Ultrasound Activated Nano TiO2 Loaded With Temozolomide Paves the Way for Resection of Chemo-resistant Glioblastoma Multiforme


 Background

Glioblastoma Multiforme (GBM) is one the most daunting issue to modern therapeutics, with a higher mortality rate post-diagnosis. Temozolomide (TMZ) is the only available treatment; however, the frequent resistance leaves the oncologists at the dead end. Therefore, new approaches to circumvent the GBM are highly desired. In this contribution, we have employed TiO2 nanosticks loaded with TMZ as nanomedicine for TMZ resistant GBM resection.
Results

The ultrasonication triple action effect could highly facilitate the tumor ablation by enhancing the TiO2 nanosticks traversing across BBB, releasing the TMZ payload from TiO2 nanosticks and Reactive Oxygen Species (ROS) generation from TiO2 nanosticks within GBM milieu. The tumor ablation was confirmed by MTT and Annexin(v)-PI assays, apoptotic proteins expression via western blot and ROS level detection in vitro, whereas tumor volume, weight, survival rate, and relative photon flux in the xenograft and orthoptic TMZ resistant GBM murine models as in vivo.
Conclusion

We found this nanomedicine-based ultrasound modality highly efficient in GBM treatment and is of future clinical application value due to employment of already FDA approved techniques and nanomedicine.


Conclusion
We found this nanomedicine-based ultrasound modality highly e cient in GBM treatment and is of future clinical application value due to employment of already FDA approved techniques and nanomedicine.

Background
Glioblastoma is a major type of glioma that primarily affects the central nervous system and known with highly engraved prognosis and postdiagnosis patient survival for less than 15 months (Xu, Chen et al. 2017, Patel, Fisher et al. 2019. Temozolomide (TMZ) is the only Food and Drug Administration (FDA) approved therapeutic agent for GBM that could merely add several months to the survival of patients and is mostly used as adjuvant therapy after surgical resection of the tumor (Chamberlain 2010). In addition, the suboptimal concentration of TMZ at the tumor site, frequent development of chemoresistance and the blood-brain barrier (BBB) selective amenability, are some of the major bottlenecks in complete resection of GBM (Haar, Hebbar et al. 2012, Casals, Gusta et al. 2017, Bahadur, Sahu et al. 2019).
The BBB is a physiological barrier comprised of endothelial cells having tight junctions, basal membrane, and podocytes of astrocytes (Pardridge 2007). The primary mandate of BBB is the central nervous system homeostasis and protection against potentially toxic substances. The BBB is only amenable to small molecules (i.e., <400 Da size and <9 hydrogen bonding, CO 2 , O 2 , Alcohol, and Glucose etc.) (Abbott, Rönnbäck et al. 2006) and it constrain 98% of all other biochemicals and drugs that may be e cient therapeutic agents in other parts of the body (Mitragotri 2013). Therefore, modalities that increase the BBB amenability for the cure of CNS ailments are highly valued. For instance, the focused ultrasound has been reported to open the BBB via micro/nanobubbles formation (Bing, Hong et al. 2018, Wu, Aurup et al. 2018) and allow maximum drug accumulation in the brain. Recently, Liu et al. employed focused ultrasound to reversibly break the BBB and enhance the TMZ localization in GBM from 6.98 to 19 ng/mg (Liu, Huang et al. 2014). Likewise, during the phase I clinical trial, Lipsman et al. employed focused ultrasound to open BBB in Alzheimer's disease (AD) patients that consequently lowered the β amyloid plaques and tau protein aggregates resulting in AD's amelioration (Lipsman, Meng et al. 2018).
The nanotechnology-based approaches have also been more e cient in drug delivery to targeted tissues (viz brain) as compared to free drugs (Srikanth and Kessler 2012, Mi, Shao et al. 2016, Rehman 2020, Younas Iqbal, Wang et al. 2020. For instance, nanoscale TiO 2 has been reported with promising biomedical applications and higher biocompatibility that has already been recognized by FDA , Youssef, Vanderesse et al. 2017 nanoparticles to arrest the growth of the HepG2 cells by 46% after sonication (Ninomiya, Noda et al. 2014).
To establish a robust nano-drug delivery system that could e ciently deliver the therapeutic cargo to GBM across the BBB and could also resect the TMZ-resistant GBM, we established the TiO 2 nanosticks based drug delivery system for TMZ delivery to GBM. Moreover, the ultrasound triple action trigger could open the BBB and further added to the release of TMZ within tumor milieu, and also generated the ROS for the re-sensitization of GBM to TMZ.

Nanomedicine properties
The SEM con rmed elongated morphology of TiO 2 nanosticks ( Fig. 1 a), whereas the DLS analyzed the average hydrodynamic size of 210±23 nm size ( Fig. 1 b). The TEM releveled the pours nature of the TiO 2 nanosticks that played an essential role in the drug loading, as shown in Fig. 1b inset. The zeta potential of TiO 2 nanosticks was 15±0.89, which shows good dispersibility of the as-prepared nanomedicine (supporting Fig. 1).
After the ultra-sonication, the TMZ release from TiO 2 nanosticks was evaluated. It was observed that after a one-hour time point, the sonication could trigger 18±4.89 % of TMZ release as compared to the non-sonicated that could only 8±4.21%, indicating that sonication could trigger TMZ release 2 x higher than non-sonicated in a given time (Fig. 1e).
In vitro anticancer effect MTT assay was performed to see the anticancer effect of ultrasound triggered TiO 2 -TMZ nanomedicine.
The western blot analysis was performed for apoptosis-related vital proteins expressions (P53 and Bcl-2) in TMZ resistant GBM cells after treatment with TiO 2 -TMZ and TiO 2 alone, post sonication. It was observed that TiO 2 -TMZ could produce a higher expression of p53, which is essential for apoptosis induction, whereas the Bcl-2 expression was signi cantly lowered as compared to TiO 2 treated cells. The Bcl-2 is known for its anti-apoptosis function, and its down regualtion indicates the apoptosis induction ( Fig. 2c).
The annexin(v)-PI assay was also performed for apoptosis induction in the U251-TMZ resistant cells. The ow cytometry data revealed that ultrasound activated TiO 2 -TMZ could produce higher apoptosis as compared to TiO 2 nanosticks alone and non-sonicated treated groups (Fig. 2d, Supporting Fig. 4).
Oxidative stress generated by nanomedicine The DCFDA was used as a ROS marker of the oxidative stress in U251-TMZ resistant cells after treatment with TiO 2 -TMZ and TiO 2 in sonicated, and non-sonicated group. The confocal uorescence microscopy revealed that TiO 2 -TMZ could generate a signi cantly higher number of ROS as compared to nonsonicated and TiO 2 alone (Fig. 3 a, b supporting 5). Besides, the ROS intensity was also evaluated via ow cytometry analysis that also exhibited higher uorescence in TiO 2 -TMZ treated group after sonication (Fig. 3 c). Meanwhile, the SOD level was also examined in the treated cells. The SOD level was signi cantly lowered in the TiO 2 -TMZ treated group after sonication as compared to non-sonicated one ( Fig. 3d).
In vitro BBB crossing ability The BBB crossing ability of TMZ-TiO 2 nanomedicine pre and post sonication was evaluated via the transwell BBB model (Fig. 4a). The EDS data revealed that TiO 2 nanosticks could readily cross the BBB after sonication and were uptaken by the U251-TMZ resistant cells, as shown in Fig. 4b, c. Moreover, the TiO 2 nanosticks were loaded with Doxorubicin as a model drug having uorescence properties. When the TiO 2 -Dox and free Dox treatment was given to the transwell BBB model, the sonication could boost the TiO 2 -Dox BBB traversing, which was evidenced by the uorescence intensity in the confocal uorescence micrographs (Fig. d).

Animal model survival post-therapy
The subcutaneous xenograft mice model was initially prepared with TMZ resistant glioblastoma cells and divided into three groups of TiO 2 -TMZ, TiO 2 , and control. After relevant treatments and sonication, it was observed that TiO 2 -TMZ could circumvent the tumor growth and signi cantly lower the tumor volume as compared to other TiO 2 alone and PBS treated groups (Fig. 5 a, b). The tumor weight was signi cantly lowered in TMZ-TiO 2 treated groups, i.e., 0.1±0.23 g, as compared to TiO 2 (0.81±0.44) and PBS group (2.0=-0.89) after sonication (Fig. 5 c). Likewise, the body weight gain was higher in TMZ-TiO 2 treated group as compared to TiO 2 and control ( Fig. 5 d).
Similarly, the tumor volume was also lower in the TIO 2 -TMZ treated models are compared to other treated groups (supporting Fig. 6). The biodistribution data of vital body organs showed higher TMZ accumulation in the liver, i.e., the elimination route for the TMZ from the body, followed by the tumor and brain ( Fig. 5 e). The vital organ histopathology also revealed no pathological lesions after treatment and sonication that vouch for the biocompatibility and inertness of the as-prepared nanomedicine (supporting Fig. 7).
The orthotopic TMZ resistant GBM tumor expressing luciferase was prepared and treated with TMZ-TiO 2 at day ten till day 21 on alternate days, as shown in the experimental layout ( Fig. 5f). Meanwhile, every next day of treatment, mice were imaged for bioluminescence, which was directly proportional to the tumor volume (Fig. 5g). The relative photon ux data showed that TiO 2 -TMZ after sonication could circumvent the tumor volume more e ciently than other treated groups (Fig. 5h). The animal treated with TMZ-TiO 2 had the highest survival rate (56 days) post-therapy as compared to other groups (Fig. 5i).

Discussion
GBM is one of the fatal brain diseases. Its complete resection is still a challenge with only TMZ as available chemotherapeutic agent. In addition, the frequent resistance development to TMZ has further complicated GBM cure (Casals, Gusta et al. 2017). In this contribution, we have employed ultrasound activated TiO 2 nanosticks loaded with TMZ to resect the TMZ resistant GBM in vitro as well as in vivo.
The ultrasound trigger has a triple action effect aiding to GBM resection by (i) opening BBB, (ii) releasing TMZ payload from TiO 2 nanosticks, and (iii) ROS generation from TiO 2 nanosticks that helps in resensitization of GBM to TMZ . Previously, the nanoscale TiO 2 sonication at 1.0 MHz frequency has been reported with e cient intratumor ROS generation that inhibited cell viability and tumor growth (Harada, Ogawa et al. 2011). Herein, we have employed 1.5 MHz frequency ultrasound that could e ciently penetrate the skull bones of orthotopic GBM models. The typical diagnostic and materials application ultrasound are ranged from 1-10 MHz frequency (Silva, Ferreira et al. 2011). Hence, the employed ultrasound modality is in line with therapeutic ultrasound in vogue.
The TiO 2 nanosticks can upload maximum TMZ at a ratio of 3:1. The TMZ has been adsorbed on the porous surface of TiO 2 nanosticks after overnight incubation, the same as previously reported . Interestingly, it was observed that ultrasonication could trigger the drug release much faster than non-sonicated TiO 2 . Likewise, Shi et al. loaded Docetaxel on the mesoporous TiO 2 nanoparticles for the treatment of cancer via sonodynamic therapy (Shi, Chen et al. 2015). It was observed that Docetaxel loaded TiO 2 could signi cantly deliver and release the drug in tumor site after sonication and exhibited excellent anticancer e cacy. Moreover, the TMZ has a natural ability to cross the BBB, as earlier reported (Brun, Carrière et al. 2012). However, when the ultrasound is employed to the BBB, the nanomedicine traversing becomes faster with maximum drug bioavailability in brain tissue.
This study has used two-step ultrasonication in orthotopic animal models; the rst one is to open the BBB for nanomedicine traversing that ensures maximum drug accumulation within the brain tissue. The second sonication ensures the swift releases of TMZ from TiO 2 nanosticks and also start ROS generation. The ultrasound activated TiO 2 can generate OH, 1 O 2 , H 2 O 2 , etc. that interferes with cell signal pathways and thus initiates apoptosis or necrosis within the target tissue (Ninomiya, Ogino et al. 2012). In this study, when TMZ resistant GBM cells were treated with as prepared nanomedicine, the sonication effect could produce less than 50% cell viability.
Moreover, the critical apoptosis markers (P53 and Bcl-2) were signi cantly in uenced by the sonicated nanomedicine. Earlier Zhang et al. reported that TMZ induces O 6 -methylguanine production that is involved in AMP-activated protein kinase (AMPK) activation via an elevated level of ROS generated. The AMPK is involved in the GBM apoptosis induction via p53 upregulation. Meanwhile, by inhibition of mTOR complex 1, the Bcl-2 protein expression is downregulated, which is further inducing TMZ mediated pro-apoptotic effect (Zhang, Wang et al. 2010). Likewise, Kim et al. also reported the tumor targeting p53 nano drug delivering system with enhanced sensitization of chemoresistant GBM to TMZ therapy (Kim, Rait et al. 2015). In analogy to TMZ, the TiO 2 also generates ROS that results in AMPK activation and apoptosis induction in TMZ resistant GBM. The sonoluminescence is considered a critical phenomenon to generate ROS from nanoscale TiO 2 (Hu, Fan et al. 2015), that on the one hand, induces apoptosis in the tumor ) and on the other hand destroy the tumor vasculature endothelial layer or blood stasis via platelet aggregation (Borsig, Wong et al. 2001, Volanti, Gloire et al. 2004).
In a recent study, Feng et al., employed the NAMPT inhibitors (FK866, CHS828) along with TMZ to sensitize the GBM cells to TMZ (Feng, Yan et al. 2016). In addition to other factors, these NAMPT inhibitors could elevate the ROS level and reduced the SOD and total antioxidants activity in GBM cells that could sensitize the GBM cells to TMZ. Likewise, Seyfrid et al. employed Smac mimetic (BV6) along with TMZ to sensitize the GBM to chemotherapy (Seyfrid, Marschall et al. 2016). The combination therapy of BV6-TMZ could orchestrate the ROS generation in the mitochondria and cytosolic contents of GBM.
These ROS could then activate the pro-apoptotic factors viz Bax protein upregulation to resect GBM. It has been investigated that TMZ could induce cytoprotective autophagy in GBM cells, thus leading to compromised TMZ sensitivity. However, when mitochondria transport chain inhibitors are combined with TMZ as adjuvant therapy, the autophagic cell death is mediated by ROS (Chen, McMillan-Ward et al. 2007), and GBM sensitivity to TMZ is signi cantly augmented (Yan, Xu et al. 2016). Our ndings are also in agreement with the aforementioned results suggesting that elevated ROS level and decreased antioxidant level (i.e., SOD) ensures the re-sensitization of GBM to TMZ. Thus, apoptosis is initiated in the GBM.

Conclusion
In summary, we report that TiO 2 nanosticks cloud signi cantly delivers the TMZ payload to brain milieu across the BBB and resect the TMZ-resistant GBM. The ultrasonication triple action effect could open the BBB, release the TMZ from TiO 2 nanosticks, and mimic the ROS generation within GBM. The generated ROS enhances the tumor sensitivity to TMZ that resulted in apoptosis induction and tumor growth arrest in vitro as well as in xenograft and orthotopic TMZ resistant GBM models. The reported modality is novel and reported for the rst time (as per our knowledge).

Methods
All the chemicals used in this study were experimental grade and purchased from Sigma Aldrich (St. Louis, Missouri, USA), otherwise mentioned. The Milli-Q deionized water with -18m /cm 2 was used to prepare chemical reagents. HyClone Inc USA, provided the cell culture media and reagents, whereas the cell culture asks were purchased from Nest biotechnologies Wuxi, China. The TiO 2 nanosticks were kindly provided by Dr. Xiao Hua Lu, College of Engineering, Nanjing University of Technology, Nanjing China.
All the animals were provided with pallet feed and water ad libitum in an environmentally controlled house. All the experiments were performed under the guidelines of Henan University animal welfare committee.

Materials Characterization
The TiO 2 nanosticks were morphologically characterized by Scanning and transmission electron microscopy (Jeol, the USA, and JEM-2010HT Japan, respectively). The size and zeta potential of materials was performed by using Zetasizer Nano (Malvern Pananalytical Ltd.). The Energy Dispersive Xray Spectroscopy (EDS) was performed by SEM to see cell uptake of TiO 2 nanosticks after crossing BBB in vitro.

Nanomedicine preparation
The TiO 2 nanosticks and TMZ were dissolved in the deionized distilled water to obtain the nal concentration of 0.5 mg/ml and 0.15 mg/ml, respectively. The two solutions were then mixed and kept at room temperature on a magnetic stirrer for overnight rotation. Then the solution was centrifuged at 8000 X g for 10 minutes to remove unbound TMZ from TiO 2 nanosticks. The TiO 2 nanosticks were then washed with deionized distilled water and used in further experiments.

Drug loading and releases
The TiO 2 nanosticks and TMZ were dissolved in deionized water and then mixed at the various ratios of 1:1, 2:1, and 3:1. The TMZ concentration was kept constant, whereas the TiO 2 nanosticks ratio was variable. The TiO 2 -TMZ mixture was kept on rotation for overnight at room temperature and then centrifuged at 8000 X g to remove untrapped TMZ. The TMZ absorption value was determined at 327 nm to determine the TMZ uploading value on TiO 2 nanosticks.
The release kinetics of TiO 2 -TMZ nanomedicine were performed at 37 o C in PBS. At various time points of 0.5, 1,2,4,8,12,16,24,36,48, and 72 hours the centrifugation was performed (8000 X g), and TMZ concentration was measured by taking absorbance value at 327 nm. For sonication effect on TMZ releases, at a time point of 1, 4, 12, 24 and 48 hours, after incubation, the sonication was performed, i.e., 1.0 Watt/cm 2 , 1.5 MHz frequency with 50% intensity by intellect mobile ultrasound (Chattanooga®, GLOBAL DJO HEADQUARTERS 2900 Lake Vista Drive Dallas, TX 75067) and then reading for TMZ concentration was performed.

Cell culture experiments
The U87 cell line of glioblastoma and b.End3 cell line of endothelial cells were provided by the Chinese Academy of Sciences, Shanghai, China. The U251 cells were TMZ resistant and transfected with luciferase were obtained from iCell Biosience Inc., Shanghai China. The cells were cultured in the 25 cm 2 tissue culture asks in DMEM high glucose medium supplemented with 10% FBS and 1% penicillinstreptomycin solution under standard incubation conditions of 95% relative humidity at 37 o C temperature, in the presence of 5 % CO 2 . When the con uency reached 90%, the cells were trypsinized with 0.25% trypsin containing EDTA and were further utilized for downstream experiments.

Western blot
The western blot technique was employed to evaluate the apoptotic protein biomarkers (Bcl-2 and p53) and housekeeping genes β-actin by the procedure mentioned earlier (Haney, Klyachko et al. 2015). The Bcl-2 and p53 primary rabbit anti-human antibodies were provided by Biolegend and diluted to a concentration of 1:5000, whereas secondary antibodies (IRDye 800CW goat anti-rabbit secondary antibodies) were provided by LI-COR Biosciences U.S. The protein bands, were visualized by Odessey® Clx western blot scanner (LI-COR Biosciences -U.S.) and processed by image studio software.
Flow cytometry for Apoptosis U251-TMZ resistant cells were cultured in 10 cm Petri dishes for 24 hours under standard conditions. The cells were divided into ve groups, i.e., PBS, TiO 2 only, TiO 2 -TMZ only, TiO 2 sonicated and TiO 2 -TMZ sonicated, and treatment was given for another 12 hours. Meanwhile, after one hour of inoculation, the two groups, i.e., TiO 2 sonicated and TiO 2 -TMZ sonicated, were sonicated for one minute. Afterward, the cells were trypsinized and treated with Annexin(v)-PI reagent (Beyotime Biotech Inc.) for cell apoptosis evaluation according to manufacturer instructions. The cells were then immediately analyzed through the BD FACSAriea ow cytometer for apoptosis detection.
MTT assay for cytotoxicity U251-TMZ resistant cells were cultured in 96 well plates at an equal concentration of 1 x10 3 cell per mL for 24 hours. The cells were treated with TiO 2 only, TiO 2 -TMZ, sonicated, and non-sonicated groups, whereas the control group was treated with PBS for 24 hours with concertation from 1 to 10 µg of nanomedicine per well. The ultrasound group was sonicated for one minute after one hour of nanomedicine inoculation by sonication procedure mentioned earlier. Afterward, 20 µl of 5 mg/mL of MTT solution was added into each well and incubated for 4 hours. Then the medium was discarded, and 200 µl of DMSO was added to each well and further incubated for 10 minutes at room temperature. The plates were then subject to an optical density (OD) reading at 492 nm wavelength by ELISA microplate reader (SpectraMax® i3x, Molecular Devices, LLC).
The following formula evaluated the cell viability: Cell viability (%) = OD value of treatment/OD value of Control X 100

Confocal microscopy
The U251-TMZ resistant cells were cultured for 24 hours on the confocal microscopy Petri dishes provided with lens at bottom. The cells were then divided into two main groups, i.e., sonicated and nonsonicated. These groups were further divided into TiO 2 , TiO 2 -TMZ, and PBS. Each group was inoculated with corresponding nanomedicine @ 10 mg/mL and further incubated for 12 hours; meanwhile, the sonicated group was ultrasound treated one-hour post nanomedicine inoculation. The cells were then treated with 2',7'-dichlorodihydro uorescin diacetate (DCFDA) reagent (Invitrogen TM ) 1% solution for one hour. Afterward, the cells were washed with PBS 3 times and added DAPI solution 1:1000 for ve minutes to stain the cell nucleus. Again, after washing, the cells were xed with paraformaldehyde 4% for 10 minutes. Then washed again with PBS and imaged under a confocal microscope (Zeiss LSM 880) at the FITC channel, whereas the cell nucleus was imaged at DAPI channel.
For quantitative ROS analysis, the DCFDA treated medium from cells was collected after one hour of incubation, and uorescence was measured at 488 nm wavelength by using SpectraMax® i3x, Molecular Devices, LLC.

In vitro BBB model preparation
The endothelial cells were cultured on the transwell membrane (Millicell®, 100 nm pore size) until the complete cell con uency was achieved, and the cells' conductivity resistance become above 300 ohms. Then U251-TMZ resistant cells were cultured on the lower chamber of the 12 well plate containing round glass coverslips and treatment of (Doxorubicin) DOX, TiO 2 -DOX, and PBS was given to sonicated and non-sonicated group @ 10 mg/mL of corresponding nanomedicine. After one hour, the sonicated group was ultrasound treated as mentioned above and incubated under standard incubation conditions with 100 rotations per minute (to generate su cient shear force) for 12 hours. Later the cell culture medium was removed and washed with PBS, then added 1:1000 DAPI solution for 5 minutes to stain the cell nucleus. Afterward, the cells were washed with PBS and xed with paraformaldehyde 4% for 10 minutes.
The glass coverslips were removed from the 12 well plates and mounted on the glass slide to observe under a confocal microscope.

SOD activity
The U251-TMZ resistant cells were cultured in six-well plates. When the cell con uence reached 90%, they were divided into two groups, i.e., sonicated and non-sonicated. Both groups were separately treated with TiO 2 and TiO 2 -TMZ @ 10 mg/mL, whereas the PBS group was kept as control. The sonicated group after one hour was treated with ultrasound, as mentioned above. After overnight incubation, the Superoxide Dismutase level was determined in each group from the cell lysate by the commercially available kit (SOD Assay Kit-WST, Dojindo Molecular Technologies, Inc.) following the manufacture instructions. Microplate reader was used to read the optical density value at 450 nm wavelength.

Xenograft and orthotopic animal models preparation
Luciferin expressing U251-TMZ resistant cells were cultured in the 75 cm 2 culture asks. After attainting 90% con uency, the cells were trypsinized (0.25% trypsin), and collected in pellet form. These cells were then injected to BALB/C athymic nude mice @ 3 x10 6 cells subcutaneously to form tumors. After two weeks, a palpable size of tumor was noticed. These mice were then used for further experiments, whereas few were euthanized to obtain the tumor for orthotopic model preparation.
The tumor was chopped to small size in normal saline and injected to a mouse brain that was exposed by skull puncture under general anesthesia of iso urane. The skull bone was then sealed with a bone sealant (CP Medical), and the wound was sealed via tissue adhesive glue (Vetbond TM , 3M, Japan). On day 3, mice were imaged for bioluminescence by IVIS-Lumina Series III, PerkinElmer Inc, after injection of D-luciferin solution (5 mg/mL). The successful models showing bioluminescence were further selected for experimental trials.

Animal treatment
The subcutaneous xenograft GBM TMZ resistant mice were divided into three groups (i.e., TiO 2 , TMZ-TiO 2 , and a control group treated with PBS. N= 5x3). At day 21 of the rst implant, the mice were treated with as-prepared TiO 2 and TMZ solution @ dose rate of 200 µl, either separately or in combination, every alternate day for 15 days. Thirty minutes post-injection, ultrasound therapy was given at the tumor site.
Then mice were euthanized, and vital organs (liver, kidney, spleen, lungs, brain) and tumor were removed and further analyzed.
The three groups of orthotopic mice were injected with the nanomedicine (200 µl) on day 10 to 22, every alternate day. Post-injection every 10 minutes, followed by 30 minutes, the ultrasound therapy at the head region was performed (N=7x3). Meanwhile, the mice were imaged for bioluminescence every next day of treatment during the experimental trial, and the relative photon ux value was recorded. Animal survival was also recorded daily.

Histopathology
The mice's vital organs were collected and preserved in the 10% formalin solution. Then para n embedding technique was used to prepare 3 µm slides and stained them with Hematoxylin & Eosin, and observed under an Olympus uorescence microscope for histopathological lesions observations.

Statistical analysis
All the data was initially recorded in the MS-Excel and then subjected to analysis of variance (ANOVA) by using statistical software named SPSS version 18 (SPSS Inc. Chicago Illinois USA). All results presented were in mean ± standard deviation (SD). The probability value < 0.05 was considered as statistically signi cant.

Declarations
Ethics approval and consent to participate Not applicable Consent for publication Oxidative stress generated by TiO2 nanosticks loaded with TMZ on TMZ resistant GBM. a) DCFDA (2',7'-Dichloro uorescin Diacetate) treated TMZ resistant cells confocal micrograph after ultrasonication.
Herein green color indicates the ROS generated, and the cell nucleus is stained with DAPI. b) Quantitative data of reactive oxygen species (ROS) generated after treatment with TiO2 nanosticks loaded with TMZ. * indicates the signi cant difference (p < 0.05) between TiO2-TMZ sonicated and non-sonicated cells ROS level c) Flow cytometry data of ROS generated by the TiO2-TMZ treatment after sonication. d) TMZ resistant GBM cells' SOD (superoxide dismutase) enzyme activity after treatment with by TiO2 nanosticks loaded with TMZ, post sonication. The * and ** are the probability value < 0.05 and 0.01, respectively.