br Cell cytotoxicity assay br A cells or T cells
2.7.3. Cell cytotoxicity assay
A549 ITF2357 or 4T1 cells were seeded in 96-well plate and cultured at
medium was replaced with 180 μL DMSO, followed by the absorbance measurement at the wavelength of 570 nm. The cell viability [Cell Viability = ODsample/ODcontrol × 100%] was calculated. ODsample or ODcontrol was the absorbance at 570 nm after the cells were treated with or without the formulations.
2.8. Establishment of A549 tumor xenograft models and 4T1 lung metastatic models
For the establishment of A549 tumor xenograft models, the male nude mice were subcutaneously injected with 1 × 107 A549 cells sus-pended in 100 μL PBS. Length (L) and width (W) of the tumor was determined by a vernier caliper. The tumor volume (V) was calculated according to the equation: V = L × W2/2.
For the establishment of 4T1 lung metastasis models, the female BALB/c mice were intravenously injected with 2 × 105 4T1 cells sus-pended in 100 μL PBS to induce the lung metastatic model following the previous report . After 14 days, all the mice were sacrificed and the lung tissues were carefully removed from each group. The isolated lung tissues were stained with Indian ink and then photographed to detect the lung metastatic nodules.
2.9. In vivo biodistribution
When the A549 tumor volume reached to about 200 mm3, the mice were intravenously injected with diﬀerent formulations: Cy5-siRNA, H-RSC and TH-s-RSC. Each group (n = 3) were administrated with Cy5-siRNA at a dose of 1 mg/kg. At diﬀerent time post-injection (1 h, 6 h, 12 h, 24 h), the images were acquired using Maestro™ in vivo imaging system (Cambridge Research & Instrumentation, USA). At 24 h post-injection, all the mice were sacrificed and the tumor and normal tissues were harvested for ex vivo imaging.
2.10. In vivo synergistic anti-tumor eﬃcacy
2.10.1. Anti-tumor eﬃcacy on A549 tumor xenograft model When the tumor reached 50 mm3, the tumor-bearing mice were intravenously administrated with TH-s-RSC/siN.C., RSC + TRAIL, H-RSC, and TH-s-RSC (1.2 mg/kg siHSP70 or siN.C. and 0.2 mg/kg TRAIL) and saline as a negative control for every three days (totally 8 injec-tions). The tumor size and body weight of the mice were monitored every 2 days. At day 22, mice were sacrificed. The tumors and normal organs were harvested and weighted. For evaluation of the in vivo gene silencing at mRNA level, the total RNA in 50 mg of tumor was extracted by TRIZOL Reagent (Life Technologies) according to the protocol of the manufacturer. The HSP70 mRNA was analyzed by qRT-PCR as de-scribed above. For analysis of the protein expression, the total protein in 50 mg of tumor was extracted by Whole Cell Lysis Assay (KeyGEN) according to the protocol of the manufacturer. The protein concentra-tion was determined using the Pierce™ BCA Protein Assay Kit. The HSP70 and c-FLIP protein was detected by Western blot as described above. For histological analysis, paraﬃn sections of tumor and normal organs were stained with hematoxylin and eosin (HE) and visualized by the optical microscope (DM5500B, Leica). For apoptosis analysis, the TUNEL staining was performed according to the protocol of the man-ufacturer and the stained tumor sections were observed by the optical microscope. For the safety evaluation, blood biochemical parameters including ALP, ALT, AST and BUN were determined, respectively. For survival studies, tumor size was monitored until experimental endpoint 50 days post tumor inoculation or until a maximal tumor determination exceeding a diameter > 1.5 cm.
or siN.C. and 0.2 mg/kg TRAIL) and saline as a negative control for every other day (7 injections total). Meanwhile, the body weight of mice was monitored every 2 days. After 14 days, mice were sacrificed and the lung tissues were carefully removed from each group, followed by staining with Indian ink and photographed to detect the lung me-tastatic nodules. The number of the lung metastatic nodules of each group were counted to estimate the inhibition eﬃciency. Moreover, paraﬃn sections of tumor were stained with hematoxylin and eosin (HE) and visualized by the optical microscope (DM5500B, Leica).