I. INTRODUCTION

Section:

ChooseTop of pageABSTRACTI. INTRODUCTION <<II. EXPERIMENTIII. RESULTS AND DISCUSSI...IV. SUMMARY AND CONCLUSIO...REFERENCESIn recent years, the electromagnetic, microwave, radiofrequency, laser, and ultrasound energy induced hyperthermia has been developed as a promising therapeutic strategy to combat cancers.1–51. Q. Wang et al., Adv. Funct. Mater. 29, 1901480 (2019). https://doi.org/10.1002/adfm.2019014802. J. Beik, Z. Abed, F. S. Ghoreishi, S. Hosseini-Nami, S. Mehrzadi, A. Shakeri-Zadeh, and S. K. Kamrava, J. Control. Release 235, 205 (2016). https://doi.org/10.1016/j.jconrel.2016.05.0623. P. B. Elming, B. S. Sørensen, A. L. Oei, N. A. P. Franken, J. Crezee, J. Overgaard, and M. R. Horsman, Cancers 11, 60 (2019). https://doi.org/10.3390/cancers110100604. E. Cazares-Cortes, S. Cabana, C. Boitard, E. Nehlig, N. Griffete, J. Fresnais, C. Wilhelm, A. Abou-Hassan, and C. Ménager, Adv. Drug Deliv. Rev. 138, 233 (2019). https://doi.org/10.1016/j.addr.2018.10.0165. J. Li, Y. Luo, and K. Pu, Angew. Chem., Int. Ed. 60, 12682 (2021). https://doi.org/10.1002/anie.202008386 The temperature above 47 °C could directly destroy the cancer tissues. The temperature ranging from 41 to 45 °C could make the cancer cells more susceptible to other treatment modalities. More importantly, nanomaterials could absorb energy originated from the aforementioned source to enhance the localized tumor thermal destruction effect. Among all hyperthermal methods, in the presence of an alternating magnetic field (AMF), the intratumoral magnetic nanomaterials induced magnetic hyperthermia (MHT) has been applied for glioblastoma multiforme (GBM) brain tumors in Germany.6–86. K. Mahmoudi, A. Bouras, D. Bozec, R. Ivkov, and C. Hadjipanayis, Int. J. Hyperth. 34, 1316 (2018). https://doi.org/10.1080/02656736.2018.14308677. R. Gupta and D. Sharma, ACS Appl. Nano Mater. 3, 2026 (2020). https://doi.org/10.1021/acsanm.0c001218. G. Hannon, A. Bogdanska, Y. Volkov, and A. Prina-Mello, Nanomaterials 10, 593 (2020). https://doi.org/10.3390/nano10030593 Also, the clinical trial for treatment of prostate and pancreatic cancer has also been approved by the U.S. Food and Drug Administration (FDA).99. K. Maier-Hauff, F. Ulrich, D. Nestler, H. Niehoff, P. Wust, B. Thiesen, H. Orawa, V. Budach, and A. Jordan, J. Neuro-Oncol. 103, 317 (2011). https://doi.org/10.1007/s11060-010-0389-0For MHT, due to the size-dependent magnetic properties, high stability, ease of functionalization with both organic and inorganic compounds, biocompatibility, minimal toxicity as well as ease of excretion, the magnetic nanoparticles (MNPs) are currently considered the favorite heating agent for MHT.10,1110. S. Spirou, M. Basini, A. Lascialfari, C. Sangregorio, and C. Innocenti, Nanomaterials 8, 401 (2018). https://doi.org/10.3390/nano806040111. I. M. Obaidat, B. Issa, and Y. Haik, Nanomaterials 5, 63 (2015). https://doi.org/10.3390/nano5010063 Upon exposure to an AMF, the MNPs with a superparamagnetic property act as a transducer to convert external electromagnetic energy onto the tumor for ablation while minimizing the adverse effects on collateral tissues. The temperature of a tumor tissue could be increased to 42 °C and above but not more than 50 °C.12–1412. P. Das, M. Colombo, and D. Prosperi, Colloids Surf., B 174, 42 (2019). https://doi.org/10.1016/j.colsurfb.2018.10.05113. K. Yu et al., Theranostics 9, 4192 (2019). https://doi.org/10.7150/thno.3415714. S. Ranoo, B. B. Lahiri, M. Nandy, and J. Philip, J. Colloid Interface Sci. 579, 582 (2020). https://doi.org/10.1016/j.jcis.2020.06.093 In order to make the MNPs achieve optimal heat performance, some strategies have been investigated to tune the well-defined structure. One is the synthetic condition related nanoparticle itself, such as its size, crystallinity, anisotropic shape, and chemical composition.15–1915. P. T. Phong, P. H. Nam, D. H. Manh, and I. J. Lee, J. Magn. Magn. Mater. 433, 76 (2017). https://doi.org/10.1016/j.jmmm.2017.03.00116. C. Guibert, V. Dupuis, V. Peyre, and J. Fresnais, J. Phys. Chem. C 119, 28148 (2015). https://doi.org/10.1021/acs.jpcc.5b0779617. H. Gavilán, S. K. Avugadda, T. Fernández-Cabada, N. Soni, M. Cassani, B. T. Mai, R. Chantrell, and T. Pellegrino, Chem. Soc. Rev. 50, 11614 (2021). https://doi.org/10.1039/D1CS00427A18. E. A. Périgo, G. Hemery, O. Sandre, D. Ortega, E. Garaio, F. Plazaola, and F. J. Teran, Appl. Phys. Rev. 2, 041302 (2015). https://doi.org/10.1063/1.493568819. V. F. Cardoso, A. Francesko, C. Ribeiro, M. Bañobre-López, P. Martins, and S. Lanceros-Mendez, Adv. Healthc. Mater. 7, 1700845 (2018). https://doi.org/10.1002/adhm.201700845 Another is the use of surface modification to form assemblies based on presynthesized MNPs.20–2220. N. K. Sahu, J. Gupta, and D. Bahadur, Dalton Trans. 44, 9103 (2015). https://doi.org/10.1039/C4DT03470H21. S. K. Avugadda et al., Chem. Mater. 31, 5450 (2019). https://doi.org/10.1021/acs.chemmater.9b0072822. I. Andreu, E. Natividad, L. Solozábal, and O. Roubeau, ACS Nano 9, 1408 (2015). https://doi.org/10.1021/nn505781f Compared with tuning the intrinsic features of MNPs, it has been found that the interparticle interactions between MNPs also have a significant effect on their MHT heat performance. However, random assemblies of MNPs may result in the occurrence of a demagnetizing effect due to the strong antiferromagnetic couplings of the MNPs within the clusters, which in turn suppresses the heat efficiency.23–2523. L. Gutiérrez, L. De La Cueva, M. Moros, E. Mazarío, S. De Bernardo, J. M. De La Fuente, M. P. Morales, and G. Salas, Nanotechnology 30, 112001 (2019). https://doi.org/10.1088/1361-6528/aafbff24. A. Singh and S. K. Sahoo, Drug Discov. Today 19, 474 (2014). https://doi.org/10.1016/j.drudis.2013.10.00525. S. Laurent, S. Dutz, U. O. Häfeli, and M. Mahmoudi, Adv. Colloid Interface Sci. 166, 8 (2011). https://doi.org/10.1016/j.cis.2011.04.003 The theoretical and experimental results have demonstrated that the noninteracting core/shell MNPs as building blocks may enhance the heating efficiency.26–2826. T. Sadhukha, T. S. Wiedmann, and J. Panyam, Biomaterials 35, 7860 (2014). https://doi.org/10.1016/j.biomaterials.2014.05.08527. R. Mejías, P. Hernández Flores, M. Talelli, J. L. Tajada-Herráiz, M. E. F. Brollo, Y. Portilla, M. P. Morales, and D. F. Barber, ACS Appl. Mater. Interfaces 11, 340 (2019). https://doi.org/10.1021/acsami.8b1845128. A. M. El-Toni, M. A. Habila, J. P. Labis, Z. A. Alothman, M. Alhoshan, A. A. Elzatahry, and F. Zhang, Nanoscale 8, 2510 (2016). https://doi.org/10.1039/C5NR07004JThe microemulsion-based polymer/phospholipid encapsulation method is an intriguing approach to control the assembly of MNPs by affecting both the miscibility and change in stability in aqueous solutions of the cluster building blocks. In this study, we have developed a repeated compression method in a sealed vial to elaborately fabricate magnetic nanobubbles (MNBs) with the γ-Fe2O3 MNPs shell in tandem with our previous study.29,3029. J. Jin, F. Yang, B. Li, D. Liu, L. Wu, Y. Li, and N. Gu, Nano Res. 13, 999 (2020). https://doi.org/10.1007/s12274-020-2732-x30. J. Li, Z. Feng, N. Gu, and F. Yang, J. Mater. Sci. Technol. 63, 124 (2021). https://doi.org/10.1016/j.jmst.2020.02.045 As shown in Fig. 1, the gaseous structure of MNBs is composed of phospholipid encapsulated γ-Fe2O3 nanoparticles self-assembled on the interface of the gas-liquid based on a temperature-regulated repeated compression self-assembly approach. The morphology, heating transferring efficiency, as well as interaction with U87MG glioma cells were investigated. The results show that the MNPs are well-distributed on the gas-liquid interface with an increased specific absorption rate (SAR) in a nanobubble structure. Further investigation on cell hyperthermia also demonstrated better anticancer effect with lower concentration of MNPs. Therefore, such MNBs have emerging potential to be a new hyperthermia agent in clinical application for glioma in the future.

II. EXPERIMENT

Section:

ChooseTop of pageABSTRACTI. INTRODUCTIONII. EXPERIMENT <<III. RESULTS AND DISCUSSI...IV. SUMMARY AND CONCLUSIO...REFERENCES

The experimental procedures and calculations must be described clearly, and enough information should be given such that they could be reproduced by other scientists.

A. Materials

1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt, chloroform) (DSPE-PEG2K-amine), and 1, 2-distearo-yl-sn-glycero-3-phosphoethanolamine-N-[carboxyl (polye-thylene glycol) 2000] (ammonium salt) (DSPE-PEG2K) were purchased from Avanti Polar Lipids, Inc. (USA). The superparamagnetic γ-Fe2O3 magnetic nanoparticles with an iron oxide core (7 nm in diameter) and a dextran (PSC) shell (20 nm in thickness) (γ-Fe2O3@PSC, MNPs, 23 mg/ml) were provided by Jiangsu Key Laboratory for Biomaterials and Devices (Nanjing, China).31,3231. B. Chen, Y. Li, X. Zhang, F. Liu, Y. Liu, M. Ji, F. Xiong, and N. Gu, Astrophys. J. 817, 93 (2016). https://doi.org/10.3847/0004-637X/817/2/9332. B. Chen et al., Nanoscale 10, 7369 (2018). https://doi.org/10.1039/C8NR00736E The gas used in the experiment was SF6 with a purity of 99.99% purchased from Anhui Qiangyuan Gas Co., Ltd (Wuhu, China). The 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Sigma Aldrich (USA). Ultrapure water (18.25 MΩ/cm), prepared with a de-ionizing water purification system (ULPURE UPH-IV-10 T, China), was used throughout the experiments. Glass vials (4 ml) with open screw caps containing Teflon-covered silicon rubber septa and a 5 ml syringe fitted with a 22 G needle (KDL, Shanghai, China) were used to produce samples.

B. Preparation of the superparamagnetic iron oxide nanoparticle shelled nanobubbles

First, after the SIPOs were mixed with a DSPE-PEG2K-amine solution, the EDC and NHS were added into the solution and incubated at 25 °C at pH 2–4 condition for 6 h, with the following pH change to 6.8–7.2. Then, the MNPs-DSPE-PEG2K product was obtained with a magnet separation.

The DSPC, DPPC, DSPE-PEG2K, and MNPs-DSPE-PEG2K at a molar ratio of 8: 1: 0.5: 0.5 were dispersed in hydration liquid consisting of 10% glycerol (V/V), 90% saline (V/V), and 4 wt. % ethanol to a total concentration of 0.5 mg lipids per ml, followed by sonication for 5 min at 60 °C. Finally, the magnetic nanobubbles were prepared according to our reported method.29,3029. J. Jin, F. Yang, B. Li, D. Liu, L. Wu, Y. Li, and N. Gu, Nano Res. 13, 999 (2020). https://doi.org/10.1007/s12274-020-2732-x30. J. Li, Z. Feng, N. Gu, and F. Yang, J. Mater. Sci. Technol. 63, 124 (2021). https://doi.org/10.1016/j.jmst.2020.02.045 The dispersion (2 ml per vial) was transferred to 3-ml glass vials sealed with plastic caps (2 ml per vial), and the ceiling air was displaced with SF6 gas. A 5-ml syringe with a needle containing 3 ml of SF6 gas was inserted into the vial with the needle tip under the dispersion surface. The MNBs with MNPs in the lipid shell were prepared by the self-assembly of lipids at a free bubble interface for 10 times of compression cycles with the pressure difference between 0.2 and 0.3 MPa at 60 °C and cooled to 4 °C for 30 min.

Nanobubbles (NBs) without magnetic nanoparticles were prepared with DSPE-PEG2K replacement of MNPs-DSPE-PEG2K under the same operation.

C. Morphology characterization of magnetic nanobubbles

The size distribution and zeta potential of magnetic nanoparticles were measured using a NanoSizer (Zeta-Sizer, Malvern Instruments, United Kingdom) at a 90° scattering angle. The concentration of the MNBs was determined by using Multisizer (Multi4e, Beckman, USA). An optical microscopy (Ti2-U, Nikon, Japan), a transmission electron microscope (TEM, JEM-2100, Japan), and a scanning electron microscope (SEM, Ultra Plus, Zeiss, Germany) were used to characterize the morphology of MNBs and NBs.

To demonstrate that the MNPs have been coupled with DSPE-PEG2K-amine by an amide bond in the lipid shell, freshly prepared MNBs were lyophilized by freeze dryer (AdVantage2.0, SP Scientific, USA). The dry powders of MNBs were scanned in the 400–4000 cm−1 wavelength range using the Fourier Transform Infrared (FT-IR) spectrophotometer (IRAffinity-1, Shimadzu Corporation, Japan). With regard to the control sample, 10 mg DSPE-PEG2K-amine powder was measured and analyzed in the same condition.

D. In vitro ultrasound imaging of MNBs

Ultrasound imaging of the MNBs was performed using microimaging systems (VisualSonics Vevo 2100, USA) with an MS-250 transducer. For the in vitro ultrasound imaging experiments, a homemade agar phantom was used.3333. F. Yang, M. Li, H. Cui, T. Wang, Z. Chen, L. Song, Z. Gu, Y. Zhang, and N. Gu, Sci. China Mater. 58, 467 (2015). https://doi.org/10.1007/s40843-015-0059-9 The sample loading hole in the gel phantom was prepared using a mold of 1.5-ml centrifuge tubes. Degassed water was scanned before sampling to confirm a clear background signal. Then, the freshly prepared MNBs and NBs with the same concentration (2 × 107 per ml) was injected into the gel phantom to be imaged under both the B-mode and the contrast-mode. The contrast-mode imaging settings for the ultrasound imaging system had a center frequency of 18 MHz, an intensity power of 4%, and a contrast gain of 35 dB. Also, the B-mode imaging settings for the ultrasound imaging system had a center frequency of 18 MHz, an intensity power of 100%, and a contrast gain of 18 dB. No parameters were changed throughout all imaging acquisition steps. The mean power intensities under the B-mode and the contrast-mode were analyzed in the region of interest (ROI).

E. Experimental setup and measurements for magnetic hyperthermia of MNBs

The magnetically induced heating efficiency of MNBs was measured and recorded by a commercial heating system equipped with a moderate radio frequency (1507 KHz), an inductive copper coil of 3 turns, an inner diameter of 41 mm, and an outer diameter of 51 mm (Shuangping SPG-06-II, China). Also, a fiber optic temperature sensor (UMI8 universal multichannel, FISO Technologies, Inc., Quebec, Canada) fitted with a precalibrated fiber optic temperature probe (FISO, model FOT-L-SD-C1-F1-M2-R1-ST) was used to monitor the real-time temperature change.

In order to understand the influence of current parameter on the increase of temperature, the prepared MNBs at a concentration of iron (Fe) 1.2 mg/ml were placed inside the copper coil under the condition of the applied alternating magnetic field (AMF) at different current parameters (6, 12, and 18 A). To optimize the iron concentration, the MNBs with different iron concentrations (0.8, 1.0, 1.2, and 1.4 mg/ml) were subjected to AMF (1507 KHz, 12 A) for 30 min.

For MHT, the specific absorption rate (SAR) represents a process in which a magnetic material absorbs electromagnetic energy and converts it to heat, which is used to quantify the heating efficiency generated per unit gram of Fe per unit time as shown in Eq. (1):3434. D. Yoo, H. Jeong, S. H. Noh, J. H. Lee, and J. Cheon, Angew. Chem., Int. Ed. 52, 13047 (2013). https://doi.org/10.1002/anie.201306557where C is the specific heat capacity of the solution (here, the heat capacity of the solvent, Cwater = 4.185 J/g °C). The dT/dt is the slope in the initial 1 min of the temperature-time curve, and the ms is the mass of the suspension. The mm is the mass of the Fe content in the suspension.

F. Cell culture and the cytotoxicity test of MNBs

Glioma cell line U87MG cells were obtained from the Shanghai Institute of Cell Biology (Shanghai, China). The mouse brain capillary endothelial cell line bEnd.3 was purchased from Jennio Biotech Co., Ltd. (Guangzhou, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 100 μg/ml penicillin/streptomycin in a humidified atmosphere of 5% CO2 at 37 °C. The culture medium was refreshed every 2–3 days. When the cells reached 70%–80% confluence, they were digested with a 0.25% trypsin–ethylenediamine tetraacetic acid (EDTA) solution, harvested by centrifugation and then resuspended in PBS for experimental use.

The cytotoxicity of MNBs for both bEnd.3 cells and U87MG cells was determined by a Cell Counting Kit-8 (CCK-8) assay based on a modified manufacturer’s protocol. Briefly, bEnd.3 cells or U87MG cells were seeded with 96-well plates at the density of 5 × 103 per well and were cultured overnight, followed by the addition of the free MNPs and MNBs at the same iron concentrations (equivalent to 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, and 1.6 mg/ml of Fe). After an additional 24 h incubation, CCK-8 (10 μl) contained medium (90 μl) was added to each well and incubated for another 1 h. The absorbance intensity in each well was measured at 450/650 nm using a multimode microplate reader Infinite M200 PRO (SpectraMax M5, Molecular Devices, CA, USA).

G. Cell uptake experiment of MNBs

To investigate the U87MG uptake capability for MNBs, the exponentially growing cells were harvested and resuspended in 24-well plates at the density of 8 000 per well and were cultured overnight. Then, the free MNPs and MNBs with the iron concentration of 1.0 mg/ml were added into the plate, respectively. After being incubated for 24 h, cells were centrifuged and counted with a live cell counter (Virtual Tour, Countess 3 and 3 FL Automated Cell Counters, Thermo, USA). When the quantity statistics were determined, the cells were treated overnight in a 12 mol/l hydrochloric acid solution, followed by ultrasonic bath treatment for 20 min to ensure that the MNPs in the cells were completely decomposed into Fe ions. The contents of Fe were measured by inductively coupled plasma optical emission spectrometry (ICP-OES, Optima 8000, PerkinElmer, USA). Each group was repeated at least three times following the same experimental protocol.

H. Cell magnetic hyperthermia experiment

For the cell magnetic hyperthermia treatment experiments, U87MG cells were collected and transferred into a 1.5 ml test tube after 24 h incubation. The magnetic hyperthermia frequency was set as 1507 kHz, and the current was set as 12 A. Under this condition, a different magnetic field treatment cycle (0, 1, 2, 3, and 4) was applied to investigate the influence of treatment number on the cell thermal therapeutic efficacy. Each treatment time was 30 min with a 2 h interval. The experiments were divided into three groups as follows: U87MG cells with MNBs without AMF treatment, U87MG cells with MNBs plus the AMF group, and U87MG cells with MNPs plus the AMF group.

Then, under the optimized parameters, the cell viability of different concentration of iron (0.6 and 1.0 mg/ml of Fe) for both MNPs and MNBs samples was investigated.

I. Statistical analysis

Statistical calculations were performed in GraphPad Prism Version 8.0, and all values were presented as mean ± SD of more than three independent experiments. Comparisons were conducted using a one-way analysis of variance (ANOVA) followed by the t-test. Values of *p < 0.05, **p < 0.01, and ***p < 0.001 were considered statistically significant. All reported p values were two-tailed.