E64 br In vitro photothermal e ciency and controlled drug re
3.2. In vitro photothermal eﬃciency and controlled drug release of PMDIs
To explore the photothermal eﬃciency of PMDIs, changes in tem-perature were tracked in vitro in the presence of laser irradiation by a thermal imaging camera. When irradiated for 5 min at 1 W cm−2, the temperatures of PBS, ICG solution, PMIs, and PMDIs maximally in-creased to 32.6, 49.2, 52.9 and 53.5 °C, respectively. The temperature was lower in free ICG solution relative to PMDIs and PMIs, because the latter displayed lower heat dissipation and higher energy eﬃcacy. As shown in Fig. 2D, these formulations were stable when heated up to approximately 54 °C, leading to irreversible destruction of tumor cells . In addition, Fig. 2C shows that only 30% of the total amount of DOX from PMDIs was released after 8 h without laser irradiation, but approximately 40% release of DOX was enabled under laser irradiation, suggesting that the laser irradiation-mediated temperature rise helped to release DOX from PMDIs.
Fig. 2. Characteristics of platelet mem-brane-cloaking PLGA NPs. (A) Photographs and TEM images of DINPs, PMDIs, PMDs and PMIs. Scale bar: 100 nm. (B) The results of SDS-PAGE analysis of PM, PMDs, PMIs and PMDIs. (C) In vitro release of DOX from PMDIs with and without laser irradiation. Error bars represent s.d. (n = 3). (D) In vitro photothermal eﬃciency of platelet-coated NPs.
3.3. Adhesion of platelets to cancer cells
Due to the strong aﬃnity between P-selectin and the CD44 receptor, we explored the adhesion of platelets to four types of cancer cells . First, we used flow cytometry to determine the expression of CD44 receptors on the cell surface. As shown in Fig. 3A, CD44 receptors were expressed on the surface of each tumor cell to a diﬀerent extent. The mean fluorescence intensity (MFI) suggested that MDA-MB-231, CAMA-1 and Hela cells overexpressed CD44. However, there was a minimal expression of CD44 receptors on the surface of MCF-7 cells. Thus, MCF-7 cells and MDA-MB-231 cells were used to represent low and high CD44 expression tumor cells, respectively.
The adhesion of DiD-labeled platelets and four types of cancer cells
were observed by confocal laser scanning microscope (Fig. 3B). The adherent DiD-labeled platelets were measured by a Varioskan Flash multimode microreader. The fluorescence intensity of attached platelet was the highest for MDA-MB-231 cells, followed by CAMA-1 and Hela cells and was the lowest for low CD44 expression MCF-7 cells (Fig. 3C).
Hyaluronic E64 (HA) was reported to represent a high-aﬃnity li-gand of the CD44 receptor [33,34], and the DiD-labeled platelet amount adherent to MDA-MB-231 cells was significantly decreased when co-incubated with free HA solution (8 mg/mL) (Figure S4). This result further confirmed that the CD44 receptor is involved in the re-cognition process of platelets and tumor cells. It has been reported that there exists a specific and strong aﬃnity between the CD44 receptor and P-selectin. Therefore, we determined the expression levels of the
Fig. 3. The expression of CD44 receptor and the adhesion of platelet-tumor cells. (A) The flow cytometric analysis of the expression of CD44 on MDA-MB-231, CAMA-1, Hela and MCF-7 cells. (B) The adhesion of DiD-la-beled platelets and four types of cancer cells as observed by confocal laser scanning mi-croscope. Scale bar: 10 μm. (C) Quantitative analysis of adhesion under Varioskan Flash multimode microreader. (D) The expres-sions of key protein P-selectin on PMIs, PMDs, PMDIs, with PM as a control. ***
Fig. 4. Synergistic eﬀect of PMDIs combination treatment of PTT and chemotherapy in MCF-7 cells and MDA-MB-231 cells. (A) The results of confocal imaging in cells after 6 h incubation with PMDIs free ICG, and free DOX with or without laser irradiation. Scale bar: 15 μm (B) Results of flow cytometry for MDA-MB-231 and MCF-7 uptake of DOX after 6 h incubation with or without laser irradiation. (C) In vitro cytotoxicity of 24 h after diﬀerent treatments against MCF-7 cells and MDA-MB-231 with diﬀerent concentrations of ICG and DOX.
key protein P-selectin on PM, PMIs, PMDs, and PMDIs and found that PM-cloaking NPs maintained the high expression of P-selectin, similarly to PM. The high aﬃnity between P-Selectin of the platelet biomem-brane and the CD44 receptors of tumor cells would contribute to the eﬀective capability of PMNPs to recognize and capture tumor cells and CTCs .
3.4. Cellular uptake and cytotoxicity assays
Confocal microscopy was used to explore the cellular internalization of PMDIs, with MCF-7 and MDA-MB-231 cells representing low and high CD44 expression tumor cells. Tumor cells were incubated with PMDIs, free ICG, and free DOX with or without laser irradiation for 6 h, respectively. As shown in Fig. 4A, a green fluorescence representing ICG was observed within the cytoplasm, while a red fluorescence re-presenting DOX was observed in the nucleus. PMDIs showed the higher