Biomaterials br examined by confocal laser scanning microsco
Biomaterials 206 (2019) 1–12
examined by confocal laser scanning microscope. Similarly, to further validate the CD44 receptor-mediated adhesion of platelets to MDA-MB-231 cells, HA (6 mg/mL) was added to the DMEM medium. The treat-ment of E 64 was the same as above, and the fluorescent intensity va-lues of adherent platelets were determined by Varioskan Flash multi-mode microreader.
2.10. Cellular uptake and cytotoxicity assays
MCF-7 and MDA-MB-231 cells, representing high and low expres-sion of CD44, were used to evaluate cellular uptake and cytotoxicity via respective assays. To evaluate the cellular uptake of formulations, these cells were incubated (at 105 cells/well) in 12-well plates for 24 h. Then, free DOX, free ICG, and PMDIs were added to the cells and incubated for 1 h with ICG and/or DOX at concentrations of 5 and 6 mg/mL, re-spectively. Subsequently, some of these cells were subjected to irra-diation for 5 min with an 808 nm laser at a power density of 2.0 W cm−2 followed by incubation for 6 h. They were then washed 3 times with ice-cold PBS, followed by counterstaining of nuclei with DAPI. Confocal laser scanning microscopy was used to observe the cells (CLSM, C2SI, Nikon, Japan). Likewise, to further explore the CD44 receptor-mediated cellular uptake of PMDIs in MDA-MB-231 cells, HA (8 mg/mL) was added to the DMEM medium. The treatment of cells was the same as above, and the cellular uptake was determined by flow cytometry (FACSCalibur, BD Biosciences) after cell collection. r> To perform viability tests for PBS, PMIs, and PMDIs after irradiation and PMDs, MCF-7 and MDA-MB-231 cells (at 5 × 103 cells) were in-cubated in 96-well plates for 24 h. This was followed by treatment with PBS, PMIs, PMDs, and PMDIs with diﬀerent DOX and ICG concentra-tions. Four hours later, the cells were subjected to laser (808 nm, 2 W cm−2) irradiation for 5 min. The cell viability was determined by the MTT assay. The absorbance of samples was measured by Varioskan Flash multimode microreader.
Mice were housed at the Animal Center of Shenyang Pharmaceutical University (China). All procedures and experiments were in conformity with the guidelines provided by the Institutional Animal Ethical Care Committee (IAEC) of our university.
To establish MDA-MB-231 tumor-bearing mice models, the female nude mice were subcutaneously implanted with 1 × 106 MDA-MB-231 cells into the right hind foot soles in 40 μL of PBS. At the tumor volume of 200 mm3, the mice were intratumorally injected with 50 μL PMDIs and DINPs (containing 60 μg/mL DOX and 50 μg/mL ICG). The noninvasive optical imaging system (IVIS) was used to acquire in vivo images at an excitation wavelength of 740 nm at 20, 40, 60, 90 and 360 min post-injection. Furthermore, 60 min after intratumoral injec-tion, their sentinel lymph nodes and primary tumors were separately subjected to a 10-min irradiation with 808 nm NIR light, with power densities of 1 W cm−2 and 0.5 W cm−2, respectively. An infrared thermal imaging camera (Fotric 226) was used to acquire the images and determine temperature changes.
1 W cm−2, respectively. The body weight was recorded every 2 d. At day 20, the lymph node volume was measured and the whole lungs were stained by 15% India ink. Lung and liver slices were collected and fixed by formalin for hematoxylin and eosin (H&E) staining after var-ious treatments. The remaining five mice in each group were normally fed for 80 d to assess survival rate.
2.13. In vivo elimination of CTCs
Mice were anesthetized with isoflurane followed by intravenous injection of 150 μL saline, DINPs and PMDIs (containing 60 μg/mL DOX and 50 μg/mL ICG) via tail vein. Thirty minutes later, the mice were injected intravenously with 1 × 107 MDA-MB-231 cells in 100 μL saline through the tail vein. Afterwards, the lung tissues were stained with India ink and excised at 50 d post injection for imaging and staining. Lung slices were harvested from all mice groups for examination.
2.14. Studies on MDA-MB-231 back tumor-bearing nude mice models
To establish the MDA-MB-231 tumor-bearing mouse model, female nude mice were subcutaneously implanted with 1 × 107 MDA-MB-231 cells in 100 μL PBS within the right back side. When the volume of tumors reached 300–400 mm3, the mice were intravenously injected with 100 μL DINPs and PMDIs (with 60 μg/mL DOX and 50 μg/mL ICG). In vivo NIRF images were recorded using the IVIS system at an ex-citation wavelength of 740 nm at 6, 12, 24 and 48 h post-injection. The mice were then sacrificed at 48 h after injection to collect the tumors and major organs for analysis. For tumor analysis, the mice were eu-thanized and tumors were removed and frozen at 6 h post-injection. Hoechst 33342 was used to stain nuclei. The stained tumor slides were observed by CLSM. Similarly, we employed an infrared thermal ima-ging camera to record the photothermal eﬀect of 100 μL DINPs and PMDIs (with 60 μg/mL DOX and 50 μg/mL ICG) with power densities of 0.5 W cm−2 for 10 min at 6 h post-injection.