*2.4. MH-Induced Apoptosis and Intracellular* •*OH Generation In Vitro*

As it is hard to distinguish the pH value of a physiological condition (pH ≈ 7.35–7.45) and a neutral condition (pH ≈ 7.0) by RPMI-1640 medium, the effect of the pH value was not considered in the cell experiment.

The Cytotoxicity experiments were studied using 4T1. Firstly, we assessed the biocompatibilities of the pH-responsive micelles and MNF, as shown in Figure 7A. It was clear that

under a physiological condition (pH ≈ 7.35–7.45), the pH-responsive micelles displayed biocompatibility, as its cell survival rates at all concentrations (0.025–1 mg mL−<sup>1</sup> ) ranged from 100% to 90%. Compared to the pH-responsive micelles, the pH-responsive MNF showed a higher cell survival rate under same the concentration, also shown in Figure 7A, indicating the excellent biocompatibility of the MNF under physiological conditions. displayed biocompatibility, as its cell survival rates at all concentrations (0.025–1 mg mL−1) ranged from 100% to 90%. Compared to the pH-responsive micelles, the pH-responsive MNF showed a higher cell survival rate under same the concentration, also shown in Figure 7A, indicating the excellent biocompatibility of the MNF under physiological conditions.

physiological environment, as shown in Figure 5, the particle size of the SPIO clusters could be increased further in neutral conditions. Therefore, the *T*2 imaging of the MNF could be enhanced by a neutral environment, indicating its application for tumor detec-

As it is hard to distinguish the pH value of a physiological condition (pH ≈ 7.35–7.45) and a neutral condition (pH ≈ 7.0) by RPMI-1640 medium, the effect of the pH value was

The Cytotoxicity experiments were studied using 4T1. Firstly, we assessed the biocompatibilities of the pH-responsive micelles and MNF, as shown in Figure 7A. It was clear that under a physiological condition (pH ≈ 7.35–7.45), the pH-responsive micelles

*Pharmaceuticals* **2023**, *16*, x FOR PEER REVIEW 10 of 17

*2.4. MH-Induced Apoptosis and Intracellular •OH Generation In Vitro* 

tion by MRI.

not considered in the cell experiment.

**Figure 7.** Cellular experiments on the biological effect of the MNF and MH, including the (**A**) biocompatibility of the PCL-*b*-pSMA micelles and MNF; (**B**) MH-induced cell death; intercellular ROS generation by (**C**) MNF (**D**) and MH. **Figure 7.** Cellular experiments on the biological effect of the MNF and MH, including the (**A**) biocompatibility of the PCL-*b*-pSMA micelles and MNF; (**B**) MH-induced cell death; intercellular ROS generation by (**C**) MNF (**D**) and MH.

Apparently, the MNF showed excellent biocompatibility; however, the MNF-based MH could also inhibit cell proliferation efficiently, as shown in Figure 7B. For these studies, the concentration of the MNF was fixed at 0.2 mg mL−1. After incubation with the MNF alone for 12 and 24 h, the 4T1 still displayed a high cell viability, which was similar as the result in Figure 7A. Nevertheless, after exposure to AMF with different *H*applied for 10 min, the cell viabilities decreased obviously. Moreover, the inhibition rate of the 4T1's proliferation under AMF exhibited a *H*applied-dependent tendency. When the *H*applied of the AMF was fixed at 21.2 kA m−1, 4T1's viabilities decreased to 81.2% at 12 h and 76% at 24 h after MH for 10 min. When the *H*applied of the AMF was increased to 31.8 kA m−1, 4T1's viabilities were suppressed exponentially, which was 21.4% for 12 h and 2.8% for 24 h. Therefore, the survival of the 4T1 under the AMF with the highest *H*applied (42.4 kA m−1) decreased to 1.7% at 24 h after MH. To determine the relationship between the *H*applied of the MH and the cell survival rate, we recorded the heating curves of the MNF under AMF with the Apparently, the MNF showed excellent biocompatibility; however, the MNF-based MH could also inhibit cell proliferation efficiently, as shown in Figure 7B. For these studies, the concentration of the MNF was fixed at 0.2 mg mL−<sup>1</sup> . After incubation with the MNF alone for 12 and 24 h, the 4T1 still displayed a high cell viability, which was similar as the result in Figure 7A. Nevertheless, after exposure to AMF with different *H*applied for 10 min, the cell viabilities decreased obviously. Moreover, the inhibition rate of the 4T1's proliferation under AMF exhibited a *H*applied-dependent tendency. When the *H*applied of the AMF was fixed at 21.2 kA m−<sup>1</sup> , 4T1's viabilities decreased to 81.2% at 12 h and 76% at 24 h after MH for 10 min. When the *H*applied of the AMF was increased to 31.8 kA m−<sup>1</sup> , 4T1's viabilities were suppressed exponentially, which was 21.4% for 12 h and 2.8% for 24 h. Therefore, the survival of the 4T1 under the AMF with the highest *H*applied (42.4 kA m−<sup>1</sup> ) decreased to 1.7% at 24 h after MH. To determine the relationship between the *H*applied of the MH and the cell survival rate, we recorded the heating curves of the MNF under AMF with the same condition as described in the cell experiment, and the results are shown in Figure S8. Apparently, under the AMF with the given *H*applied in this study, MH could not induce a sufficient temperature (>45 ◦C) to suppress cell death efficiently. Therefore, we evaluated the intercellular ROS level, another possible mechanism related to cell death, after different treatments.

As 2',7'-dichlorodihydrofluorescin diacetate (DCFH-DA) can be metabolized within the cell by intercellular ROS, forming a fluorescent compound, 2',70 -dichlorofluorescein (DCF), flow cytometry was utilized to quantify the intercellular ROS level by detecting the fluorescent intensity of the DCF. In order to correspond to the cytotoxicity of the MNF for different culture times, we studied the intercellular ROS level after incubating with MNFs for 12, 24 and 48 h. The result confirmed the excellent biocompatibility of the MNF, again, as shown in Figure 7C. The fluorescent intensities of the DCF for all predetermined times overlapped with that of 4T1 under standard culture conditions for 48 h. Although the MNF showed a high biocompatibility, MNF-mediated MH could boost the intercellular ROS level, as shown in Figure 7D. Further, the intercellular ROS level under MH presented a similar trend as that of its counterpart for the cell inhibition rate. For the group of MH-1, the low *H*applied (21.2 kA m−<sup>1</sup> ) limited the increase in the DCF fluorescent intensity, resulting in a high cell viability, as shown in the biocompatibility for the MNF. For the groups of MH-2 (31.8 kA m−<sup>1</sup> ) and MH-3 (42.2 kA m−<sup>1</sup> ), they showed similar intercellular ROS levels, corresponding to their cell mortality rate efficiencies on inhibiting 4T1 proliferation. Therefore, the SPIO biocompatibility did not conflict with the SPIO-mediated CDT. The occurrence of CDT should be triggered by a certain stimulation, such as MH [41], photothermal treatment [42], photodynamic therapy [43], and tumor microenvironment [44]. On the basis of the pH responsiveness under neutral conditions, the MNF possessed many advantages for tumor microenvironment-enhanced MH, catalytic activity and MRI. This novel MNF should be a competitive candidate for tumor diagnosis and treatment.

#### **3. Materials and Methods**

#### *3.1. Materials*

The Sn(Oct)<sup>2</sup> (92.5–100%), 4,4'-Azobis(4-cyanovaleric acid) (V501, 98%), 1,2-hexadecanediol (97%) and Oleylamine (>70%) were purchased from Sigma Aldrich (Steinheim, Germany). ε-Caprolactone (ε-CL, 99%), N,N'-dicyclohexylcarbodiimide (DCC, 98%), 4 dimethylaminopyridine (DMAP, 99%) and sulfadimethoxine (SM, 98%) were purchased from Tokyo Chemical Industry (TCI, Tokyo, Japan). Iron(III) acetylacetonate [Fe(acac)3], benzyl ether (99%) and oleic acid (90%) were purchased from Alfa-Aesar (Heysham, England). Benzyl alcohol (BaOH, 99%, safe dry), acryloyl chloride (98%), tetrahydrofuran (THF, 99%) and MB (95%) were purchased from Admas (Shanghai, China). The dialysis tubing (8000–14,000 Da), dichloromethane (DCM, 99.9%), dioxane (99%), ethyl ether (99.5%), methanol (99.5%) and H2O<sup>2</sup> (30%) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) was purchased from Beyotime Biotech. Co., Ltd. (Shanghai, China).

The SMA was synthesized by the procedure described in the relevant literature [45]. The DDMAT was synthesized according to [27], and the monodisperse superparamagnetic Fe3O<sup>4</sup> nanoparticles were synthesized according to [46], with minor modifications. The typical synthetic procedure is described as follows: A certain amount of Fe(acac)3, 1,2 hexadecanediol, oleic acid and oleylamine with molar numbers of 2, 10, 2 and 2 mmol were dispersed successively in benzyl ether (20 mL). After deoxidizing by argon at 50 ◦C for 30 min, the mixture was heated to 200 ◦C for 2 h under an argon atmosphere and then heated further to reflux (≈300 ◦C) for two and a half hours. The product, monodispersed Fe3O<sup>4</sup> nanoparticles, was precipitated by excess ethanol and then collected by centrifugation. The purified process was repeated three times. The purified Fe3O<sup>4</sup> nanoparticles were dried by a high-purity argon flow. Finally, the magnetic nanoparticles were dispersed in THF with a concentration of 10 mg mL−<sup>1</sup> for storage under −20 ◦C.

The other reagents were used as received. The water used in all experiments was deionized with a Millipore Milli-Q system (Billerica, USA).

#### *3.2. Synthesis of the pH-Responsive Amphiphilic Copolymer*

The pH-responsive amphiphilic copolymer, PCL-*b*-pSMA, was synthesized by the reaction procedure, as shown in Figure S1, which was described as follows.

In the first step, the PCL was synthesized by ROP using BaOH as an initiator and ε-CL as monomers with a corresponding molar ratio of 1:50. The reaction was heated to 110 ◦C under an argon (Ar2) atmosphere for 24 h. The product, PCL, was purified by precipitating in excess ethyl ether three times from its DCM solution. Finally, the PCL was dried until reaching a constant weight in a vacuum oven at an ambient temperature.

The second reaction was to prepare the macro-CTA, PCL-DDMAT, by esterification between the PCL and DDMAT, according to the established method [25]. In the reaction,

a certain amount of PCL, DDMAT, DCC and DMAP with a corresponding molar ratio of 1:5:5:1 was weighted accurately and dissolved in anhydrous DCM by magnetic stirring. The esterification was carried out under an Ar<sup>2</sup> atmosphere for 48 h. To purify the PCL-DDMAT, the supernatant of the reaction was collected and successively precipitated in excess cold ethyl ether. The process for the purification was repeated several times until the supernatant was without any DDMAT. Finally, the purified PCL-DDMAT was dried in a vacuum oven at an ambient temperature and preserved in Ar<sup>2</sup> under a low temperature (−20 ◦C).

The third reaction was the synthesis of the PCL-*b*-pSMA by a RAFT reaction. In the reaction, the PCL-DDMAT (0.02 mmol, 110 mg), SMA (2 mmol, 728 mg) and V501 (0.004 mmol, 1.1 mg) were dissolved in dioxane of 5 mL under magnetic stirring. Then, the mixture was thoroughly degassed by three freeze–pump–thaw cycles. The reaction was carried out at 70 ◦C for 10 h. The final product, PCL-*b*-pSMA, was purified by repeated precipitation in excess methanol, followed by freeze-dried treatment and also preserved in Ar<sup>2</sup> under a low temperature (−20 ◦C).

## *3.3. Characterization of the pH-Responsive Amphiphilic Copolymer*

All products were characterized by their <sup>1</sup>H NMR spectrum, with CDCl<sup>3</sup> or DMSO-*d<sup>6</sup>* as the solvent, and their chemical shifts relative to tetramethylsilane (TMS) were identified. In order to identify the pH responsiveness of the PCL-*b*-pSMA, we prepared micelles of the PCL-*b*-pSMA first, where the THF solution of the PCL-*b*-pSMA (20 mg mL−<sup>1</sup> ) was dialyzed against an alkaline solution (pH = 9.13) for 48 h. After, the obtained micelle solution of the PCL-*b*-pSMA with a high concentration (>3 mg mL−<sup>1</sup> ) downregulated its pH value by the gradual addition of a small amount of hydrochloric acid solution (1 M). By using a UV-Vis spectrophotometer (Shimadzu, UV2600, Tokyo, Japan), we recorded its light transmittance at different pH values (9.13~5.18). The p*K*<sup>a</sup> value of the PCL-*b*-pSMA was defined as the surrounding pH value, producing a 50% decrease in the optical transmittance at 500 nm [29]. At the same time, the p*K*<sup>a</sup> value of the PCL-*b*-pSMA was also identified by DLS (Malvern, Nano ZS90, Worcestershire, UK), which measured the size distribution of the dilute PCL-*b*-pSMA micelles (0.3 mg mL−<sup>1</sup> ) with varied surrounding pH values. Moreover, the morphologies of the PCL-*b*-pSMA micelles at pH values under physiological and neutral conditions were observed directly by TEM (Hitachi, HT-7800, Tokyo, Japan), in which the samples were stained by phosphotungstic acid (2%).

#### *3.4. Preparation of the Tumor Microenvironment-responsive MNF*

The tumor microenvironment-responsive MNF was prepared by ultrasound-assisted self-assembly. In a typical procedure, the PCL-*b*-pSMA (210 mg) and Fe3O<sup>4</sup> (90 mg) were dissolved in THF (10 mL) completely by oscillation. The mixed solution was then slowly added into an excess alkaline solution (pH ≈ 9, 50 mL) under sonication, followed by dialyzing against the same alkaline solution for 48 h. The dialysis solution was purified by centrifugation (2000 RPM, 10 min), and the supernatant was collected. Finally, the tumor microenvironment-responsive MNF was purified from the supernatant by high-speed centrifugation (100,000× *g*, 20 min). The obtained sediment was collected by lyophilization and stored at 4 ◦C.

#### *3.5. Physicochemical Properties of the Tumor Microenvironment-responsive MNF*

First, the morphologies of the Fe3O<sup>4</sup> nanoparticles and MNF were characterized by TEM (Hitachi, HT-7800, Tokyo, Japan) directly, in which the MNF was dispersed into an alkaline solution (pH ≈ 9). Their particle sizes were measured by DLS (Malvern, Nano ZS90, Worcestershire, UK) under an ambient condition. In order to confirm the pH responsiveness of the MNF, we dissolved the MNF into an alkaline solution (pH ≈ 9) and downregulated the surrounding pH value to physiological (pH ≈ 7.43), neutral (pH ≈ 7.02) and weak acidic (pH ≈ 6.67) conditions. Then, we studied their colloidal stability by qualitative observation (digital imaging and TEM) and quantitative analysis (DLS). In

addition, the zeta potentials of the MNF in the corresponding buffers (pH ≈ 7.43, pH ≈ 7.02 and pH ≈ 6.67) were characterized simultaneously by DLS. The long colloidal stability of the MNF was also assessed by DLS from 1 to 13 d. The content of the Fe3O<sup>4</sup> in the tumor microenvironment-responsive MNF was measured by TGA (NETZSCH STA 449 F3, Weimar, Germany).

#### *3.6. Enhancements of the MNF for MH, FR and MRI under a Neutral Condition*

In order to determine the magnetocaloric effect of the MNF, the concentration of Fe ([Fe]) was fixed at 100 µg mL−<sup>1</sup> , which was identified by an inductively coupled plasma mass spectrometer (ICP-MS, Thermo scientific, Xseries II, Waltham, USA). Then, the heating curves of the MNF under physiological (pH ≈ 7.45), neutral (pH ≈ 6.97) and weak acidic (pH ≈ 6.64) conditions were plotted. In this study, an AMF generator (SPG-20AB, ShuangPing Tech. Ltd., Shenzhen, China) with the corresponding frequency (*f*, 114 kHz) and strength (*H*applied, 89.9 kA m−<sup>1</sup> ) was employed. The inner diameter of the heating coil was 28 mm. The increasing temperature was recorded by a computer-attached fiber optic temperature sensor (FISO, FOT-M, Québec, Canada). Finally, the SAR was calculated by the formula described in a relative study [23].

In addition to the MH, the catalytic potential of MNF under physiological (pH ≈ 7.45), neutral (pH ≈ 6.97) and weak acidic (pH ≈ 6.64) conditions was also studied by detecting the generation of •OH. As the •OH can induce the degradation of MB [47], the study was divided into five groups: MB, MB + H2O2, MB + H2O<sup>2</sup> + MNF (pH = 7.45), MB + H2O<sup>2</sup> + MNF (pH = 6.97) and MB + H2O<sup>2</sup> + MNF (pH ≈ 6.64). In this study, the concentrations of MB, H2O<sup>2</sup> and MNF were 50 µg mL−<sup>1</sup> , 1 mM and 250 µg mL−<sup>1</sup> . Then, the degradation of MB was observed using digital imaging and monitored using a UV-Vis spectrophotometer (Shimadzu, UV2600, Tokyo, Japan) 2 h later.

The MRI studies were performed with a 3.0-T clinic MRI imaging system (Siemens Trio 3T MRI Scanner, Erlangen, Germany), which was equipped by a micro coil for the transmission and reception of the signal. After dissolving the MNF in an alkaline solution (pH ≈ 8.45), the concentrations of Fe ([Fe]) were identified by ICP-MS first. Then, the study was divided into two groups: physiological condition (pH ≈ 7.42) and neutral condition (pH ≈ 6.99). All groups possessed identifiable [Fe] from 1360 to 42.5 µM.

For the *T*2-weighted images, two groups with a series of [Fe] gradients were scanned under these conditions, listed as the following: TR = 5000 ms, TE = 10–90 ms, slice thickness = 3 mm and flip angle = 150◦ .

#### *3.7. Cellular Studies on MH and MH-Induced ROS Generation*

In this study, we used a mouse-derived breast cancer cell line, 4T1, purchased from the Chinese Academy of Sciences (Shanghai, China). The 4T1 was cultured using RPMI-1640 medium (Hyclone) containing 10% fetal bovine serum (FBS, every green, Hangzhou, China) and then placed in an incubator at 37 ◦C with 5% CO<sup>2</sup> and humidified conditions. The cytotoxicity in vitro was performed by the standard MTT assay. In the research, the biocompatibilities of the PCL-*b*-pSMA and MNF with varied concentrations from 0.25–1 mg mL−<sup>1</sup> under physiological conditions for 48 h were studied.

To evaluate the efficiency of the MH in vitro, the MNF was dissolved in RPMI-1640 medium (containing 10% FBS and 1% penicillin–streptomycin) at a concentration of 0.2 mg mL−<sup>1</sup> . Considering the effect of *H*applied on the MH, the study was divided into three group, MH-1, MH-2 and MH-3, which were operated under the corresponding *H*applied as 21.2, 31.8 and 42.4 kA m−<sup>1</sup> , respectively. The 4T1 was incubated in a culture dish (35 mm) at a density of 4 <sup>×</sup> <sup>10</sup><sup>5</sup> cells per dish in 2 mL of corresponding medium. After incubating for 24 h, the culture medium was replaced by the corresponding medium containing MNF (0.2 mg mL−<sup>1</sup> ). Then, the 4T1 was placed in a heating coil with an inner diameter of approximately 38 mm. The applied AMF possessed a constant frequency (*f* = 114 kHz) and varied *H*applied (21.2, 31.8 and 42.4 kA m−<sup>1</sup> ). The exposure time under AMF was fixed at 10 min. After MH, the cells were cultured for a prolonged 12 or 24 h. Finally, the cell viabilities were quantified by MTT. After the cell experiment, the heating curves of the MNF (dissolved in RPMI-1640 medium) were recorded in a culture dish (35 mm) under AMF with the same conditions as described for the MH study.

For detecting the ROS generation, DCFH-DA was used following the instructions of the ROS Assay Kit (Beyotime Biotech. Co., Ltd., Shanghai, China). Prior to treatment of the DCFH-DA, 4T1 was incubated in a culture dish (35 mm) at a density of <sup>2</sup> <sup>×</sup> <sup>10</sup><sup>5</sup> cells per dish in 2 mL of corresponding medium. Then, the 4T1 was treated by the MNF (0.2 mg mL−<sup>1</sup> ) alone for different culture times (12, 24 and 48 h) or MH for 12 h under the established conditions, as described in the MH study. After pretreatment, the culture medium of the 4T1 was replaced by serum-free medium containing DCFH-DA (10 µM) for 40 min. Finally, 4T1 was collected and analyzed by flow cytometry.

## **4. Conclusions**

We successfully prepared tumor microenvironment-responsive MNFs by the selfassembly of pH-responsive PCL-*b*-pSMA and superparamagnetic hydrophobic Fe3O<sup>4</sup> nanoparticles for targeted tumor theranostics. As the PCL-*b*-pSMA possessed a p*K*<sup>a</sup> within a pH range of the tumor microenvironment, the MNF exhibited a great potential for tumor targeting, by maintaining a high colloidal stability in a physiological environment for a long time, forming large, agglomerated clusters in the tumor microenvironment rapidly. Because the tumor microenvironment-responsive MNF showed larger agglomerated clusters in a tumor microenvironment, the close-packed multiple SPIOs with a larger diameter exhibited better performance in reducing the *T*<sup>2</sup> and increasing SAR simultaneously. Meanwhile, due to the agglomeration of the MNF in the tumor tissue, the encapsulated Fe3O<sup>4</sup> nanoparticles could be exposed from the inner core of the micellar MNF, which induced efficient ER by the interaction between Fe2+ and endogenous H2O2. The further cell experiments confirmed that the tumor microenvironment-responsive MNF displayed excellent biocompatibility, corresponding to its high stability in a physiological environment. However, after applying AMF, the tumor microenvironment-responsive MNF exhibited high efficiency in inhibiting cell proliferation, because of the MH-induced CDT. According to these results, the tumor microenvironment-responsive MNF not only showed high safety in medical areas but also showed itself as a powerful tool to enhance tumor contrast by MRI and improve tumor treatment by combing MH and CDT specifically.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ph16020166/s1, Figure S1: Synthetic scheme of PCL-*b*-pSMA; Figure S2: <sup>1</sup>H NMR spectrum of PCL; Figure S3: <sup>1</sup>H NMR spectrum of PCL-DDMAT; Figure S4: Content of Fe3O<sup>4</sup> in the tumor microenvironment-responsive MNF; Figure S5: Time-dependent hydrodynamic diameters of the tumor microenvironment-responsive MNF in physiological conditions; Figure S6: Particle sizes of the tumor microenvironment-responsive MNF in solutions with different pH values; Figure S7: Zeta potentials of the tumor microenvironment-responsive MNF in solutions with different pH values; Figure S8: Time-dependent temperature curves of the tumor microenvironment-responsive MNs under AMF with different *H*applied.

**Author Contributions:** Conceptualization, Y.L.; methodology, Y.X.; software, Y.Y.; validation, M.S.; formal analysis, Y.L. and Y.X.; investigation, L.S. and X.Z.; resources, Y.L. and Y.X.; data curation, Z.L. and M.S.; writing—original draft preparation, L.S. and X.Z.; writing—review and editing, Y.L. and Y.X.; supervision, Y.L. and Y.X. 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 numbers: 81871343 and 51703086; China Postdoctoral Science Foundation, grant number: 2019M651730; Nature Science Foundation of Jiangsu Province, grant number: BK 20160496; Key Research and Development Project of Jiangsu Province, grant number: BE2021693; Postdoctoral Science Foundation of Jiangsu Province, grant number: 2018K057C.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article and supplementary material.

**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.
