Pharmacological Research br the sample was measured by
Pharmacological Research 143 (2019) 178–185
the sample was measured by dynamic light scattering (DLS) by using the Malvern Zetasizer Nano ZS-90 instrument. The zeta-potential measurement was also performed by the same instrument. The optical properties of ACA nanocomplex were recorded using the Rayleigh UV-1601 instrument.
2.3. Tumor induction
CT26 cell line derived from mouse colon adenocarcinoma and male BALB/c mice (5–8 weeks old and weighing 20–25 g) were obtained from Pasteur Institute of Iran. Cells were cultured in RPMI 1640 medium with 10% FBS, 100 units/ml penicillin, and 100 μg/ml strep-tomycin at 37 °C in 5% CO2. Cells were harvested by trypsinizing cul-tures with 1 mM EDTA/0.25% Trypsin (w/v) in PBS. After three pas-sages, 2 × 106 CT26 FLAG tag Peptide suspended in 200 μl RPMI 1640 solution were injected subcutaneously on the right flank of BALB/c mice. All the animal experiments were conducted in accordance with guidelines es-tablished by the Institutional Animal Care Committee.
2.4. Tumor accumulation and tissue distribution of ACA nanocomplex
A tumor uptake study was performed by using inductively coupled plasma-mass spectrometry (ICP-MS) in order to determine the optimum time after injection for maximal tumor accumulation of ACA nano-complex. Tumor bearing mice were intraperitoneally injected with ACA (Au concentration: 5 mg/kg) and then were scarified at diﬀerent time points after injection (6, 12 and 24 h). The tumors were dissected, weighed and dissolved in aqua regia (HCl:HNO3 = 3:1). Finally, ICP-MS (ELAN DRC-e spectrometer; PerkinElmer SCIEX, Concord, Ontario, Canada) was performed to determine the Au content in the tumors (μg of Au per gram tissue). To further investigate the biodistribution of ACA nanocomplex in living mice, the Au contents of the major organs (heart, kidney, liver, spleen and lung) were also measured.
2.5. In vivo antitumor study
The in vivo antitumor examination was started when the average tumor volume reached to approximately 150 mm3. BALB/c mice bearing CT26 colon cancer were randomly divided into six groups (n = 7) and received various treatments including: control (untreated), cisplatin (1 mg/kg), ACA nanocomplex (containing 1 mg/kg cisplatin and 5 mg/kg Au), laser, alginate coated-AuNPs (5 mg/kg Au) + laser and ACA nanocomplex + laser. For laser irradiation groups, mice were exposed with a continuous laser at wavelength of 532 nm and power density of 1.1 W/cm2 for 15 min (Changchun New Industries Optoelectronics Tech, China). For the mice treated with AuNPs + laser and ACA + laser, a time interval of 24 h was adopted between the in-jection and exposure according to ICP-MS results. In each group, the treatment regime was administered for three sessions with one day in between. To estimate the antitumor eﬀect of the various treatments, the tumor volume was monitored every three days. The tumor volume was calculated as: π × length × (width)2. Finally, the tumor growth profile
6 of the various treatments was plotted during 21 days of study span. Moreover, the body weight and general condition of mice were re-corded.
2.6. In vivo thermometry and thermal dose calculations
To evaluate the photothermal eﬀect of ACA nanocomplex, the transient temperature variations of the tumor loaded with ACA were monitored with an infrared (IR) thermal camera (Testo 875–1i, Germany) during laser irradiation. The thermometry results were then analyzed based on the thermal dose parameter Rolling circle provides a mean for quantizing hyperthermia treatments. This concept presents an expres-sion named CEM 43 °C that normalizes any combination of time-
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temperature to an equivalent time at reference temperature of 43 °C.