Theranostic nanosensitizers for highly efficient MR/fluorescence imaging‐guided sonodynamic therapy of gliomas

Abstract Glioma is the most frequent primary brain tumour of the central nervous system. Its high aggressiveness and deep‐seated brain lesion make it a great challenge to develop a non‐invasive, precise and effective treatment approach. Here, we report a multifunctional theranostic agent that can integrate imaging and therapy into a single nano‐platform for imaging‐guided sonodynamic therapy (SDT). The SDT agents were fabricated by encapsulation of sinoporphyrin sodium (DVDMS) chelating with manganese ions into nanoliposomes (DVDMS‐Mn‐LPs). DVDMS‐Mn‐LPs are physiologically stable and biologically compatible, and they can produce singlet oxygen upon ultrasound irradiation to kill cancer cells. Both cell and animal studies demonstrated that SDT with DVDMS‐Mn‐LPs can significantly improve the antitumour growth efficiency even in the presence of skull. In addition, DVDMS‐Mn‐LPs are good for MR and fluorescence imaging. Thus, DVDMS‐Mn‐LPs reported here may provide a promising strategy for imaging‐guided modality for glioma treatment.


| INTRODUCTION
Glioma is the most common primary malignant brain tumour and considered one of the most difficult curable tumours because of its typical characteristics such as rapid proliferation, strong invasiveness and unsatisfactory prognosis. [1][2][3] The median survival for patients is only 12-18 months. 4 In clinic practice, surgery is traditionally the initial definitive treatment option for glioma patients. Although radiotherapy, chemotherapy or their combination treatments are also widely used for consolidating the surgical effect, they indeed cause a range of side effects and seriously lower the quality of the patient's life due to their non-selective killing of malignant and normal cells. 5,6 Therefore, it is desirable to develop alternative or complementary glioma treatment modalities for safer and more efficient killing of tumour cells that may help to extend the patient survival.
To date, massive tumour treatment strategies have emerged, including photothermal therapy (PTT), photodynamic therapy (PDT), high intensity-focused ultrasound (HIFU), radiofrequency ablation (RFA) and cryoablation. [7][8][9] Most of them exhibit satisfactory efficacy against various types of tumours and actually some patients are benefiting from them, such as HIFU for uterine fibroids and RFA for liver cancer. 10,11 However, most of these modalities are not so effective for glioma treatment. An important reason may attribute to the violent histologic change from these treatments. 12,13 These changes are usually hazardous and disabling for patients because glioma is seated in the brain, with a huge number of nerve cells around the tumour. Clinical evidence has shown that heating of brain tissue in the sonication path resulted in a secondary focus outside the target causing neurological deficit. 12 Photodynamic therapy is a mild treatment strategy, killing tumour cells through reactive oxygen species (ROS) generated from lowintensity laser irradiation on photosensitizers. 14 However, light cannot penetrate the skull, largely limiting its application in glioma treatment. Compared to PDT, sonodynamic therapy (SDT) is another approach for cancer treatment which uses ultrasound to activate sonosensitizers to generate ROS to kill cancer cells. [15][16][17] Different from invasive RFA and cryoablation, ultrasound is featured with noninvasiveness and high tissue penetrability, making it possible to reach and treat deep-seated tumours, especially for glioma. More importantly, ultrasound can be focused into the brain in a small volume, achieving a precise position on tumours. [18][19][20][21] Although ultrasound can pass through the skull, it is of great challenge to precisely activate the sonosensizers accumulated in the glioma for efficient SDT.
Imaging-guided therapy has provided a new solution to solve this issue. [22][23][24][25][26][27] The imaging guidance plays unique roles and shows its intrinsic advantages in cancer treatment, especially for glioma. On the one hand, it could provide useful information such as size and location of the tumour, as well as the relationship of the tumour with surrounding tissues, which can help ultrasound for determining the irradiation position and scope. On the other hand, it could also provide the optimal time window for treatment so that the ultrasound irradiation could be conducted when the sonosensitizer reaches the peaked level in the targeted lesion. On this ground, we herein report a multifunctional theranostic agent that integrates imaging and therapy functionalities into a single nano-platform for highly efficient imaging-guided SDT. Organic sinoporphyrin sodium (DVDMS) molecules were initially used as the sonosensitizer since they have been widely used in PDT and SDT for the therapy of many human tumours. 28

DVDMS-Mn-LPs
To chelate manganese with DVDMS, manganese chloride (MnCl 2 ) was added to the DVDMS solution at a molar ratio of 2:1 (MnCl 2 : DVDMS) for 1 hour. Excess MnCl 2 was removed using a dialysis process (MWCO = 1 kD). To prepare DVDMS-Mn-LPs, DPPC, Cholesterol and DSPE-PEG 2000 were dissolved in chloroform in a 55:40:5 molar ratios. The chloroform was removed under a nitrogen flow until uniform phospholipid films were formed. The phospholipid films were further dried for over 3 hour under vacuum, followed by hydration at 65°C with double-distilled water containing DVDMS-Mn solution (DVDMS = 1 mg/mL) to obtain a final total lipid concentration of 20 mg/mL. After hydration, the mixed solutions were sonicated in a bath ultrasonic oscillator until it became pellucid.
To remove the unbound fraction of DVDMS-Mn, the liposomes were ultracentrifuged at 100 000 g for 30 minutes at 4°C.

| Characterization of DVDMS-Mn-LPs
The resulting DVDMS-Mn-LPs were subjected to TEM scanning

| Cellular uptake
The U87 human glioma cells were cultured in high glucose DMEM with 10% (v/v) foetal bovine serum, 1% (v/v) penicillin and 1% (v/v) streptomycin. Cells were incubated in a humidified incubator at 37°C with 5% CO 2 . For the cellular uptake experiment, U87 cells (2 × 10 5 cells per well) were seeded in 6-well plates and incubated overnight, and then incubated with DVDMS-Mn-LPs or free DVDMS (DVDMS = 1 μg/mL). After incubation for predetermined time, cells were rinsed with PBS three times, and fixed with 4% paraformaldehyde solution and incubated for 20 minutes each. The nuclear dye DAPI was used as a control to stain nuclei in the experiment. Images of cells were acquired using a biological inverted microscope (Olympus IX71, Olympus Corporation, Olympus, Japan). For quantitative analysis, these cells were harvested and determined by Accuir C6 flow cytometer using CFlow Plus software (BD, Ann Arbor, MI, USA).

| Biocompatibility assay
U87 cells were cultured in standard cell media. Cells were first seeded into 96 well plates and then incubated with free DVDMS or DVDMS-Mn-LPs with different concentrations for 24 hour. The standard CCK8 assay was used to determine relative cell viabilities.

| In vitro cell SDT
Six groups were involved in this study, including the untreated control

| In vitro cellular ROS detection
As for single oxygen detection, the probe reagent (DCFH-DA) was dissolved in DMSO at the concentration of 10 mmol/L, and then diluted with PBS at the final concentration of 25 μmol/L for experiment use. week-old female BALB/c athymic nude mice were maintained under aseptic conditions in a small animal isolator and were housed in standard cages at 12 hour/12 hour light/dark cycle with free access to food and water. All animals were acclimated to the animal facility for at least 3 days before experimentation. All possible parameters that may cause social stress, like group size, type (treated and non-treated), etc., among the experimental animals were carefully monitored and avoided.
Tumour models were established by subcutaneous injection of 5 × 10 6 U87 cells into the right flank of each mouse. Animals were observed daily for any behavioural abnormalities and weighed every 3 days.
Tumour-bearing mice were used when the volume of tumours reached to 100 mm 3 . BALB/C athymic nude mice orthotopic glioma models were established as follows. Animals were anesthetized intraperitoneally with chloral hydrate (40 mg/kg). After being immobilized in a stereotaxic apparatus (RWD68003; RWD Life Science Co, Shenzhen, China), the dorsal surface of the skull was sterilized with an iodine swab. A linear skin incision was placed over the bregma and a burr hole was drilled into the skull approximately 1.8 mm lateral and 1 mm posterior to the bregma. A 25 μL gas-tight syringe was used to inject 5 × 10 5 U87 cells suspended in 5 μL DMEM into the right frontal lobe at a depth of 3.5 mm relative to the dural surface of the brain. The cell suspension was slowly injected over 10 minutes. And then, the needle was slowly retracted over an additional 5 minutes. The wound was rinsed with 0.9% NaCl solution and the burr hole occluded with sterile bone wax to prevent leakage of the cerebrospinal fluid. The skin was then closed with absorbable sutures and the mice were allowed to recover from anaesthesia under observation. Each animal was given a one-time dose by IP administration of antibiotics. MR images of the brain were acquired 7 days following implantation.  were randomly divided into three groups: the control group, the SDT group and the PDT group. The treatment strategies of each group were the same as subcutaneous tumour. MRI images of the brain were acquired at different times to monitor tumour growth. Animals were followed until death, up to 33 days. Total survival times from tumour implantation until death were recorded.

| Histological analysis
After therapy, the mice were sacrificed by standard decapitation, and the tumours and major organs (heart, liver, spleen, lung and kidney) were harvested, fixed with 10% formalin and embedded in paraffin. 7-μm sections were cut with a paraffin slicing machine, fol-

| Statistical analysis
Statistical analysis was carried out with SPSS version 19.0. All of the data represent mean values ± standard deviation of independent measurements. Statistical analysis was performed with a one way ANOVA. Differences were considered significant at *P < 0.05 and very significant at **P < 0.01.

Mn-LPs
As shown in Figure 1A

| Cellular uptake and in vitro SDT
The cell uptake behaviour of DVDMS-Mn-LPs was investigated on U87 cancer cells through fluorescence microscopy and flow cytometry. As shown in Figure 5A respectively. In marked contrast, the combination treatments, regardless of the SDT and SDT plus skull, were found to be highly effective in destructing cancer cells, resulting in 54.82 ± 8.55% or 64.91 ± 11.02% cell viability, respectively. As expected, the PDT plus skull group showed 85.95 ± 5.60% cell viability ( Figure 6B). The levels of intracellular ROS detected by ROS probe further revealed the cytotoxicity mechanism of SDT. As shown in Figure 6C, the tumour cells treated with SDT caused a significantly increased level of ROS, with 1.98-fold, 2.15-fold and 2.65-fold higher than that of the tumour cells treated with only DVDMS-Mn-LPs, only ultrasound and PBS control, respectively. Comparatively, the treatment with PDT plus skull produced much less ROS than that of SDT plus skull.

| Dual-modal imaging in vivo
The cross-section T 1 -weighted MR images were acquired at different time intervals after intravenous injection of DVDMS-Mn-LPs into subcutaneous tumour-bearing mice ( Figure 7A). The brighter T 1 MR signals in the tumour appeared at the third hour and gradually reached the plateau after 6 hour ( Figure 7B). The in vivo T 1weighted MR imaging of orthotopic glioma-bearing mice was also carried out. The T 1 signals of tumour on mice strengthened with the increase in time interval, and reached a peak after 3 hour injection of DVDMS-Mn-LPs ( Figure S3).
In vivo fluorescence imaging was performed on IVIS Spectrum system using a 430 nm excitation wavelength and a 680 nm filter at different time points after injection of free DVDMS or DVDMS-Mn-LPs. As shown in Figure S4A

| Imaging-guided in vivo SDT
The SDT effect in vivo was shown in Figure 7E,F, for tumours receiving DVDMS-Mn-LPs or PDT plus skull treatments, their growths were not obviously inhibited, almost the same as the untreated control. Only ultrasound treatment produced partial tumour growth inhibition. Importantly, both the SDT treatment and SDT plus skull treatment with the assistance of DVDMS-Mn-LPs could inhibit the growth of tumours. No significant difference of the therapeutic efficacies was found between the SDT and SDT plus skull groups. A significantly longer survival period was also observed for the groups treated with SDT and SDT plus skull than that of the other groups (P < 0.01). Moreover, neither obvious body weight loss nor pathological damage was found in the six groups of mice (Figures S5 and S6), indicating no significant acute toxicity for the theranostic agent.
To further examine the treatment efficacy against orthotopic glioma, DVDMS-Mn-LPs were intravenously administrated to the orthotopic glioma mouse model followed by SDT process. The experimental scheme over time was shown in Figure 8A 27,[34][35][36][37] In our study, the r 1 value for DVDMS-Mn-LPs was about 4-fold higher than that of the clinical approved Magnevist due to increased local concentration of Mn 2+ and lowered molecular tumbling rate in those nanocarrier systems. [34][35][36][37] Given that the generation of ROS plays an important role in SDT of tumour treatment, the potential of DVDMS-Mn-LPs to generate ROS was determined using 2′, 7′-dichlorofluorescin diacetate as a typical probe. 23,8 The probe will be deacetylated by cellular esterases to non-fluorescent 2′,7′-Dichlorodihydrofluorescin (DCFH), which is rapidly oxidized to highly fluorescent 2′,7′-Dichlorodihydrofluorescein (DCF) by ROS. These results clearly revealed that the DVDMS-Mn-LPs could act as an effective SDT sonosensitizer for the generation of toxic ROS (Figure 4).
The low cytotoxicity is especially important for a SDT agent to guarantee its biological safety when it is intravenously injected into animal or human body before receiving ultrasound irradiation. Additionally, the previous reports have shown that free DVDMS had obvious cytotoxicity to some cells, even if at lower concentrations (0.5 μg/mL). 24,29 Our study provides a safer DVDMS formulation which has no obvious cell damage below 20 μg/mL DVDMS concentration before they receive laser or ultrasound irradiation ( Figure 5).
Although a large amount of literatures available suggested that PDT has promising applications in superficial tumours, our results imply that PDT is much less effective in the treatment of tumours seated in deep tissues while SDT is featured with high efficiency for solving this critical issue, especially in the treatment of brain tumours.  Because low frequency ultrasound has a strong penetrability through the mouse skull and focus into the brain in a small volume, achieving a precise position on tumours. [18][19][20][21] DVDMS-Mn-LPs not only could be used as dual-modal contrast agents for MRI and fluorescence imaging in vitro but also in vivo.
Free DVDMS could be quickly excreted with a low tumor accumulation amount, but DVDMS-Mn-LPs accumulated in tumour effectively ( Figure 7). The reasons may be attributable to the passive tumour targeting by enhanced permeability and retention effect. In fact, the previous reports had demonstrated that nanoparticles could permeate into brain tumours due to the destruction of blood brain barrier. 39,40 All these results showed that the DVDMS-Mn-LPs successfully integrated two imaging modalities into a single nanoscale system, which provided the basis for further imaging-guided treatment experiments.
With the assistance of imaging guidance, the ultrasound beam could be precisely and flexibly positioned to the whole tumour. Both the SDT treatment and SDT plus skull treatment could inhibit the growth of tumours, supported that SDT but not PDT has greater antitumour efficacy for glioma treatment. The SDT and SDT plus skull groups had no significant difference of the therapeutic efficacies, suggesting that the low frequency ultrasound has a strong penetrability through the mouse skull. All these results indicate that the SDT with DVDMS-Mn-LPs is a safe and effective glioma treatment technique.