Hyperthermia‐Triggered On‐Demand Biomimetic Nanocarriers for Synergetic Photothermal and Chemotherapy

Abstract Nanoparticle‐based drug delivery systems with low side effects and enhanced efficacy hold great potential in the treatment of various malignancies, in particular cancer; however, they are still challenging to attain. Herein, an anticancer drug delivery system based on a cisplatin (CDDP) containing nanogel, functionalized with photothermal gold nanorods (GNRs) which are electrostatically decorated with doxorubicin (DOX) is reported. The nanoparticles are formed via the crosslinking reaction of hyaluronic acid with the ancillary anticarcinogen CDDP in the presence of DOX‐decorated GNRs. The nanogel is furthermore cloaked with a cancer cell membrane, and the resulting biomimetic nanocarrier (4T1‐HANG‐GNR‐DC) shows efficient accumulation by homologous tumor targeting and possesses long‐time retention in the tumor microenvironment. Upon near‐infrared (NIR) laser irradiation, in situ photothermal therapy is conducted which further induces hyperthermia‐triggered on‐demand drug release from the nanogel reservoir to achieve a synergistic photothermal/chemo‐therapy. The as‐developed biomimetic nanocarriers, with their dual‐drug delivery features, homotypic tumor targeting and synergetic photothermal/chemo‐therapy, show much promise as a potential platform for cancer treatment.


Materials and instruments
Ascorbic acid, chloroauric acid (HAuCl 4 ), sodium borohydride, cetrimonium bromide CD11c-FITC, CD80-PE and CD86-APC monoclonal antibody were purchased by the eBioscience. The All reagents for cell culture were bought from Gibco. The purified deionized water was prepared by the Milli-Q plus system (Millipore, USA).
The structure of the formed nanoparticles was analyzed with a JEM 1400 Transmission Electron Microscope (TEM) with an acceleration voltage of 120 kV (JEOL, Japan). Zeta potential and size measurements were performed on a Zetasizer Nano ZS (Malvern, UK).
Hydrodynamic diameters were investigated with Nanoparticle Tracking Analysis (NTA) by Zetaview (PMX, Germany). The UV-vis absorption was measured with an UV-2600 spectrophotometer (SHIMADZU, Japan). Fluorescence spectra were analyzed with a RF-6000 fluorescence spectrophotometer (SHIMADZU, Japan). Cell morphologies were captured on a Ti2-A Inversion Fluorescence Microscope (Nikon, Japan). The mean fluorescence intensity of cells was analyzed with cytoFLEX (BECKMEN, USA). The thermographic images were acquired by an infrared thermal camera (FLIR, USA). Photothermal therapy on cells or mice were carried out with a 808 nm laser (Stone, China). All the parameters of blood biochemistry were analyzed by Pointcare M3 (MNCHIP, China). The in vivo imaging was performed by In-Vivo FX PRO (BRUKER, Germany). The fluorescence image of cell uptake was performed by the CLSM of FLUOVIEW FV1000 (OLYMPUS, Japan)

Cell Membrane Proteins Analysis
The SDS-PAGE analysis was performed to estimate the retained proteins of the 4T1 cell membrane vesicles, HANG-GNR-DC and 4T1-HANG-GNR-DC. Briefly, equal amounts of proteins from different samples (quantitafied with BCA protein assay kit) were used for SDS-PAGE analysis. The gel was treated with Coommassie brilliant bule for stained and imaged.
RBCs membranes and RBC-HANG-GNR-DC were also analyzed according to the same method.

DOX and CDDP Loading Capacity
The prepared 4T1-HANG-GNR-DC were centrifuged (6500 g, 20 min, 20 °C) and the supernatant containing free DOX and CDDP was collected for further quantification. To quantify the loading efficiency, the lyophilized 4T1-HANG-GNR-DC were then weighed precisely. UV-Vis spectrometer and ICP-OES were used to measure the amount of the DOX and CDDP. The LE/LC of DOX and CDDP were calculated respectively.

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The formulations of HA-GNR, HANG-GNR-DC, 4T1-HANG-GNR-DC with same concentration of Au (85 μg/mL) were added into EP tube. All the samples were treated with NIR laser (808 nm, 1 W/cm 2 ) for 8 min, following with infrared imaging at each 30 s. To study the photostability of the resulting materials, 4T1-HANG-GNR-DC (800 μg/mL) were irradiated with NIR laser (808 nm, 1 W/cm 2 ) for 4 cycles, followed by infrared imaging recording.

Cell Culture and Cell Uptake
The 4T1 cell was used in this work and cultured in RPMI 1640 supplemented with 10% FBS, penicillin (50.0 IU mL −1 ) and streptomycin (50.0 IU mL −1 ). The cells were incubated at 37 °C in a atmosphere with 5% CO 2 .
The cells were seeded in the 24-well plates with a density of 5 ×10 5 per well and cultured for 24 h. Then the original medium was replaced by free DOX, HANG-GNR-DC or 4T1-HANG-GNR-DC solution in RPMI 1640 with a same DOX concentration of 50 μg/mL. After incubation for 8 h, the cells were washed and stained with Hoechst 33342 (blue) for 12 min, and all the groups were observed with fluorescence microscope.
To evaluated the uniqueness of the homologous targeting ability of the 4T1 cancer cell membranes, the non-tumor cell (RBCs) were isolated from the femal BALB/c mice. The whole blood was centrifuged at 3000 rpm for 5 min and then washed with saline for three times. The obtained RBCs were then re-suspended in DI water to induce membrane rupture.
Subsequently, the solution was centrifuged at 13500 rpm for 8 min at 4 °C. The collected RBCs membranes were co-extruded with HANG-GNR-DC through the 400 nm polycarbonate film to obtained RBC-HANG-GNR-DC.
The 4T1 cells were seed in the 24-well plates with a density of 5 ×10 5 per well and cultured for 24 h. Then the original medium was replaced repectively by 4T1-HANG-GNR-DC and RBC-HANG-GNR-DC solution in basic medium with the concentration of 400 μg/mL. After incubation for 3 h and 6 h, the cell were washed and stained with Hoechst 33342 (blue) for 12 min, and all the groups were observed with the confocal fluorescence microscope.

In Vivo Temperature Measurement on Tumor region
6 The in vivo photothermal effects were also determined in the orthotopic breast tumor model.
The femal BALB/c mice were injected subcutaneously with cell suspension (1.5×10 6 4T1 cells). After two weeks, the tumor bearing mice were injected intravenously with PBS, HA-GNR, HANG-GNR-DC and 4T1-HANG-GNR-DC (the GNR concentration was 10 mg/kg per mouse) respectively. After 24 h, the mice were anesthetized and the tumors were exposed to 808 nm laser at 1 W/cm 2 for 2 min, followed by near-infrared thermal imaging. The dendritic cells were harvested and cultured according to the previous reports. [2] On day 6, the semi-mature DCs were collected and seeded on the 24-well plates with a density of 8 ×10 5 cells per well and cultured for 24 h. The 4T1 cells were seeded onto the Transwell inserts with 2×10 5 cells per well. After 18 h, the medium of 4T1 cells were replaced by PBS, HA-GNR, HANG-GNR-DC and 4T1-HANG-GNR-DC solution (100 μL) in basic medium with the same gold concentration of 150 μg/mL, followed by 6 h incubation. After that, the cells were washed with fresh 1640 medium and then irradiated with the NIR laser (808 nm, 1 W/cm 2 ) for 8 min. Finally, the Transwell inserts were then placed to the DCs cultured plates to induce the maturation. After 12 h, the DCs with different treatments were centrifugated at 1500 rpm for 5 min respectively and then re-suspended in the HBSS containing 10 % FBS to stain with fluorescent antibody (CD11c-FITC, CD80-PE and CD86-APC ) for followed flow cytometry analysis.

In Vivo Tumor Growth Inhibition and Safety Analyses
To perform the in vivo tumor therapy, the BALB/c mice with subcutaneous 4T1 xenografts

Statistical Analyses
Data analyses were conducted using the software GraphPad Prism 7.0. The mean ± SD were determined for all the treatment groups. Statistical analysis was performed by Student's t-test (two-tailed). P < 0.05 was considered representative of a statistically significant difference between two groups.