Page 75 - ARNM-3-3

Advances in Radiotherapy
& Nuclear Medicine CS@LGG for acute radiation-induced bowel injury alleviation

2.2. Cell culture 2.6. Mice model

The MC38 tumor cells and IEC-6 epithelial cells were 2.6.1. Acute radiation-induced bowel injury mice
cultured in DMEM (Gibco) growth medium (10% FBS, 1% model
penicillin-streptomycin) at 37°C in a 5% carbon dioxide Male C57BL/6 mice (5 weeks old; body weight 18–20 g
(CO ) humidified atmosphere (Thermo Fisher, USA). each) were used in this study. All animal experiments
2
Using light microscopy, cells were passaged into a new were approved by the Institutional Animal Care and
100 mm culture dish or T25 flask after trypsinization and Use Committee of Nanfang Hospital, Southern Medical
centrifugation (200 × g, 8 min) for subsequent experiments University. The experimental group received 12 Gy lower
when they were in the logarithmic growth phase.
abdominal IR. Approximately 12 h after IR, the mice
2.3. Cultivation of the bacterial strain of LGG were administered CS@LGG (1 UI, 100 μL), CS (10 mg/
mL, 100 μL), or LGG (1×10 CFUs, 100 μL) individually
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LGG (ATCC 53103) was cultured under strictly anaerobic through daily gavage for 5 days. The mice were then
conditions (80% nitrogen [N ], 10% hydrogen [H ], scarified for sample collection.
2
2
and 10% CO ) at 37°C in the MRS culture medium. The
2
bacterial concentration was determined by plate colony 2.6.2. Subcutaneous tumor model and intratumor
counting and spectrophotometer (Thermo Fisher, USA) at injection
optical density (OD) . Cultures were preserved at −80°C 5
600
in 20% glycerol cryopreservation solution. MC38 cells were cultured to 5 × 10 in a cell incubator
(37°C, 5% CO ) and collected after trypsinization in PBS
2
6
2.4. The preparation of CS@LGG hydrogel to a final concentration of 1 × 10 cells/mL. The mice were
To synthesize the biomaterial, the pre-fabricated CS then subcutaneously injected with 100 μL cell suspension
powder was completely dissolved in glacial acetic acid on both sides of the lower back. Each injection formed a
(1%, v/v) and stirred in a 37℃ water bath for at least small subcutaneous mass using a 1 mL syringe. Changes
30 min until it turns into a hydrogel structure, ensuring of tumor volume were observed every 2–3 days until the
a final concentration of 10 mg/mL. Then, the resuscitated tumors were measurable. Afterward, these mice were
LGG was separated from the MRS medium after 24 h of exposed to 12 Gy whole-abdomen radiotherapy (WART)
incubation at 37℃. Next, the bacterial growth fraction was and received PBS or CS@LGG intratumor injection every
measured by spectrophotometer (Thermo Fisher, USA) at other day on either the left or right side of the back, serving
OD , and the bacterial suspension was diluted with CS as a self-controlled experiment. After 16 days, the tumors
600
hydrogel for 1 × 10 colony-forming units (CFUs). The were harvested, and the tumor weight was recorded. Tumor
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suspension was then mixed thoroughly with CS hydrogel volumes were measured by length (a) and width (b) with
2
in a 37℃ incubator (Thermo Fisher, USA). Because LGG a vernier caliper and calculated as tumor volume = ab /2.
has a negative surface charge and CS is positively charged, 2.6.3. Colon orthotopic tumor model and gavage
stable electrostatic adsorption occurred under weakly
acidic conditions. After incubation in a shaker (Thermo MC38 cell suspension was prepared as described above. The
Fisher, USA) at 250 rpm and 37℃ for more than 2 h, CS@ mice were then anesthetized with sodium pentobarbital
LGG was fully prepared for further experiments. (1%, v/v) and placed on a surgical benchtop. Next, a
longitudinal incision with a length of approximately 0.5 cm
2.5. Characterization of CS@LGG probiotic was made along the lower abdomen midline. Afterward, the
biomaterial cecal pouch was located and detached, then placed on the

Scanning electron microscopy (SEM) images of CS, LGG, abdominal surface with forceps. Finally, the mesocecum
and CS@LGG were obtained using a Hitachi SU8010 was clamped and 30 μL cell suspension was aspirated with
(Hitachi, Japan) at the School of Pharmaceutical Sciences, a microsyringe. After completing the above manipulation,
Southern Medical University. The zeta potential was the mice were sutured and disinfected. After approximately
analyzed with a Malvern Zetasizer Nano ZS90 (Malvern 2 weeks, once the tumor had grown and become palpated
Panalytical, United Kingdom) at the School of Laboratory on the surface, the mice were divided into four groups,
Medicine and Biotechnology, Southern Medical University. such as IR + PBS, IR + CS@LGG, PBS, and CS@LGG. The
The chemical structure of CS@LGG was tested by X-ray “IR” groups received 12 Gy lower abdominal IR, and the
photoelectron spectroscopy (XPS, Thermo Fisher, USA) other groups served as controls. The mice were then given
and Fourier transform infrared spectroscopy (FTIR, CS@LGG (1 UI, 100 μL, daily) or PBS (100 μL, daily) for
Thermo Fisher Scientific Nicolet iN10, USA) in Zhengzhou 18 days, while their weight gain and general condition
Feynman Biotechnology Co., Ltd. were recorded.

Volume 3 Issue 3 (2025) 67 doi: 10.36922/ARNM025230026