br intracellular fluorescence intensities of
intracellular fluorescence intensities of ICG and DOX in MDA-MB-231 Digitoxin versus those in MCF-7 cells, suggesting that P-Selectin and CD44 receptor interactions contribute to the enhanced cellular uptake of PMDIs in MDA-MB-231 cells. The fluorescence intensity of DOX was further increased by laser irradiation in the MCF-7 and MDA-MB-231 cells when treated with free ICG and PMDIs (Fig. 4A), probably due to the increased molecular pervasion elicited by the photothermal eﬀect of ICG. In contrast, tumor cells treated with free DOX alone in the presence or absence of laser irradiation showed similar fluorescence intensities (Fig. 4A).
We further used flow cytometry to quantitatively determine the cellular uptake of DOX for PMDIs and free DOX solution in MDA-MB-231 and MCF-7 cells. PMDIs showed enhanced cellular uptake of DOX in MDA-MB-231 cells as compared to that in MCF-7 cells. Laser irra-diation further increased the cellular uptake of PMDIs in MDA-MB-231 and MCF-7 cells, but not for free DOX solution. Moreover, the uptake eﬃciency of PMDIs was reduced by nearly 80% in MDA-MB-231 cells when co-incubated with 8 mg/mL HA solution, a finding which is consistent with the specificity and competition for CD44 receptors on MDA-MB-231 cells (Figure S4).
The in vitro cytotoxicity of PMDIs towards MDA-MB-231 and MCF-7 cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT) assay. Under laser irradiation, PMDIs exhibited significantly enhanced cytotoxicity compared with PBS, PMIs, and PMDs at all studied concentrations (Fig. 4c). Notably, PMDIs exhibited significant cytotoxicity against MDA-MB-231 cells, with a low IC50 value of 159.9 ng mL−1 (DOX dose), which was 4.5-fold lower than PMDs, 3.0-fold reduced as opposed to PMIs (versus ICG concentration), and 4.9-fold decreased versus PMDIs against MCF-7 cells (versus DOX dose). Collectively, based on the higher cellular uptake and photothermal eﬀect of PMDIs, these results showed that PMDIs conferred high cytotoxicity to the CD44 receptor-overexpressed MDA-MB-231 cells. Therefore, we postulated that a combination of DOX and ICG with laser irradiation and platelet membrane coating would provide higher anticancer eﬃciency.
3.5. Anti-metastasis eﬃcacy in the footpad MDA-MB-231 tumor-bearing model
To explore the CTC tracking performance of PMDIs in the lymphatic circulation, we used the footpad MDA-MB-231 tumor-bearing metas-tasis model, with DINPs as a negative control. DINPs could move to adjacent sentinel lymph nodes after being injected into the primary tumor, but PMDIs could move to sentinel lymph nodes and spread further into the distal subiliac lymph node. These results revealed that PMDIs have the greater capacity to penetrate into deeper lymph nodes through lymphatic circulation for tracking CTCs (Fig. 5A).
When subjected to laser light irradiation, the sentinel lymph node and the primary tumor displayed significant temperature increases as a result of the lymphatic drainage of intratumorally injected PMDIs (Fig. 5B). However, in saline-injected mice, changes in temperatures at both locations were relatively lower at the same power intensities, le-veling to below 40 °C, which could not suﬃciently destroy tumor cells (Fig. 5D) .
The photothermal ablation eﬃcacy of sentinel lymph nodes (the first site for metastatic tumors) and the primary tumors were subse-quently determined in BALB/c-nu mice inoculated with MDA-MB-231 tumors in the right hind paws for 15 d. Notably, the mice treated with PMDIs demonstrated longer survival periods, and only 1 out of 5 mice died within 56 d (Fig. 5G). The sizes of sentinel lymph nodes of mice in the PMDIs-treated group were 6.3-fold smaller than those in the saline-treated group (Fig. 5C, E). Moreover, as illustrated by hematoxylin and eosin (H&E) staining of lung slices and India ink staining of whole lung tissue, there were no micrometastasis sites in the lungs of mice after treatment with PMDIs. However, there were many micrometastasis sites within the lung and liver in other treatment groups (Fig. 5F). This Biomaterials 206 (2019) 1–12
indicated that PMDIs could penetrate into deeper lymph nodes via the lymphatic circulation and that they might capture and clear the CTCs from the lymphatic circulation. PMDIs are therefore therapeutically eﬀective and display good biocompatibility for clinical applications (Fig. S3).
3.6. In vivo elimination of CTCs in blood circulation
To confirm the ability of PMDIs to clear CTCs in blood, nude mice were injected with MDA-MB-231 cells (106 cells/100 μL saline). Many CTCs could metastasize to diﬀerent organs, especially lung . Fig. S2 shows that mice given saline injections had many lung micro-metastases. However, there was only a slight decrease in the amount of lung micrometastases in DINPs-treated mice relative to the saline group. Surprisingly, there was a significant decrease in metastatic no-dules in the lungs of PMDIs-treated mice in comparison with saline- and DINPs-treated mice. The results suggested that PMDIs could capture the CTCs in blood circulation via specific P-Selectin-CD44 receptor inter-actions and destroy the CTCs by the sustained released of DOX from PMDIs.