Synopsis

Sonodynamic therapy (SDT) is a non-invasive and effective therapeutic modality for solid cancerous tumors. In this study, to improve the effectiveness of SDT, we fabricated a high-performance multifunctional albumin nanoassembies (HSA-Ce6-Mn NCs) having higher cell uptake efficiency and the tumor accumulation. It also exhibited an excellent ability of MR/ fluorescence (FL) dual-modality imaging so that glioma margin and HSA-Ce6-Mn NCs metabolism could be clearly visualized. The SDT has been demonstrated for suppressing the tumor growth in-vivo under a MRI-guided FUS system and it is highly expected to have a great potential in clinical translation.

Introduction

Methods

Preparation and characterization of HSA-Ce6-Mn NCs

HSA-Ce6-Mn NCs were formulated using HSA dissolved in water and added Ce6 into the solution, then mixed with MnCl₂. The relationship of the T₁ relaxation rate and the concentration of HSA-Ce6-Mn NCs were investigated through the inversion recovery TSE sequence. The generation of ROS was determined using fluorescence spectrometer after samples mixed with DCFH-DA and sonicated by ultrasound with different intensities (0.5MHz, 0.25~1.5W/cm², 50% duty cycle,1min). For cellular uptake, the U87 cells were incubated with samples and stained with DAPI and DiO, then observed by confocal microscope.

Animal model

Six-to-seven week old male BALB/c athymic nude mice were used for experiment. To develop the subcutaneous tumor models, U87MG cells were injected into the back of mouse. To develop the orthotopic glioma models, animals were immobilized in a stereotaxic apparatus, a burr hole was drilled into the skull and U87MG cells was injected into the right frontal lobe, then the incision was sewed up.

In-vivo dual-modality imaging

After injection HSA-Ce6-Mn NCs, animals were anesthetized. The FL images were acquired by IVIS Spectrum system using a 605 nm excitation and 635 nm filter. MR images were acquired on a 3.0 T clinical MR scanner (TIM TRIO, Siemens, Germany). T1-weighted TSE sequence was used with parameters: TR=700ms, TE=13ms, resolution was 0.3*0.3*2mm.

MR guided In-vivo treatment

When the volume of tumors reached to 50-100mm³_, the animals were selected for treatment. With the injection of HSA-Ce6-Mn NCs, the anesthetized animals were fixed in a customized MR compatible ultrasound platform. The position of the tumor was determined from the pre-scan T₁w images. The ultrasound transducer was moved so that the ultrasound focus was inside the target. The tumor target was sonicated under FUS (f=0.8 MHz) to release the ROS in the local area. During the sonication, MR temperature imaging was used to monitor the temperature change with parameters: GRE sequence, TR=29ms, TE=10ms, resolution=0.6*0.6*3mm, with the acquisition time 4.6 second/measurement. A feedback temperature PID control algorithm was implemented to keep local temperature constant at 42℃ for 20min.

Results

The schematic process of HSA-Ce6-Mn NCs was described in Figure1a. The T₁w MR images and longitudinal relaxivity (r₁) of HSA-Ce6-Mn NCs compared to the clinical approved Magnevist are shown in Figure 1b. The experiment apparatus schematic of detection the ROS is showed in Figure 1c and the ROS intensity of HSA-Ce6-Mn NCs exhibited an intensity-dependent enhancement under ultrasound sonication (Figure1d).The cell uptake behavior of HSA-Ce6-Mn NCs was further evaluated through confocal microscopy shown in Figure 2.

In-vivo FL imaging of glioma-bearing mice showed higher fluorescence intensity than free Ce6 (Figure 3a-b). In-vivo T₁w MR images of mice were all showed signal enhancement in tumor area with the increase of time post injection of HSA-Ce6-Mn NCs up to 24h (Figure 4a-b).

For in-vivo treatment, the MR guided ultrasound platform configuration is illustrated in Figure 5a, the target temperature of US focal spot was set to 42℃and maintained about 20 minutes successfully under the temperature control algorithm (Figure 5b-c). The tumor growth showed significantly suppressed in the HSA-Ce6-Mn NCs combined US group compared to the other three groups (Figure 5d), the H&E and TUNEL stained section of this group showed the cells have suffered necrosis and apoptosis(Figure 5e).

Discussion and conlustions

Acknowledgements

References

1. Huang, P., et al., Metalloporphyrin-Encapsulated Biodegradable Nanosystems for Highly Efficient Magnetic Resonance Imaging-Guided Sonodynamic Cancer Therapy. J Am Chem Soc, 2017. 139(3): p. 1275-1284.

2. Deepagan, V.G., et al., Long-Circulating Au-TiO2 Nanocomposite as a Sonosensitizer for ROS-Mediated Eradication of Cancer. Nano Lett, 2016.