Chapter 3. Dual Targeting System by Supramolecular Complex of Folate-conjugated Methyl--
3.10. Summary
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Conclusion
Colorectal cancer represents the fourth most commonly diagnosed and one of the main causes of cancer death worldwide. In spite of evolution of cancer chemotherapy, low therapeutic effects, serious adverse effects, drug resistance, and high cost are important issues in the clinical field.
Therefore, development of new kinds of antitumor agents is strongly required. In this study, we designed and evaluated the novel M-β-CyD systems as antitumor agents for colon cancer. Firstly, we evaluated the potential of FA-M-β-CyD for colorectal cancer (Chapter 1). Secondly, we newly prepared a supramolecular complex of Ad-HA/M--CyD, and evaluated its in vitro antitumor activity (Chapter 2). Thirdly, we investigated the effect of Ad-HA/FA-M-β-CyD as dual ligand system that can target FR- (+) and CD44 (+) in HCT116 cells (Chapter 3). The overall findings are summarized as follows:
Chapter 1. Involvement of Mitophagy-mediated Cell Death in Colon Cancer Cells by Folate-appended Methyl-β-cyclodextrin (FA-M-β-CyD)
1) FA-M-β-CyD showed potent cytotoxic activity in HCT116 cells via FR--mediated cytotoxic activity.
2) FA-M-β-CyD was well associated with HCT116 cells, compared with M-β-CyD through FR-.
3) FA-M-β-CyD mainly distributed in the cytoplasm after cellular uptake in HCT116 cells, and then provided the potent cytotoxic activity.
4) FA-M-β-CyD induced formation of autophagic vacuoles and provided mitophagic cell death in HCT116 cells.
5) FA-M-β-CyD showed greater inhibitory effects on growth of tumor polyps than 5% mannitol, 5-FU and M-β-CyD in AOM/DSS colorectal cancer model mice.
6) Intravenous administration of FA-M-β-CyD negligibly changed in the body weight and blood chemistry values of AOM/DSS colorectal cancer model mice.
Chapter 2. Supramolecular Complex of Adamantane-grafted Hyaluronic Acid with Methyl-β-cyclodextrin (Ad-HA/M-β-CyD) as a Novel Antitumor Agent
1) Ad-HA/M-β-CyD was successfully prepared to achieve CD44 binding ability. In addition, it formed a complex with M-β-CyD.
2) Ad-HA/M-β-CyD showed a potent cytotoxic activity in CD44-expressing tumor cells HCT116 cells, not in NIH3T3 cells (CD44 (-)).
3) Ad-HA/M-β-CyD induced CD44-mediate cytotoxicity in HCT116 cells.
4) Ad-HA/M-β-CyD was highly associated with HCT116 cells rather than M-β-CyD through CD44-mediated endocytosis.
5) Ad-HA/M-β-CyD was endocytosed into HCT116 cells through the clathrin-independent pathway and caveolae dependent pathway.
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6) Ad-HA/M-β-CyD was internalized into HCT116 cells through CD44-mediated endocytosis and colocalized into the mitochondria.
7) Ad-HA/M-β-CyD induced a potent cytotoxicity without extracting cholesterol from plasma membrane of HCT116 cells.
8) Ad-HA/M-β-CyD induced mitochondria-dependent apoptosis in HCT116 cells.
Chapter 3. Dual Targeting System by Supramolecular Complex of Folate-conjugated
Methyl--cyclodextrin with Adamantane-grafted Hyaluronic Acid (Ad-HA/FA-M-β-CyD) for the Treatment of Colorectal Cancer
1) Ad-HA prepared in this study possessed CD44 binding ability.
2) Ad-HA/FA-M--CyD had favorable physicochemical properties for antitumor agents.
3) The superior cytotoxic activity of Ad-HA/FA-M--CyD to FA-M--CyD was observed in HCT116 cells (FR- (+), CD44 (+)).
4) FR-- or CD44-mediated cellular association of Ad-HA/FA-M--CyD was observed in HCT116 cells.
5) Ad-HA/TRITC-FA-M--CyD was internalized into HCT116 cells via FR-- and CD44-mediated endocytosis.
6) Ad-HA/FA-M--CyD induced mitophagic cell death in HCT116 cells.
7) Ad-HA/FA-M--CyD has the potent antitumor activity after intravenous injection to HCT116 cells-bearing mice with less systemic side effect.
The results mentioned above suggest that 1) FA-M-β-CyD had the antiproliferative effects in colorectal cancer, due to the FR--mediated endocytosis and mitophagy induction, 2) Ad-HA/M-β-CyD showed the superior cytotoxic activity to M-β-Ad-HA/M-β-CyD via CD44-mediated cytotoxicity and induced an apoptotic cell death in HCT116 cells (CD44 (+)), and 3) Ad-HA/FA-M-β-CyD was internalized into HCT116 cells through FR-- and CD44-mediated endocytosis and showed mitophagy-mediated antitumor activity. Therefore, it can be concluded that these M-β-CyD systems have the potential as novel tumor-selective antitumor agents for the treatment of colorectal cancer.
These findings may be useful information for preparation of M-β-CyD based tumor-selective antitumor agent.
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Acknowledgement
Firstly, I would like to express my sincere gratitude to my greatest teacher Prof. Hidetoshi Arima for the continuous support of my Ph.D. study and related research, for his patience, motivation, and immense knowledge. His guidance helped me in all the time of research and writing of this thesis. I could not have imagined having a better teacher and mentor for my Ph.D. study.
I am enormously grateful to Associate Professor Keiichi Motoyama for his continuous encouragement, kindly advice throughout my study and I am thankful Research Associate Taishi Higashi for kind advice and help.
Besides my teachers, I would like to thank the rest of my thesis committee: Professor Masami Ohtsuka, Prof. Toru Maruyama and Dr. Hirofumi Jono, for their insightful comments and encouragement.
I am especially thankful for Dr. Nayouki Kawahara for his appreciable recommendation to continue my postgraduate studies in Japan especially at Kumamoto University.
My sincere thank goes to Prof. Mustafa Elbashir who provided me both academic and moral support to continue my Ph.D. research.
My gratitude expands to Japan Ministry of Education, Culture, Sports, Science and Technology (MEXT) for providing me monbukagakusho scholarship to study Ph.D. at Kumamoto University.
I wish to express my deep thanks to all the members of Department of Physical Pharmaceutics, including my dearest friends Ahmed and Nasrul for their kindness and helps to my livings and studies in Japan.
Finally, I am thankful to my beloved father and my beloved mother, for their continuous praying to get the Ph.D. and my lovelies and adored wife Sara for her kind motivation, and my dear son Amin who gave me the energy I need every day, my lovelies sisters Sawsan, Nesreen and my beloved brother Ahmed for their continuous motivation support and encouragement for my pursuit.
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Materials and methods
1. Materials
AOM, 1-Adamantane methyl amine and 5-FU (purity: 98.5%) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Folic acid (purity: >98%), D-mannitol (purity: >98%), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 1-hydroxybenzotriazole (HoBt), and Annexin V-FITC Apoptosis Detection Kit were purchased from Nacalai (Kyoto, Japan).
Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Nissui Pharmaceuticals (Tokyo, Japan) and Nichirei (Tokyo, Japan), respectively. DSS was purchased from MP Biomedicals (CA, USA). Tetramethylrhodamine isothiocyanate (TRITC) was purchased from Funakoshi (Tokyo, Japan). Cyto-ID® Autophagy Detection Kit and MitoTracker® Green were purchased from Enzo Life Sciences (Farmingdale, NY) and Invitrogen (Tokyo, Japan), respectively. HA (M.W. 50 kDa or 200-500kDa) were supplied by Kewpie Corporation (Tokyo, Japan). M-β-CyD having a degree of substitution (DS) of methyl group of 12.2 was obtained from Tokyo Kasei (Tokyo, Japan). A Cell Counting Kit (WST-1) was purchased from Wako Pure Chemical Industries (Osaka, Japan). CD44 siRNA was purchased from Hokkaido System Science.
(Hokkaido, Japan). FR-α siRNA (Sc39969) was purchased from Santa Cruz Biotechnology.
LipofectamineTM2000, cytochrome c monoclonal antibody-FITC conjugate and 5-(4,6-dichlorotriazinyl)aminofluorescein (5-DTAF) were obtained from Thermo Fisher Scientific (Tokyo, Japan). Sephadex® G-15 was obtained from GE Healthcare UK. (Buckinghamshire, UK). FA-M--CyD (M.W. 1738) and TRITC-FA-M-β-FA-M--CyD were synthesized and purified as reported previously
50, 108) All other chemicals and solvents were of analytical reagent grade, and deionized double-distilled water was used throughout the study.
2. Synthesis and characterization of Ad-HA conjugate 2.1. Synthesis of Ad-HA
HA (200 mg) was dissolved in 50 mL of water by agitation for 3 h. Then, EDC (122 mg), HOBt (4 mg) and 1-adamantne methyl amine (18 mg) were dissolved in 50 mL of DMSO, and mixed with the HA solution. After agitation for 24 h at 45°C, 500 mL of acetone was added to yield the white precipitates, and the precipitates were collected by centrifugation (12,000 rpm, 10 min). After washing three times with n-hexane (50 mL), the product was dried overnight under the reduced pressure. The resulting Ad-HA was dissolved in deuterium oxide (D2O), and characterized by 1 H-NMR (JEOL JNM-R 500 instrument, Tokyo, Japan), operating at 500 MHz for protons at 25°C.
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2.2. Stability constant between Ad-HA and M-β-CyD
The stability constant (Kc) of Ad-HA/M--CyD was determined by the analysis of peak shifting in 1H-NMR at different concentration of M--CyD (0-5 mM) in D2O. The Kc value was obtained from the following Benesi-Hildebrand Equation, assuming the 1:1 guest/host interaction (Ad moiety of Ad-HA/M--CyD).
Where Δδ is the change in the 1H-NMR shifting, Δδ max is the maximum possible change in 1H-NMR shift, [H]0 is the total M--CyD concentration, and Kcis the stability constant.
2.3. Physicochemical properties of Ad-HA/M-β-CyDs
The particle size, polydispersity index and ζ-potential of Ad-HA/M-β-CyDs were measured using a Zetasizer Nano analyzer (Malvern Instruments, Worcestershire, UK). The complex was prepared in 5% mannitol solution at a molar ratio of 1:1 (Ad moiety of Ad-HA/M-β-CyDs). The concentrations of Ad-HA and M-β-CyD were 4.2 nM and 102 nM, respectively. In case of Ad-HA/FA-M-β-CyD, the concentrations of Ad-HA and FA-M--CyD were 0.2 g/mL and 0.125 g/mL, respectively.
3. Cell culture
HCT116 cells (FR- (+), CD44 (+)), a human colon cancer cell line, KB cells (FR- (+), CD44 (+)), a subline of the HeLa cells, and Ihara cells (FR- (+), CD44 (+)), a human melanoma cell line, were grown in DMEM containing penicillin and streptomycin supplemented with 10% FBS at 37ºC in a humidified 5% CO2 and 95% air atmosphere.
4. Cytotoxic Activity
4.1. Cytotoxic activity of FA-M-β-CyD in HCT116 cells
The in vitro cytotoxic studies in HCT116 cells were followed by the Kameyama’s report.51) HCT116 cells (2×104 cells/96-well microplate) were treated with 5 mM M-β-CyD or 5 mM FA-M-β-CyD for 2 h at 37°C in the absence and presence of 4 mM FA. After washing twice with PBS, the cells were incubated with 10% WST-1 reagent in HBSS at 37°C for 30 min, then the absorbance was measured at 450 nm against the reference wave length 620 nm using 96-well microplate reader (Bio-Rad Model 550, Tokyo, Japan).
4.2. Cytotoxic activity of Ad-HA/M-β-CyD in HCT116 cells or NIH3T3 cells
𝟏
∆𝛅
=
𝟏𝐊𝐜
𝟏
∆𝜹 𝐦𝐚𝐱[𝑯]𝟎
+
𝟏∆𝛅𝐦𝐚𝐱
61
HCT116 cells or NIH3T3 cells were seeded at 2×104 cells onto 96-well microplate (Iwaki, Tokyo, Japan), and then incubated for 24 h in a humidified atmosphere of 5% CO2 and 95% air at 37°C.
Cells were washed twice with DMEM, and then incubated with 150 µL of DMEM containing 0-5 mM M-β-CyD with or without Ad-HA for 2 h at 37°C. The concentration of Ad-HA was 14 mg/mL for each 5 mM M-β-CyD. For the competition study, the cells were pretreated for 1 h with DMEM containing 10 mg/mL of HA, and then incubated for 2 h with DMEM containing 5 mM M-β-CyD with Ad-HA at 37°C. After washing twice with PBS (pH 7.4), cells were incubated with 10% WST-1 in HBSS for 30 min at 37°C, and then the absorbance was measured at 450 nm against the reference wave length 620 nm using a microplate reader (Bio-Rad Model 550, Tokyo, Japan).
4.3. Cytotoxic activity of Ad-HA/FA-M-β-CyD in HCT116 cells
HCT116 cells were seeded at 2×104 cells onto 96-well microplate (Iwaki, Tokyo, Japan), and then incubated for 24 h in a humidified atmosphere of 5% CO2 and 95% air at 37°C. Cells were washed twice with DMEM, and then incubated with 150 L of DMEM containing 0-5 mM FA-M--CyD with or without Ad-HA for 2 h at 37°C. The concentration of Ad-HA was 14 mg/mL for each 5 mM FA-M-β-CyD. For the competition study, cells were pretreated for 1 h with DMEM containing 10 mg/mL of HA or 4 mM FA, and then incubated for 2 h with DMEM containing 5 mM FA-M--CyD with Ad-HA at 37°C. After washing twice with PBS (pH 7.4), cells were incubated with 10% WST-1 reagent in HBSS for 30 min at 37°C, and then the absorbance was measured at 450 nm against the reference wave length 620 nm using a microplate reader (Bio-Rad Model 550, Tokyo, Japan).
4.4. Effect of siCD44 or siFR-α knockdown in HCT116 cells on the cytotoxic activity of Ad-HA/M-β-CyDs in HCT116 cells
HCT 116 cells (5×104 cells/24-well microplate) were transfected for 1 h with LipofectamineTM2000 containing siFR-, siCD44 or siGL3 (siControl) as a control siRNA. After adding 10% FBS, the cells were incubated for 24 h in a humidified atmosphere of 5% CO2 and 95%
air at 37°C. Cells were washed twice with DMEM, and then incubated for 2 h with 300 L of DMEM containing 5 mM M--CyDs with Ad-HA at 37°C. Then, the cytotoxic activity was measured by the WST-1 method.
5. Cellular association of M-β-CyDs in HCT116 cells 5.1. Synthesis of 5-DTAF-M-β-CyD
M--CyD (15 M) and 5-DTAF (7.6 M) were dissolved in 0.2 M NaOH aqueous solution, and then stirred at room temperature for 24 h. The crude product was fractionized by Sephadex®-G15 to
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remove the unreacted 5-DTAF, and then lyophilized. The conjugate was characterized by TLC using 1-butanol/ethanol/water 5/4/3 as eluents using anisealdehyde as indicator.
5.2. Cellular association of FA-M-β-CyDs in HCT116 cells
HCT116 cells were seeded at 2×105 cells onto 24-well microplate, and then incubated for 24 h in a humidified atmosphere of 5% CO2 and 95% air at 37°C. Then, the cells were washed twice with DMEM, and then incubated for 2 h with 300 L of DMEM containing 10 µM TRITC-FA-M-β-CyD at 37°C for 1 h with DMEM containing 10 mg/mL of HA or 4 mM FA, and then incubated for 2 h with 300 L of DMEM containing TRITC-FA-M-β-CyD (15 M) with or without Ad-HA. After washing twice with PBS, the cells were detached, suspended witjh PBS, and kept on ice. Then, the cells were collected and performed a flow cytometry analysis by FACS Calibur flow cytometer using CellQuest software (Becton-Dickinson, Mountain View, CA, USA).
5.3. Cellular association of Ad-HA/5-DTAF-M-β-CyD in HCT116 cells
HCT116 cells were seeded at 1×105 cells onto 24-well microplate, and then incubated for 24 h in a humidified atmosphere of 5% CO2 and 95% air at 37°C. Cells were washed twice with DMEM, and then incubated for 2 h with 300 L of DMEM containing 5-DTAF-M--CyD (15 M) with or without Ad-HA. For the competition study, cells were pretreated for 1 h with DMEM containing 10 mg/mL of HA, and then incubated for 2 h with 300 L of DMEM containing 5-DTAF-M-β-CyD (15
M) with or without Ad-HA. For the endocytosis inhibition, the cells were pretreated for 1 h with DMEM containing amiloride (25 g/mL), chloropromzine (10 g/mL), genistein (200 g/mL) or filipin III (5 g/mL), then treated with 300 L of DMEM containing 5-DTAF-M-β-CyD (15 M) with Ad-HA.
6. Intracellular distribution of M-β-CyDs in HCT116 cells
6.1. Intracellular distribution of TRITC-FA-M-β-CyDs in HCT116 cells
HCT116 cells were seeded at 1×105 cells onto 35 mm glass bottom dish, and then incubated for 24 h in a humidified atmosphere of 5% CO2 and 95% air at 37°C. Then, the cells were washed twice with DMEM, and then incubated for 2 h with 150 L of DMEM containing TRITC-FA-M--CyD (10 M). For the competition study, the cells were pretreated for 1 h with DMEM containing 4 mM FA, and then incubated for 2 h with 150 L of DMEM containing TRITC-FA-M--CyD (10 M).
In the case of Ad-HA/TRITC-FA-M-β-CyD, HCT116 cells were seeded at 2 × 105 cells onto 35 mm glass bottom dish, and then incubated for 24 h in a humidified atmosphere of 5% CO2 and 95% air at 37°C. After that, the cells were washed twice with DMEM, and then incubated for 2 h with 150
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L of DMEM containing TRITC-FA-M--CyD (15 M) with or without Ad-HA. Then, the cells were washed with PBS, incubated with Hoechest33342 (10 μg/mL) for 10 min. After washing with PBS (pH 7.4), the culture medium was added. A fluorescence microscope (KEYENCE Biozero BZ-8000, Osaka, Japan) was used for the detection of TRITC and Hoechest33342.
6.2. Intracellular distribution of Ad-HA/5-DTAF-M-β-CyD
HCT116 cells were seeded at 2×105 cells onto 35 mm glass bottom dish, and then incubated for 24 h in a humidified atmosphere of 5% CO2 and 95% air at 37°C. Cells were washed twice with DMEM, and then incubated for 2 h with 150 L of DMEM containing 5-DTAF-M--CyD (15 M) with or without Ad-HA. For the competition study, cells were pretreated for 1 h with DMEM containing 10 mg/mL of HA, and then incubated for 2 h with 150 L of DMEM containing 5-DTAF-M--CyD (15 M) with Ad-HA. Then, the cells were washed with PBS, and incubated with 10
M Hoechest33342 for 10 min. To observe the colocalization with mitochondria, 10 M rhodamine 123, a mitochondria marker, was incubated for 30 min before the treatment with Hoechest33342, a nucleus marker. The cells were fixed with 4% paraformaldehyde in PBS for 15 min, and observed by a Biozero BZ-8000 fluorescent microscope (KEYENCE, Osaka, Japan).
7. Efflux of cholesterol from HCT116 cells
HCT116 cells were seeded in 12-well plate (5×105/well) for 24 h. The cells were incubated with 500 L of M-β-CyD (5 mM) with Ad-HA at 37°C for 1 h. After centrifugation (10,000 rpm, 5 min), the supernatant was recovered, and total cholesterol level was determined using a Cholesterol E test® Wako (Wako Pure Chemical Industries, Osaka, Japan).
8. Cell death mechanism
8.1. Detection of autophagosome formation
HCT116 cells were seeded at 5×105 cells onto 35 mm glass bottom dish, and then incubated for 24 h in a humidified atmosphere of 5% CO2 and 95% air at 37°C. After that, the cells were washed twice with DMEM, and then treated with 150 µL of DMEM containing 5 mM FA-M-β-CyD with or without Ad-HA for 2 h, and then the cells were incubated with Cyto-ID® Autophagy Detection Kit for 30 min. Then, the cells were washed with PBS, incubated with Hoechest33342 (10 μg/mL) for 10 min. After washing with PBS (pH7.4), the culture medium was added. A fluorescence microscope (KEYENCE Biozero BZ-8000, Osaka, Japan) was used for the detection of Cyto ID® and Hoechest33342.
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8.2. Detection of mitophagy
HCT116 cells were seeded at 5×105 cells onto 35 mm glass bottom dish, and then incubated for 24 h in a humidified atmosphere of 5% CO2 and 95% air at 37°C. After that, the cells were treated with 100 nM Mtphagy Dye® for 15 min. After washing with the medium, the cells were treated 150 µL of DMEM containing 5 mM FA-M--CyD or Ad-HA/FA-M--CyD for 2 h. After washing with the medium, 1 M Lyso Dye® was added and further incubated for 10 min. After washing with HBSS, the cells were scanned with Biozero BZ-8000, a fluorescence microscope (Keyence, Osaka, Japan).
8.3. Annexin V-FITC assay
HCT116 cells were seeded at 2×105 cells onto 24-well microplate, and then incubated for 24 h in a humidified atmosphere of 5% CO2 and 95% air at 37°C. Cells were washed twice with DMEM, and then incubated for 2 h at 37°C with 300 L of DMEM containing 5 mM M--CyD with Ad-HA in the absence and presence of 10 mg/mL of HA. After that, the cells were trypsinized, centrifuged and washed twice with PBS. Then the cells were incubated with Annexin V-FITC and propidium iodide (PI) solution according to the Annexin V Kit protocol, and the fluorescence intensity was measured using a FACS Calibur flow cytometer with CellQuest software (Becton-Dickinson, Mountain View, CA, USA).
8.4. Cytochrome c assay
HCT116 cells were seeded at 2×105 cells onto 24-well microplate and then incubated for 24 h in a humidified atmosphere of 5% CO2 and 95% air at 37°C. Cells were washed twice with DMEM, and then incubated for 2 h at 37°C with 300 L of DMEM containing 5 mM M--CyD with Ad-HA in the absence and presence of 10 mg/mL of HA. After that, the cells were trypsinized, centrifuged and washed twice with PBS, and then suspended in 100 L of PBS containing 1 g of anti-cytochrome c, and the fluorescence intensity was measured using the FACS Calibur flow cytometer.
9. In vivo studies
9.1. Evaluation of the antitumor activity of FA-M-β-CyD in AOM/DSS colorectal cancer mice model
Male ICR mice (5 weeks old) were intraperitoneally injected AOM at a dose of 10 mg/kg body.
One week later, the mice were received 2% DSS in the drinking water for 7 days, and then no further treatment was performed for 9 weeks. After 10 weeks of AOM injection, the mice were classified
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into five groups; control (healthy mice), AOM/DSS with 5% mannitol, AOM/DSS with 5-fluorouracil (5-FU), AOM/DSS with M-β-CyD, and AOM/DSS with FA-M-β-CyD. The AOM/DSS mice were administered 5% mannitol, 5-FU (40 mg/kg), M-β-CyD (5 mg/kg) or FA-M-β-CyD (0.5, 2.5 and 5 mg/kg) from tail vein of the mice, once a week for 4 consecutive weeks. After 15 weeks of AOM injection, the serum was collected. Then, all the mice were sacrificed and large bowels were excised. Their length from the ileocaecal junction was measured, cut open longitudinally along the main axis. The whole colon was microscopically inspected; the number of tumor polyps and tumor size were calculated using ImageJ software.
9.2. Evaluation of the antitumor activity of Ad-HA/FA-M-β-CyD in HCT116 cells tumor bearing mice
HCT116 cell suspension (2.5 × 106 cells/100 µL) was subcutaneously xenografted to BALB/c nu/nu mice (male, six-weeks-old, and ca. 20 g, Japan SLC, Shizuoka, Japan). Fourteen days later, 10 mg/kg of FA-M-β-CyD or Ad-HA/FA-M-β-CyD in mannitol solution (5%) were administered via a single intravenous injection to HCT116 cell-xenografted mice. The tumor volumes were determined as reported previously108). The tumor volume and body weight changes of the tumor-bearing mice were monitored for 24 days. All animal experiments described above were carried out in accordance with the guidelines approved by the Ethics Committee for Animal Care and Use of Kumamoto University composed of a third party (Approval ID: 27-141).
9.3. Blood chemistry values analysis
Twenty four h after intravenous injection of Ad-HA/FA-M--CyD (10 mg/kg) in mannitol solution (5%) to HCT116 cells-xenografted BALB/c nu/nu mice, the blood were collected. The blood chemistry data of lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CRE) and blood urea nitrogen (BUN) in serum were analyzed using JCA-BM2250, a clinical chemistry analyzer (JEOL, Tokyo, Japan).
10. Statistical analysis
The data are presented as the mean ± standard error (S.E.). The statistical significance of the mean coefficients of the experimental data was performed using an analysis of variance (ANOVA) followed by Scheffe's test. In addition, p-values for significance were set at 0.05.
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