*3.5. Characterization of Molecular Structures*

The structure of small molecular prodrug Pt(IV)-COOH was characterized by FT-IR (Thermo Scientific, Nicolet iS50, Madison, WI, USA) and <sup>1</sup>H NMR spectrum (Bruker, AVANCE II, Zurich, Switzerland). The molecular weight of Pt(IV)-COOH was measured by electrospray ionization mass spectrometer (ESI-MS), which was performed by a highresolution time-of-flight mass spectrometer (MS, Bruker, MaXis, Karlsruhe, Germany) equipped with electrospray ionization (ESI). The composition and structure of the polymer were characterized by a <sup>1</sup>H NMR spectrum with CDCl<sup>3</sup> or DMSO-d6 as the solvent and analyzed by their chemical shifts relative to tetramethylsilane (TMS). The Pt content in polymeric Pt(IV) was determined by ICP-MS (Thermo scientific, Xseries II, USA).

#### *3.6. Preparation of Polymeric Pt(IV) Micelles (PPMs) and MTNs*

All micelles were prepared by self-assembly technology. For the preparation of PPM, polymeric Pt(IV) was dissolved in THF completely with the concentration of 5 mg mL−<sup>1</sup> . The mixed solution was then slowly added into excess deionized water under sonication and dialyzed against water for 48 h to remove THF and other impurities, in which a dialysis bag (8–14 KD) was used. The dialysis solution was purified by centrifugation (2000 rmp). After that, PPMs were collected from the supernatant by lyophilization and stored at −20 ◦C.

For the preparation of MTNs, a certain amount of magnetic nanoparticles Mn0.6Zn0.4Fe2O<sup>4</sup> and polymeric Pt(IV) were dispersed in THF adequately with the same concentration as 3 mg mL−<sup>1</sup> . Then, the MTN was prepared by the same procedure.

#### *3.7. General Properties of PPM and MTN*

The morphologies of PPM and MTN were characterized by high-resolution transmission electron microscopy (HRTEM, JEOL, JEM-2100, Tokyo, Japan). Particle diameters of PPM and MTN were measured by DLS (Malvern, Nano ZS90, Worcestershire, UK). The content of Mn0.6Zn0.4Fe2O<sup>4</sup> nanoparticles in MTNs was characterized by ICP-MS. The magnetic property of MTN was measured by a vibrating sample magnetometer (VSM, Lake Shore Cryotronics, Inc., LakeShore 7404, Westerville, OH, USA) at 300 K.

We also estimated particle size and colloidal stability of PPMs and MNTs by DLS after they dispersed in phosphate buffer solution (PBS) for 7 d. The concentration of these samples was fixed as 0.5 mg mL−<sup>1</sup> .

The magnetocaloric effect of MTNs was evaluated by calculating its SAR, according to our previous study [18]. MTNs were dispersed into water with a concentration of Mn0.6Zn0.4Fe2O<sup>4</sup> as 0.1 mg mL−<sup>1</sup> , which was determined by ICP-MS. The colloidal solution (4 mL) was placed in AMF, which was generated by an alternating magnetic field generator (SPG-20AB, ShuangPing Tech. Ltd., Shenzhen, China). Then, the temperature change of the sample was recorded by a computer-attached fiber optic temperature sensor (FOT-M, FISO, Québec, Canada). Finally, the SAR was calculated by the formula described in our previous study [18].

In this part of the study, the frequency (*f*) and strength (*H*applied) of the AMF was fixed at 114 kHz and 63.6 kA m−<sup>1</sup> . The inner diameter of the heating coil was 20 mm.

#### *3.8. Drug Release Studies*

In a typical experiment, a certain amount of MTNs (20 mg) was dispersed into 2 mL of the acidic buffer. Then, the solution was placed into dialysis tubing (3 kDa) against 98 mL of the corresponding buffer. The process of drug release was performed at 37 ◦C in a sharking incubator. At every predetermined time interval, 1 mL sample was withdrawn from outside of the dialysis tubing and measured by ICP-MS to determine the content of Pt. At the same time, an equal volume corresponding buffer was added as a release medium. According to Pt content, the percentage of cumulative Pt release from the MTNs was calculated and determined finally by averaging three measurements.

Furthermore, we also investigated the influence of MH on drug release. In this part, the inner diameter of the heating coil was 40 mm, resulting in constant f (114 kHz) and reduced strength (15.9 kA m−<sup>1</sup> ). Because of the limitation of the heating coil, the study was performed in a 50 mL centrifuge tube. Briefly, MTNs of 10 mg were dispersed into 1 mL of the acidic buffer with 1 mM GSH, followed by 49 mL of the corresponding buffer. To simulate conditions of actual MH in the research, 20 min MH per 1 h was applied and repeated 8 times in a typical study. During the study, the 50 mL centrifuge tube was placed in a sharking incubator at 37 ◦C after MH until the next MH. At every time interval of 0.5 h, a 0.5 mL sample was withdrawn from the centrifuge tube, and an equal volume of the corresponding buffer was added as a release medium. Finally, the percentage of cumulative Pt release from the MTNs was measured and calculated as we described above.

#### *3.9. Cell Viability Assay In Vitro*

The 4T1 was gifted by my colleague, Rui Mengjie, who purchased the cell line from the Chinese Academy of Sciences (Shanghai). 4T1 was cultured in RPMI-1640 medium (Gibco) containing 10% fetal bovine serum (FBS, Biological Industries, Israel), then placed in an incubator at 37 ◦C, 5% CO2, and humidified circumstance.

The cell viability in vitro was performed by standard MTT assay in the study. Biocompatibility of magnetic nanoparticle Mn0.6Zn0.4Fe2O<sup>4</sup> has been confirmed by our previous study. In the research, cytotoxicity of hydrophilic copolymer mPEG-*b*-pHEMA was studied and shown in supporting information (SI).

To evaluate the chemotherapy efficiency of PPMs and MTNs, 4T1 was seeded in 96-well plates at a density of 2 <sup>×</sup> <sup>10</sup><sup>4</sup> cells per well in 100 µL of corresponding medium and incubated under standard culture conditions for 24 h. After that, the medium was replaced by a medium containing various drug formations of cisplatin, Pt(IV), PPMs, and MTNs, respectively, in which the final equivalent Pt concentration was varied from 0.625 to 20 µg mL−<sup>1</sup> . After incubating for 24 and 48 h, cell viabilities were investigated by MTT.

Furthermore, to assess the assistance of MH, 4T1 was seeded in a culture dish (35 mm) at a density of 2 <sup>×</sup> <sup>10</sup><sup>5</sup> cells per dish in 2 mL of the corresponding medium. After incubating for 24 h, the culture medium was replaced by a medium containing MTNs (20, 10, 5, 2.5, 1.25, 0.625 µg mL−<sup>1</sup> ). To make sure constant concentration of Mn0.6Zn0.4Fe2O<sup>4</sup> (60 µg mL−<sup>1</sup> ) in the study, we prepared additional magnetic nanocluster fluid according to our previous study [17], then complement shortage of SPIO in groups of Pt concentration as 10, 5, 2.5, 1.25, 0.625 µg mL−<sup>1</sup> . Afterward, cells were exposed to AMF (20 min per 24 h, 114 kHz, and 15.9 kA m−<sup>1</sup> ) at the beginning of the 24, following culture in an incubator under standard culture conditions. When total culture time reached 24 and 48 h, cell viabilities were also quantified by MTT.

As the culture medium was selected as a negative control, cell survival rates of different treatments were calculated as the percentage of negative control values.

#### *3.10. Cellular Uptake Studies*

4T1 were also seeded in culture dish (35 mm) with density of 2 <sup>×</sup> <sup>10</sup><sup>6</sup> cells per dish. After incubating for 24 h, cells were treated with cisplatin, PPM, MTN, and MTN plus MH (20 min), respectively. For each formulation, the equivalent Pt content was fixed at 2.5 µg mL−<sup>1</sup> (2 mL); meanwhile, the SPIO concentration of MTN plus MH was fixed

at 60 µg mL−<sup>1</sup> (2 mL). The drug uptake of Pt was performed by incubating cells with the above-mentioned drug formulations for 1 and 4 h. The cellular uptake of MTN plus MH was treated by exposure to AMF (20 min, 114 kHz, and 15.9 kA m−<sup>1</sup> ) firstly, then incubated under standard culture conditions for prolonged 40 and 220 min, respectively. After removing the supernatant and washing three times, the cells in each culture dish were digested and counted to collect 1 <sup>×</sup> <sup>10</sup><sup>6</sup> cells. After repeated freezing and thawing, the intracellular Pt content was determined by ICP-MS.

#### *3.11. Animal Protocol*

BALB/c mice (female, 5–7 weeks old, 18–20 g weight) were purchased from the laboratory animal center of Jiangsu University and maintained in the laboratory animal center of Jiangsu University under specific pathogen-free conditions. The orthotopic breast tumor was established by injection of 4T1 cells (1 <sup>×</sup> <sup>10</sup><sup>7</sup> cells per mL, 100 µL per mice) into the fourth mammary fat pads of mice. After eight days, the tumor can grow to 100 mm<sup>3</sup> . Animals were treated according to the ethical guidelines of Jiangsu University. The animal experiments (approval code: UJS-IACUC-2021092703) were carried out according to the regulations for animal experimentation issued by the State Committee of Science and Technology of the People's Republic of China.

#### *3.12. Biodistribution of MTNs and MRI of Tumor In Vivo*

When tumors grew to 200–300 mm<sup>3</sup> , these mice were divided into two groups (*n* = 5): MTN alone and MTN plus MT. MRI studies were performed with a 3.0-T clinic MRI imaging system (Siemens Trio 3T MRI Scanner) by using a micro coil for transmission and reception of the signal. The T2-weighted images were acquired by these conditions, which was listed as following: TR = 5000 ms, TE = 10–90 ms, slice thickness = 3 mm, flip angle = 150◦ , matrix size = 256 × 256, FOV = 100 mm, echo length = 8. Before injection of MTN, tumor-bearing mice were scanned by MRI. Then, the MTN solution prepared by dissolving into PBS at a dose of 4.2 mg kg−<sup>1</sup> (mFe + mMn + mZn) was injected with 0.1 mL MTN solution from tail-vein for each tumor-bearing mouse. For the group of MT, the button magnet with a surface magnetic intensity of 0.18 T (diameter of 10 mm and thickness of 4 mm) was placed on the tumor area after injection immediately and maintained for 4 h. At 20 h later, after injecting MTNs, an MRI scan was performed to observe the T2 signal of the tumor site.

After that, the influence of MH on the penetration of MTNs within the tumor was also observed by MRI. After the second scanning, mice were treated with MH (20 min of each mouse, 114 kHz, and 15.9 kA m−<sup>1</sup> ), then scanned by MRI following. In reality, the last scanning was performed at 22 h after MTNs injection.

To ensure the distribution of MTNs in vivo, a histological examination was performed after the last MRI scanning. The main organs (heart, liver, spleen, lung, and kidney) and tumors were extracted and fixed in 10% formalin, following treatment with nuclear fast red and Prussian blue stain.

### *3.13. In Vivo Tumor Inhibition Studies*

When tumors grew to 150 mm<sup>3</sup> , these mice were divided into 6 groups (*n* = 6), injected with PBS, cisplatin, MTN, MTN plus MT (MTN + MT), MTN plus MH (MTN + MH), and MTN plus MT plus MH (MTN + MT + MH), respectively, in which, the dosage of Pt was calculated as 3 mg kg−<sup>1</sup> cisplatin. In these groups, the injection was performed every three days, and MH was operated under AMF (114 kHz and 15.9 kA m−<sup>1</sup> ) for 20 min at 20 h after each injection. The MT was carried out as we described in the MRI study. During the treatment, we injected 5 times in each group. Tumor volume and body weight of tumor-bearing mice were measured and recorded every three days, in which tumor volume was calculated by a formula described in a relative study [43]. The TGI rates of different treatments were calculated to assess corresponding antitumor efficacies by a formula described in a relative study [48].

## *3.14. Statistical Analysis*

First of all, all data were presented with mean ± standard deviation (SD). Secondly, a one-way analysis of variance (ANOVA) was used in the research to determine significant differences between pairs of two groups. *p* < 0.05 was considered statistically significant, and *p* < 0.01 was considered as significant extremely.

#### **4. Conclusions**

In the study, we developed a versatile MTN successfully with high stability, MHfacilitated accumulation, high-sensitivity MRI, MH-enhanced penetration, and efficacy of Pt(IV) by self-assembly of monodispersed Mn0.6Zn0.4Fe2O<sup>4</sup> and amphiphilic polymeric Pt(IV). First of all, we synthesized macromolecular cisplatin prodrug with Pt content as high as 22.5% polymeric Pt(IV), then used it to prepare MTN by encapsulating superparamagnetic Mn0.6Zn0.4Fe2O<sup>4</sup> nanoparticles. The MTN did not only exhibit the potential for passive targeting by combining a suitable diameter around 151 nm and high stability under physiological conditions but also displayed more abilities on MT, MH, and MRI because it possessed high *M*<sup>s</sup> (103.1 emu g−<sup>1</sup> ) and SAR (404 W g−<sup>1</sup> ). More importantly, the drug release of MTN was sensitive to the intracellular environment, especially for low pH, indicating its low side effects in circulation. According to these properties, polymeric Pt(IV) based formulations, PPM and MTN alone displayed low cytotoxicity, but the combination of MTN and MH showed as high cytotoxicity as cisplatin. After intravenous injection, targeted accumulation of MTN in tumors could be improved under MT, which facilitated observation of tumor by MRI. The further MH enhanced MTN penetration in tumors, which also could be reflected by MRI directly. By integrating MT and MH, MTN displayed the highest TGI at 88.38% and reduced the side effects of cisplatin simultaneously. Although we did not observe tumor extinction by cascade effects of MT, MH, and chemotherapy of polymeric Pt(IV), the MTN was still a competitive candidate for diagnosis and therapy of tumors in clinics because of its advantages in efficient targeting, high-sensitivity MRI, low toxicity, and high efficacy simultaneously.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ph15040480/s1, Figure S1: Synthetic scheme of polymeric Pt(IV); Figure S2: FT-IR spectrum of Pt(IV)-COOH; Figure S3: <sup>1</sup>H NMR spectrum of Pt(IV)-COOH; Figure S4: Mass spectrum of Pt(IV)-COOH; Figure S5: <sup>1</sup>H NMR spectra of DDAT and mPEG-DDAT; Figure S6: TEM result of monodispersed Mn0.6Zn0.4Fe2O<sup>4</sup> nanoparticles; Figure S7: The time-dependent hydrodynamic diameter of PPM and MTN in PBS; Figure S8: Magnetization curve of the Mn0.6Zn0.4Fe2O<sup>4</sup> at 300 K; Figure S9: The biocompatibility of mPEG-*b*-pHEMA.

**Author Contributions:** Conceptualization, Y.Q.; methodology, Y.Q. and Y.X.; software, X.Z.; validation, M.S.; formal analysis, X.D. and M.Z.; investigation, Z.W. and T.Z.; resources, Y.X. and H.L.; data curation, Z.W. and M.S.; writing—original draft preparation, Y.Q.; writing—review and editing, Y.Q.; supervision, Y.Q., Y.X. and H.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of China, grant number 81602656; Nature Science Foundation of Jiangsu Province, grant number BK 20160546; Postdoctoral Science Foundation of Jiangsu Province, grant number 2018K272C; Jurong Science & Technology program, grant number YF202004 and ZA42109; 2021 Zhenjiang sixth "169 project" scientific research project; and Graduate Research and Innovation Projects of Jiangsu Province, grant number KYCX20-3094.

**Institutional Review Board Statement:** The animal study protocol was approved by the Institutional Review Board (or Ethics Committee) of Jiangsu University (protocol code UJS-IACUC-2021092703 and 3 September 2021).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors would like to thank Mengji Rui, who donated the 4T1 cell line.

**Conflicts of Interest:** The authors declare no conflict of interest.
