarily because of low mucosal permeability and the instability of the gastrointestinal environment. To overcome this problem, chemical modification of drug carriers with particular synthetic polymers has been frequently employed in attempts to increase in vivo longevity. Polyethylene glycol (PEG) is a hydrophilic nonionic polymer and has no toxic and antigenic properties and reduces immunogenicity and prolongs the protein permanence in blood.17−19 Furthermore, PEG is biocompatible and widely used in graft-forming polymers, where it functions as a crosslinker and forms interconnected channels to improve protein stability toward pepsin protease in an oral delivery system.20−22 Another method of actively targeting cancer cells is by using nanoparticles that are conjugated with molecules that bind to antigens or receptors on cancer cells. Fucose, a type of Received: July 7, 2015 Revised: August 8, 2015
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DOI: 10.1021/acs.biomac.5b00907 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules
Figure 1. Representation of the prepared FCS/PCS/Gel/EGCG nanoparticles and the strategy and observation against gastric carcinoma using the nanoparticles. available CS, was used to prepare PCS. CS (MW 50000 Da) with approximately 85% deacetylation was treated with sodium perborate (NaBO3) and continuously stirred at 50 °C for 12 h, then depolymerized CS was precipitated with aqueous sodium hydroxide at pH 7.2−7.4, and the precipitated CS was washed three times with deionized water. The average molecular weight was determined by a gel permeation chromatography system equipped with a series of PL aquagel−OH columns (one Guard 8 μm, 50 × 7.5 mm and two MIXED 8 μm, 300 × 7.5 mm, PL Laboratories, UK) and a refractive index detector. Polysaccharide standards (molecular weights range from 180 to 788000 Da) were used to construct a calibration curve. The synthesis procedure for PCS was as follows: 0.1 g of CS was dissolved in 10.0 mL of dimethyl sulfoxide (DMSO, 5.0 mL) and 1.0% acetic acid (5.0 mL); 4.0 mL of DMSO containing 0.05 g of methoxyl PEG succinimidyl ester (mPEG−NHS, MW 5000 Da) was added with continuous stirring at room temperature for 12 h. To remove the unconjugated mPEG−NHS, the sample was dialyzed in 5 L of deionized water, which was replaced six times per day. The resultant PCS was lyophilized in a freeze-dryer. The degrees of grafting density on PCS were determined by the potassium polyvinylsulfate titration method, and the purified PCS was confirmed by NMR and FTIR analyses.30 Preparation of FCS/PCS/Gel/EGCG Nanoparticles. The FCS/ PCS/Gel/EGCG nanoparticles were prepared by dropping aqueous EGCG into an aqueous FCS/PCS/Gel mixed solution. The fucoseconjugated CS (FCS) was synthesized essentially as described in our previous study.31 The 0.5 g of CS was dissolved in 50 mL of a 1:1 mixture of methanol and 1.0% acetic acid; 10 mL of deionized water containing 0.5 g of fucose was added with continuous stirring. Then, 1.0 g of sodium cyanoborohydride was added into a FCS solution and allowed to react for 6 h. To remove unconjugated fucose, the sample was dialyzed against 5 L of deionized water, and the resultant fucosechitosan was lyophilized in a freeze-dryer. A mixture of aqueous FCS/ PCS at distinct concentrations (2.50:2.50, 5.00:5.00, and 7.50:7.50 mg/mL, 0.50 mL) was added into an aqueous Gel solution in deionized water (10.00 mg/mL, 0.50 mL) at FCS/PCS/Gel = 1.25:1.25:5.00, 2.50:2.50:5.00, and 3.75:3.75:5.00 mg/mL. The aqueous EGCG solution in deionized water (4.00 mg/mL, 1.0 mL) was added to 1.0 mL of aqueous FCS/PCS/Gel mixed solution and stirred at room temperature for 30 min to form nanoparticles at FCS/ PCS/Gel/EGCG = 0.625:0.625:2.500:2.000, 1.250:1.250:2.500:2.000, and 1.875:1.875:2.500:2.000 mg/mL. The nanoparticles were collected by centrifugation at 15000g for 50 min. The nanoparticles were resuspended in deionized water, and their size distribution and zeta potential were evaluated with a zetasizer (Malvern Instruments Ltd., UK). FTIR spectra of the prepared nanoparticles were recorded
deoxyhexose sugar, physiologically assists in modifying various types of molecules in mammal cells. Yoshida et al. utilized fucose-bound nanoparticles as vehicles in anticancer drug delivery to pancreatic cancer cells.15 Increased levels of fucose are frequently found in the sera and urine of patients with various types of cancers, including gastric cancer. This suggests that fucosylation increases inside cancer cells.23−25 Overall, oral administration of a drug through site-specific and targetactivated release in the stomach would allow for oral drug delivery treatment of gastric carcinoma. We developed nanoparticles composed of fucose-conjugated chitosan (fucose-chitosan; FCS) and PEG-conjugated chitosan (PEG-chitosan; PCS) complexes with a gelatin (Gel) of encapsulated green tea polyphenol extract of EGCG (Figure 1). Chitosan (CS), a linear polymer with the charged amino group of D-glucosamine residues, may interact with Nacetylnuraminic acid in gastric mucus through electrostatic interaction. This would provide a longer residence time in the stomach.26 Gel is a biodegradable polymer that contains a large numbers of glycine residues, including proline and 4hydroxyproline residues, both of which are major amino acids.27,28 Physicochemical characteristics were studied by Fourier transform infrared (FTIR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, dynamic light scattering, and transmission electron microscopy (TEM). Drug effects and mechanism of interaction with human gastric cancer cells (MKN45 cells) were investigated by a field emission scanning electron microscope (FE-SEM), confocal laser scanning microscopy (CLSM), and Western blotting of growth factor proteins. An in vivo experiment to verify the inhibition of gastric tumor growth after treatment with fucosechitosan/PEG-chitosan/gelatin/EGCG nanoparticles (FCS/ PCS/Gel/EGCG nanoparticles) was performed in an orthotopic bioluminescent expression gastric tumor mouse model. Furthermore, histological examination was performed to detect gastric and liver tissue inflammation levels.
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EXPERIMENTAL SECTION
Preparation and Characterization of PCS. PCS was synthesized as previously described, with some modification.29 A low-molecular weight CS, obtained from the depolymerization of a commercially B
DOI: 10.1021/acs.biomac.5b00907 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules
°C into the donor compartment of the cells, and the samples were then aspirated after incubation. After three PBS washes, the cells were fixed in 3.7% paraformaldehyde, washed again three times with PBS, and permeabilized with 0.2% triton X-100 for 15 min at 37 °C. The washes were repeated, and the cells were stained with DAPI, which binds to nuclei specifically; the stained cells were observed by CLSM with excitation at 340, 488, 543, and 633 nm. The 2 × 105 cells were then incubated into matrigel-coated global eukaryotic microcarrier GEM (Global Cell Solution, USA) in specialized culture vessels (LeviTubes) in the bioWiggler at 37 °C and 5% CO2 for 7 days to track the nanoparticle internalization of three-dimensional gastric cancer cells. The EGCG-loaded nanoparticles were added to the cells for 2 h, then the samples were aspirated. The cells were washed with PBS, fixed in 4.0% glutaraldehyde, and dehydrated with a series of ethanol solutions (35%−100%), then finally soaked in 100% ethanol. Before visual inspection via FE-SEM, each sample was subjected to supercritical carbon dioxide drying and sputter coated with 60/40 gold−palladium. Confocal Microscopy Image and Flow Cytometry Analysis of Cell Apoptosis. To assay cell apoptosis expression, the cells were seeded onto glass coverslips at a density of 3 × 105 cells/cm2 and incubated for 24 h. EGCG-loaded nanoparticles (EGCG concentrations at 10, 20, and 40 mg/L) were added to the cells for 2 h. After incubation, the test samples were aspirated. After two PBS washes, the cells were incubated in a growth medium for an additional 22 h, then washed twice with PBS again before 5 μL of FITC Annexin V solution was added from a commercial kit (BD Biosciences, USA). After 15 min, the stained cells were examined with excitation at a 488 nm laser line and the emission recorded at 510 nm under CLSM. In addition, MKN45 cells were cultured at 37 °C for 24 h, followed by treatment with medium (control group) and EGCG-loaded nanoparticles for 2 h. The treatments were removed, replaced with culture medium, and incubated for an additional 22 h. The treated cells were then harvested and stained with the standard detection kit (FITC Annexin V/ Propidium Iodide apoptosis detection kit; BD Biosciences), according to the manufacturer’s protocol. The stained cells were analyzed with a FACSCalibur flow cytometer (Becton Dickinson, USA). Data were collected using 20,000 cells from each sample and analyzed by Cell Quest software WinMDI (Verity Software House, Inc., USA). All of the samples were examined in triplicate from three independent experiments. Western Blotting and Immunofluorescence Staining. Western blotting and immunofluorescence staining were conducted to assay the expression of vascular endothelial growth factor (VEGF) protein after treatment with the prepared EGCG-loaded nanoparticles in a coculture with MKN45 cells for 2 h. The test samples were aspirated after incubation, and the cells were washed twice with PBS and incubated in growth medium for an additional 22 h. The cells were washed three times with PBS and lysed in lysis buffer with a protease inhibitor cocktail (Roche, Germany) to collect the required protein. The Bradford method was used to measure the obtained protein concentration, which was then boiled for 5 min in sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) buffer. The protein samples were then transferred onto polyvinylidene difluoride membranes after being resolved by 12% SDS−PAGE. Following electrotransfer, the membranes were incubated in PBST buffer at 4 °C with the appropriate primary antibodies [rabbit anti-VEGF antibody or mouse antiglyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) (Chemicon Europe Ltd., UK)] overnight. Then, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies, and the proteins were visualized by enhanced chemiluminescence Western blotting detection reagents (GE Healthcare, USA) and detected by X-ray films (Kodak, NY). Immunofluorescence staining was used to observe the interaction of the prepared EGCG-loaded nanoparticles on VEGF protein expression. Further, the prepared fluorescent nanoparticle (FITC− FCS/PCS/Gel/Rh6G−EGCG nanoparticles)-treated cells were fixed in 3.7% paraformaldehyde and stained for VEGF protein by the immunofluorescence method. After PBS washing, the fixed cells were permeabilized with 0.5% triton X-100 and 100 μg/mL ribonuclease for
with an FTIR spectrometer (Shimadzu Scientific Instruments, USA). The amount of free EGCG in the supernatant was analyzed using high-performance liquid chromatography (HPLC) with a UV detector and a reversed phase C18 column. It was eluted with acetonitrile− 0.005 M citric acid (14:86; v/v) at a flow rate of 1.0 mL/min. EGCG loading efficiency and loading content of the nanoparticles was calculated from the following equation: loading efficiency =
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loading content =
total amount of EGCG − free EGCG total amount of EGCG
total amount of EGCG − free EGCG weight of nanoparticles
Characterization of the Prepared Nanoparticles and EGCG Release Profiles. The prepared nanoparticles of their size distribution and zeta potential were measured by a zetasizer at pH 1.2 (0.1 N hydrochloric acid with 0.8 mg/mL pepsin) and pH 5.5 (5 mM acetate buffer) and pH 6.8 (10 mM phosphate-buffered saline; PBS), simulating gastric acid, gastric mucosa, or gastric tumor tissue environments.32−34 Additionally, continuous EGCG release profiles from EGCG-loaded nanoparticles were investigated in a simulated dissolution medium (pH 1.2 with pepsin for 120 min, then pH 5.5 for another 120 min, and pH 6.8 for the other 360 min) during the 600 min of EGCG release experiment at 37 °C. At specific time intervals, the samples were removed and centrifuged, and the supernatants underwent HPLC. The percentage of cumulative amount of EGCG released was determined from a standard calibration curve. Viability of MKN45 Cells Treated with EGCG Solution and FCS/PCS/Gel/EGCG Nanoparticles. The Japanese Collection of Research Bioresources Cell Bank provided the MKN45 cell line, which were initially grown in cell culture flasks with RPMI 1640 medium containing 10% fetal calf serum, then harvested, and used in cytotoxicity experiments. After seeding at a concentration of 1 × 104 cells/well in 96-well plates, the MKN45 cells were allowed to adhere overnight. The growth medium was substituted by an HBSS solution (pH 6.0) that contained the aqueous EGCG solution in deionized water and EGCG-loaded nanoparticles at various EGCG concentrations (0, 10, 20, 30, and 40 mg/L) for 2 h. Then, the test samples were aspirated, and cells were washed twice with PBS. The cells were then cultured in growth medium for an additional 22 h, followed by incubation in the growth medium containing 1 mg/mL MTT for an additional 4 h. To ensure solubilization of resulting formazan crystals, DMSO (100 μL) was added to each well. A microplate spectrofluorometer (Molecular Devices SpectraMax M2e, USA) was used to measure optical density at a wavelength of 570 nm.35,36 In Vitro Cellular Uptake and CLSM Visualization. The fluorescent FITC−FCS/Cy5−PCS/Gel/Rh6G−EGCG nanoparticles were produced according to the procedure described in the preparation of FCS/PCS/Gel/EGCG nanoparticles section; fluorescein isothiocyanate (FITC)-labeled FCS and rhodamine 6G (Rh6G)labeled EGCG were synthesized as described elsewhere.22 The fluorescent cyanine 5 (Cy5)-PCS was produced based on the reaction between free amines on PCS and N-hydroxysuccinimide on Cy5− NHS. A Cy5−NHS solution in DMSO (1 mg/mL) was gradually added to aqueous PCS in deionized water with stirring overnight in the dark. Cy5−PCS was dialyzed with 5 L of deionized water that was replaced daily to remove unconjugated Cy5. The Cy5−PCS resultant was then lyophilized in a freeze-dryer. Furthermore, Rh6G−EGCG solution or Rh6G−EGCG-loaded FCS/PCS/Gel nanoparticles were used for specific durations to treat MKN45 cells plated in 12-well plates. The cells were rinsed three times with PBS and solubilized with 0.5% triton X-100 in 0.2 M NaOH (1 mL). By analyzing the cell lysates in a microplate spectrofluorometer, the cell-associated test samples were quantified. At a density of 5 × 105 cells/insert, MKN45 cells were grown on Costar Transwell plates. The cell culture medium was added to the donor and acceptor compartments, then the cultures were kept in an incubator and used for 24−30 days. Furthermore, the medium (pH 6.0) containing the Rh6G−EGCG solution and FITC−FCS/Cy5− PCS/Gel/Rh6G−EGCG nanoparticles was introduced for 2 h at 37 C
DOI: 10.1021/acs.biomac.5b00907 Biomacromolecules XXXX, XXX, XXX−XXX
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Figure 2. Nuclear magnetic resonance of PCS and fluorescent FCS/PCS/Gel/EGCG nanoparticles incubated in three-dimensional MKN45 cells and noninvasive in vivo images of micee after oral treatment of near-infrared fluorescent nanoparticles.
Figure 3. FTIR spectra of PCS, FCS, Gel, EGCG, and FCS/PCS/Gel/EGCG nanoparticles. nanoparticles and EGCG solution were studied in vivo using athymic nude mice xenografts. Five-week-old male BALB/c nude mice were obtained from the National Laboratory Animal Center, Taiwan. The mice were treated in accordance with the Animal Care and Use Guidelines for China Medical University, under a protocol approved by the Institutional Animal Care Use Committee. The mice were allowed to acclimatize to local conditions for a single week before being injected with gastric cancer cells. The luminescent expression MKN45 cells (Luc MKN45) were obtained from JCRB and grown in RPMI 1640 medium containing 10% fetal calf serum. For development of the orthotopic gastric tumor model, Luc MKN45 cells were
15 min at room temperature. The permeabilized cells were blocked in 5% normal goat serum in PBS for 60 min, then treated with rabbit anti-VEGF antibody at a 1:50 dilution for 60 min at 37 °C. After three PBS washes, they were incubated in Cy5-conjugated goat antirabbit IgG at a 1:100 dilution (Jackson ImmunoResearch Laboratories, USA), then uniformly mounted on slides and examined under CLSM. In Vivo Orthotopic Gastric Tumor Mouse Models. Animal care and its use complied with the 1996 revision of the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources, National Research Council and published by the National Academy Press. The effects of oral FCS/PCS/Gel/EGCG D
DOI: 10.1021/acs.biomac.5b00907 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules
Table 1. Effect of Different Fucose-Chitosan/PEG-Chitosan Concentrations on Particle Sizes, Polydispersity Indices (PDI), Zeta Potential Values, and Loading Efficiency of the Fucose-Chitosan/PEG-Chitosan/Gelatin/EGCG Nanoparticles (n = 5)a
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a
fucose-chitosan: PEG-chitosan/gelatin/EGCG (mg/mL)
mean particle size (nm)
zeta potential value (mV)
PDI
loading efficiency (%)
loading content (%)
0.625:0.625:2.500:2.000 1.250:1.250:2.500:2.000 1.875:1.875:2.500:2.000
758.8 ± 16.4 409.7 ± 9.8 257.5 ± 5.1
12.6 ± 4.3 20.3 ± 2.6 28.9 ± 1.9
0.72 ± 0.11 0.49 ± 0.09 0.16 ± 0.04
32.3 ± 4.3 45.1 ± 3.6 53.7 ± 1.8
19.1 ± 2.4 24.8 ± 1.8 29.7 ± 1.1
PEG, polyethylene glycol; EGCG, (−)-epigallocatechin-3-gallate.
Furthermore, the signal observed at δ 3.35 ppm corresponds to a methyl group, and δ 3.52 and 3.85 belongs to H-2 to H-4 of PEG, respectively (Figure 2). The chemical structure characterized by FTIR spectra of PCS is shown in Figure 3. The characteristic peak of -NH bending (amide II) and -CO stretching (amide I) on CS was at 1552 and 1651 cm−1, respectively. In addition, 848, 1241, and 958 cm−1 characteristic peaks were clearly seen, which were contributions from C−C and C−O stretching on PEG. These results showed that PEG became connected to CS in preparation of the conjugate. The degree of substitution value of 0.34 ± 0.09 was determined by potassium polyvinylsulfate titration. Nanoparticles were produced by the gelation of aqueous EGCG solution mixed with FCS/PCS/Gel mixture solution. As shown in Table 1, FCS/PCS/Gel/EGCG nanoparticles were prepared with different mixtures of aqueous FCS/PCS concentrations, with particle sizes ranging from 250−760 nm and positive zeta potentials, depending on the FCS/PCS used. Furthermore, EGCG percentage loading efficiency and loading content were as follows: 32.3 ± 4.3% and 19.1 ± 2.4% (for FCS/PCS 0.625:0.625 mg/mL), 45.1 ± 3.6% and 24.8 ± 1.8% (for FCS/PCS 1.250:1.250 mg/mL), and 53.7 ± 1.8% and 29.7 ± 1.1% (for FCS/PCS 1.875:1.875 mg/mL). Therefore, FCS/ PCS concentration of 1.875:1.875 mg/mL with higher loading efficiency and better particle size/distribution was a relatively desired concentration for the subsequent experiments. Figure 3 shows the FTIR spectra of FCS/PCS/Gel/EGCG nanoparticles. For FCS, the spectra showed transmission peaks at 1418 cm−1 for the −CH3 bending on fucose and at 1553 cm−1 for protonated amino groups (−NH3+) on CS. As shown in the spectrum of gelatin and EGCG, characteristic peaks were observed at 1548 cm−1 for -NH bending vibration on Gel and 1093 and 1543 cm−1 for C−OH alcohols and a CC aromatic ring on EGCG. In the spectrum of the FCS/PCS/Gel/EGCG complex, the characteristic peak was at 843, 1259, and 973 cm−1 for C−C and C−O stretching on PEG and 1421 cm−1 for the −CH3 bending on fucose. Furthermore, the characteristic peak at 1553 cm−1 for -NH3+ on CS and 1548 cm−1 for amide II on Gel disappeared. In its place, new peaks at 1556 and 1541 cm−1 emerged, and the characteristic C−OH deformation peak on EGCG at 1093 cm−1 shifted to 1084 cm−1. These observations can be attributed to hydrogen bond interactions between N atoms in Gel or CS chains and H atoms in the EGCG (C−N···HO−C). Further, FCS, PCS, Gel, and EGCG complexes can be segregated into colloidal nanoparticles (Figure 1). Characterization of the Prepared Nanoparticles and EGCG Release Profiles. The particle sizes, morphologies, and EGCG release profiles of FCS/PCS/Gel/EGCG nanoparticles were examined via zetasizer, TEM, and HPLC. At pH 1.2 with pepsin (simulating the pH of gastric acid), the nanoparticles formed particle sizes of 268.2 ± 4.1 nm and a zeta potential of 31.9 ± 1.7 mV (Table 2), and the morphology became slightly
inoculated by the orthotopic implantation method. Further, after the abdomen of BALB/c nude mice was sterilized with alcohol, an incision was made to expose the stomach, and Luc MKN45 cells (1 × 107/100 μL) mixed in a 50% matrix gel (BD Biosciences, USA) suspension were injected subserosally into the gastric wall. The stomach was then returned into the peritoneal cavity, and the abdominal wall and skin were closed with surgical suture. Evaluation of Antitumor Activity of Bioluminescent Imaging and Histological Examinations. For analysis of antitumor activity, BALB/c nude mice were inoculated by orthotopic injection of Luc MKN45 cells. Tumors were allowed to grow for 14 days. The mice were then randomly divided into three groups. Each group contained six mice and received different EGCG formulations with an equal 0.5 mL at a fixed 40.0 mg/kg EGCG dose in the form of EGCG solution in deionized water and FCS/PCS/Gel/EGCG nanoparticles or FCS/ PCS/Gel solution for the control group once daily for 15 consecutive days. In vivo bioluminescent imaging was carried out with a highly sensitive CCD camera by a noninvasive in vivo imaging system (IVIS) and viewed in real time on a computer screen by a color scale expressed as total flux (photons per second per square centimeter per steradian). The mortality and body weight of each mouse was measured. One day after the final bioluminescent observation, the mice were sacrificed, and their stomach and livers were removed for histological examination. Histologic analysis of tissue biopsies was carried out utilizing a light microscope; biopsies were fixed in buffered paraffin and embedded in paraffin wax. These biopsies which were placed on approximately 5 μm tissue slides and stained with hematoxylin−eosin or immunohistochemistry staining. The tissue sections were dewaxed and rehydrated. After blocking with 3% bovine serum albumin, rabbit monoclonal antibodies against cleaved PARP (Cell Signaling Technology, USA) and Ki-67 (Thermo Fisher Scientific, USA) were added to the tissue sections for 24 h at 4 °C. After washing, the samples were probed with peroxidase-labeled goat antirabbit secondary antibody (Epitomics, USA) and detected with an ABC kit (Vector Laboratories, USA). Microdistribution and tissue inflammatory reaction of the stained sections were then examined at different magnifications under a light microscope. Statistical Analysis. Statistical analysis of the differences in the measured properties of the groups was performed with one-way analysis of variance and the determination of confidence intervals, with the statistical package Statistical Analysis System, version 6.08 (SAS Institute, NC). All data are presented as the means and standard deviations, indicated as “mean ± SD”. Differences were considered statistically significant when the p values were
