Deep Penetrating and Sensitive Targeted Magnetic Particle Imaging and Photothermal Therapy of Early‐Stage Glioblastoma Based on a Biomimetic Nanoplatform

Abstract Early diagnosis can effectively improve the survival of glioblastoma multiforme (GBM). A specific imaging technique that is simultaneously deep penetrating and sensitive to small tissue changes is desired to identify GBM. Due to its excellent features in signal contrast, detection sensitivity, and none or little attenuation in tissue, magnetic particle imaging (MPI) possesses great potential in cancer diagnosis, especially when the imaging modality is equipped with specifically targeted nanoprobes. However, when gliomas are small, the blood–brain barrier (BBB) is complete and prevents nanoprobes from entering the brain, which negates the theranostic effect. This study proposes a biomimetic nanoplatform that assist the MPI tracers in breaking through the BBB and then demonstrate a targeted and sensitive diagnosis of GBM. Afterward, the photothermal therapy and immune regulation show an excellent therapeutic effect on the GBM. It is experimentally confirmed that the MPI signal does not decay with tissue depth and shows excellent sensitivity for thousands‐cells. Only small animals are conducted in this study due to the limitations of the current commercial MPI scanner, however, this research theoretically enables large animal and human studies, which encourages a promising pathway toward the noninvasive diagnosis of early‐stage GBM in clinics.

Cell culture and animal model GL261,LO2,b.End.3, and HUVEC cells were cultured separately in DMEM supplemented with 10% (v/v) FBS and 1% penicillin-streptomycin solution under a 5% CO2 atmosphere and at 37°C.
For animal models, male BALB/c nude mice and male C57/6N mice were purchased from Charles River (Beijing, China). Male BALB/c nude mice were used for MPI, MRI, and fluorescence imaging, and male C57/6N mice were used for in vivo antitumor tests. To create glioma model, Luciferase-tagged GL261 cells in a volume of 5 µL were injected into the right caudate nucleus of the BALB/c nude mice by a stereotaxic device. For C57/6N mice, 2 × 10 5 GL261 cells in about 50 μL PBS were subcutaneously injected into the hind leg positions of the mice to set up the tumor model. All animal procedures were carried out according to the guidelines approved by the Animal Ethics Committee of the Hong Kong Polytechnic University. The approval number for animal experiments is A0043201. In vitro BBB model assay b.End.3 cells (1.0 × 10 5 cells/well) were seeded in the 12-well transwell plate with a membrane with a mean pore size of 0.4 µm to simulate the BBB environment. The transendothelial electrical resistance (TEER) values were recorded by the Millicell ERS-2 Epithelial Volt-Ohm Meter voltohmmeter (Millipore, USA) to assess the cell monolayer integrity during cell culture [1] . When the TEER value reaches 200 Ω cm 2 or above, it can be considered as an in vitro BBB model. Next, the fresh DMEM with uncoated SPIO and CCM-SPIO was replaced in the cells, respectively. Afterward, the medium in both apical and basolateral chambers was collected for ICP assays after another 6hour incubation.

In vitro targeting studies
The cells were co-incubated with uncoated SPIO and CCM-SPIO for biological scanning electron microscopy. Following the incubation, DMEM was poured off from the dish without rinsing. The cells were immediately covered with electron microscope fixative and collected into a centrifuge tube by gently scraping them off the dish. After further addition of electron microscope fixative, the cells were fixed for 2 hours at room temperature and then transferred to a 4 °C refrigerator to be ready for subsequent experiments.

Cytotoxicity assay
The cytotoxicity of the nanoprobes was tested on GL261 and LO2 cells. Cells were seeded at a density of 1×10 4 /well in 96-well cell culture plates and incubated at 37°C under a 5% CO2 atmosphere for 24 hours. Then, cells were treated with various concentrations (0, 5, 10, 20, 30, and 40 μg mL -1 ) of CCM-SPIO (100 μL/well) for 24 hours. Finally, 100 μL/well of CCK-8/culture medium (10 μL/100 μL) was supplemented in each well and the plates were incubated for an additional 1 hr under the same conditions. A Synergy HT microplate reader (BioTek, Winooski, VT, USA) was used to measure the absorbance of each well at 450 nm (OD 450). The following formula was used to calculate the cell viability: Cell Viability (%) = [(As-Ab)/(Ac-Ab)] × 100%, where As, Ac, and Ab represent the OD 450 of the treatment group, control group, and blank, respectively.

Photothermal performance assay
Continuous 785-nm NIR laser illumination with a spot size of 5 mm was used to test the photothermal effect. The power density was 0.5 or 0.8 W cm -2 . Before irradiation, the samples were dissolved in deionized water to achieve Fe concentrations of 0, 5, 10, 20, 30, and 40 μg mL -1 , respectively. Then, 100 μL of each sample was used for photothermal measurements. The temperature variations of all samples were recorded using a FLUKE Ti25 infrared thermal imaging camera (Everett, WA, USA) at 30-second intervals, with an accuracy of 0.1°C. For optical stability detection, 20 and 40 μg mL -1 Fe concentrations in CCM-SPIO solutions were analyzed at 5-min intervals by fits and starts using the same laser conditions stated above. All experiments were conducted in triplicate.

In vitro PTT assay
We used calcein-AM and propidium iodide (PI) staining to visually test the photothermal effect of CCM-SPIO. GL261 cells were cultured in 6-well plates at 37°C under a 5% CO2 atmosphere for 24 hours. The original medium was washed off, followed by adding a medium containing 10 µg/mL or 20 µg mL -1 Fe in CCM-SPIO, whereas the medium without nanoprobes was used as a control. After 4 hours of continuous incubation, the cells were irradiated with a 785-nm laser (0.8 W cm -2 ) for 5 mins in an already outlined area. Afterward, the cells were washed gently twice with 1× Assay buffer, followed by the addition of Calcein-AM and PI were added. The cells were incubated for another 15 mins and visualized by an inverted Leica M205 FA fluorescence microscope (Leica, Jena, Germany).
For quantitative analysis, GL261 cells were seeded at a density of 1×10 4 /well in a 96-well plate at 37°C under a 5% CO2 atmosphere for 24 hours. Then, the cells were randomly divided into four groups: control group, CCM-SPIO group, laser group, and CCM-SPIO with laser group. In the control group, the cells were replaced with a new routine culture medium as stated before. In the CCM-SPIO group and CCM-SPIO with laser group, the cells were treated with various concentrations (5, 10, 20, 30, and 40 μg mL -1 of iron) of CCM-SPIO for 4 hours. Subsequently, cells in the CCM-SPIO with laser group were washed three times with PBS and then exposed to 785-nm laser illumination at 0.8 W cm -2 for 3 mins. The same laser treatment procedure was also applied to the laser group but in the absence of CCM-SPIO. After that, CCK-8 was used as discussed earlier.

Statistical analysis
GraphPad Prism 8 software (GraphPad Software, San Diego, CA, USA) was used for statistical analysis. A t-test was performed to determine the differences among different groups. *p < 0.05, **p < 0.01, ***p < 0.001, and ***p < 0.0001 were considered statistically significant, whereas n.s. indicated no significant difference between the two selected groups.