Purpose(s)
1) Establishing biodegradable nanosystem for desirable cancer immunotherapy via framework-incorporation induced swelling based on hydrogen bond/electrostatic-assisted co-assembly strategy;
2) Incorporating carbon nanodots (CD) into mesoporous silica framework for enhanced photothermal effect and elevated targeting accumulation to achieve photothermal imaging-guided photothermal therapy (PTT);
3) Exploring the preliminary mechanism of debris-mediated photothermal synergistic immunotherapy and providing an innovative insight into the biodegradable nanoparticles-associated anticancer immunity.
Method(s)
Preparation of CD@MSN and their derivatives.
CD@MSN were prepared by a hydrogen bond/electrostatic-assisted co-assembly strategy. Typically, 2 mg CD were dispersed in 24 mL CTAB-deionized water solution (8.3 mg mL-1). After adjusting the pH to 9.5 with appropriate amount of TEA, 1.1 mmol TBOS was added and the solution was stirred at 80 °C for 6 h. The precipitation was recovered by centrifugation (12,000 rpm, 5 min) and repeatedly washed with water and ethanol. To remove residual CTAB, the black precipitate was stirred in NH4Cl ethanol solution (0.6 wt.%) at 70 °C for 3 h. Finally, CD@MSN were obtained by freeze-drying. Accordingly, pristine MSN were prepared without the addition of carbon dots.
Characterization
3D-TEM, EDS-mapping and ELLS profiling and spectrum imaging were performed by Talos F200X TEM (ThermoFisher, USA) with Velox software. NIR-mediated photothermal images were recorded with thermal imaging equipment (FLIR A65, USA). The photothermal effect was measured under NIR irradiation (808 nm) by a semiconductor laser unit (Changchun Laser Optoelectronics Technology Co., Ltd., China).
Biodegradation of CD@MSN and the antigen adsorption
For the characterization of biodegradation, CD@MSN were shaken in simulated physiological solutions (PBS 7.4, 37 °C) for different periods before the typical measurements including TEM, DLS particle size distributions, UV-vis-NIR spectra, and photothermal heating curves. To probe the intracellular biodegradation, the nanoparticles (MSN or CD@MSN) were firstly co-incubated with the same quantity of 4T1 cells for different periods. Then, the biodegraded nanoparticles were recovered for TEM observation. Correspondingly, BCA assay and Western Blot were conducted for the analysis of antigens adsorbed on these debris.
Analysis of the proliferation and differentiation of immune cells
For the detection of immune activation, mice bearing 4T1 tumors were randomly divided into two groups (4 in each group): (a) MSN group and (b) CD@MSN group. On day 7 after inoculation of 4T1 cells, pristine MSN (the same concentration with the silica spheres in CD@MSN) and CD@MSN (10 mg kg-1, 2 mg mL-1) were injected intravenously. 4 hours after injection, all the mice (after anesthesia) were subjected to therapeutic laser irradiation for 5 min (808 nm, 0.75 W cm-2). Finally, on the 7th day after injection, the mice were sacrificed and cells were harvested from spleen, liver, lung and tumor site. The blocking and staining on cells were conducted according to the manufacturer's instructions, and then CD45, CD49b, NKG2G, NKp46, F4/80, CD86 and CD206 expressing cells were analyzed by flow cytometer (BD FACAria, USA).
All animal experiments were performed in accordance with the guidelines evaluated and approved by the Ethics Committee of Fudan University.
Result(s)
Figure 1. (A) STEM image, (B-D) EDX mappings of silica, oxygen, carbon, and (E) merged three-dimensional reconstructed mapping image of CD@MSN. (F-J) TEM images of CD@MSN incubated in physiological solutions for 1 d, 2 d, 4 d, 6 d and 12 d, respectively. The inset in (H) showed the leach out (red arrows) of the incorporated CD from CD@MSN (yellow dotted line) due to the biodegradation. (K) Photothermal heating curves of PBS 7.4, free CD and CD@MSN containing the same concentration of CD under an 808 nm laser irradiation of (2.0 W cm−2). (L) Particle size, (M) UV absorption and (N) photothermal heating effect of CD@MSN during the simulated degradation process in vitro with or without laser irradiation. The numbers in the columns represent the duration of laser exposure.
Figure 2. (A) Confocal images of 4T1 cells after different treatments. Green: live cells; red: dead cells. Bar = 200 μm. (B) Cytotoxicity of 4T1 cells after different treatments with or without an 808 nm laser irradiation (2.0 W cm−2, 5 min). Data were represented as mean ± SD (n = 4). (C) Photothermal images of tumor-bearing mice via an 808 nm laser irradiation within 5 min (2.0 W cm−2) under their corresponding optimum post-injection time points with maximal photothermal heating effects, respectively. (D) Tumor apoptosis results of mice after different treatments on 7th day post-injection. Blue: DAPI-stained nuclei; green: FITC-labeled apoptosis cells. Bar = 500 μm. (E) Changes of tumor volume over time after corresponding treatments. Data were represented as mean ± SD (n = 4). Statistical significance was calculated by one‐way ANOVA using the Tukey post‐test (***p < 0.001). (F) Photos of the lungs harvested from different groups of tumor-bearing mice 14 days after administration (the yellow dotted line circled the metastatic tumor focis) and corresponding H&E stained sections (the blue dotted line presented the border of the metastatic tumors), respectively. Bar = 100 μm.
Figure 3. (A) Fluorescence images of main organs on different days after intravenous injection of MSN and CD@MSN, respectively. Fluorescence signal: Cy5-labeled nanoparticles. (B-D) Semi-quantification of the fluorescence intensity in spleen, liver and lung from corresponding groups at different time points post-injection. (E) Concentrations of granzyme B and IFN-γ in mice plasma from different groups. (F, G) Representative flow cytometric analysis images and the corresponding quantification of proliferation and differentiation of NK cells (F) and macrophages (G) gating on CD45+ cells harvested from different organs. In dot plots, data were presented as mean ± SD. While in box-and-whisker plots, center lines, box limits and whiskers indicated medians, upper and lower quartiles, and the highest and lowest observations, respectively. Statistical significance was calculated by Student’s T-test. P‐value: *, P < 0.05; **, P < 0.01; ***, P < 0.001 (n = 4).
Conclusion(s)
1) Carbon nanodots-incorporated mesoporous silica nanoparticles (CD@MSN) were smartly synthesized by a hydrogen bond/electrostatic-assisted co-assembly strategy;
2) The obtained CD@MSN exhibited an enhanced photothermal performance via gathering the dispersed carbon nanodots, which therefore exhibited an elevated targeting accumulation in tumor and achieved photothermal imaging-guided PTT both in vitro and in vivo;
3) The biodegraded debris could in-situ acquire cancer immune antigens from photothermally lethal tumor cells, and subsequently carried these antigens to escape from the necrotic tissues and selectively entered the immune organs for immunotherapy via stimulating the proliferation and activation of NK cells and macrophages, and simultaneously upregulating the secretion of corresponding cytokines. As a result, effective inhibition of distant tumor metastasis was achieved via the CD@MSN-based PTT.