Biocompatible Polymer-Grafted TiO2 Nanoparticle Sonosensitizers Prepared Using Phosphonic Acid-Functionalized RAFT Agent

Sonodynamic therapy is widely used in clinical studies including cancer therapy. The development of sonosensitizers is important for enhancing the generation of reactive oxygen species (ROS) under sonication. Herein, we have developed poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC)-modified TiO2 nanoparticles as new biocompatible sonosensitizers with high colloidal stability under physiological conditions. To fabricate biocompatible sonosensitizers, a grafting-to approach was adopted with phosphonic-acid-functionalized PMPC, which was prepared by reversible addition–fragmentation chain transfer (RAFT) polymerization of 2-methacryloyloxyethyl phosphorylcholine (MPC) using a newly designed water-soluble RAFT agent possessing a phosphonic acid group. The phosphonic acid group can conjugate with the OH groups on the TiO2 nanoparticles. We have clarified that the phosphonic acid end group is more crucial for creating colloidally stable PMPC-modified TiO2 nanoparticles under physiological conditions than carboxylic-acid-functionalized PMPC-modified ones. Furthermore, the enhanced generation of singlet oxygen (1O2), an ROS, in the presence of PMPC-modified TiO2 nanoparticles was confirmed using a 1O2-reactive fluorescent probe. We believe that the PMPC-modified TiO2 nanoparticles prepared herein have potential utility as novel biocompatible sonosensitizers for cancer therapy.


Introduction
Ultrasounds with wavelengths beyond human hearing have been widely used in diagnosis and therapy because they can penetrate deep into tissues without radiation damage. Sonodynamic therapy (SDT) has been widely used in clinical studies, including cancer therapy, owing to its non-invasiveness and temporal-spatial controllability with great depth [1][2][3][4][5][6][7]. In SDT, reactive oxygen species (ROS) such as singlet oxygen ( 1 O 2 ) and hydroxyl radicals are generated under ultrasound irradiation, and the ROS induce oxidative damage to target tissues. Furthermore, the generated ROS breaks the redox balance in living cells, which induces effective treatment on the hypoxic tumor [8]. The mechanism of ROS generation is the cavitation effect induced by ultrasounds which causes sonoluminescence, and the phenomenon is attributed to generate ROS from sonosensitizer. For efficient SDTs, sonosensitizers are of great importance to initiate a sonochemical reaction when producing ROS. In the past, many sonosensitizers, such as TiO 2 [9,10], porphyrin and its derivatives [11][12][13][14], BaTiO 3 [15], and PtCu 3 [16,17] have been developed to enhance ROS generation and therapeutic effects. TiO 2 nanoparticles have the potential for targeted delivery to tumors through enhanced permeability and retentivity effects [18,19] owing to their nanometer size [5]. TiO 2 nanoparticles have high chemical and physical stabilities; however, the colloidal stability of TiO 2 nanoparticles under physiological conditions is poor. TiO 2 nanoparticles have an isoelectric point at pH 6.2 when TiO 2 is formed as an anatase crystal [20]. Thus, TiO 2 nanoparticles have an anionic surface charge at neutral pH and are colloidally stabilized via electrostatic repulsion owing to their anionic charge. However, the electric bilayer on the particles becomes thinner in media containing high ionic concentrations, resulting in poor colloidal stability of the TiO 2 nanoparticles. An important requirement to ensure the efficacy of the sonodynamic therapy is the high colloidal stability of TiO 2 nanoparticles under physiological conditions. Several approaches have been reported to prepare TiO 2 nanoparticles with high colloidal stability for SDT under physiological conditions. Poly(ethylene glycol) (PEG) modification of TiO 2 nanoparticles is used to create highly stable TiO 2 nanoparticles for various therapies because PEG induces the steric repulsion of the PEG-modified nanoparticles [5,21]. Polysaccharide (e.g., dextran and hyaluronic acid)-modified TiO 2 nanoparticles have also been prepared for sonodynamic cancer therapy [10,22]. In our previous studies, polyion complex (PIC) micelles incorporating TiO 2 nanoparticles were developed as novel sonosensitizers possessing high colloidal stability under physiological conditions [23]. The PIC micelles were prepared from cationic polyallylamine-grafted poly(ethylene glycol) (PAA-g-PEG) and anionic TiO 2 nanoparticles at neutral pH, where the micellar structure was stabilized via electrostatic and van der Waals interactions between the polyallylamine and TiO 2 nanoparticles. The PIC micelles possessed high colloidal stability owing to the steric repulsion derived from the PEG grafted on the PIC micelles. Furthermore, we confirmed the sonosensitizing effect of PIC micelles incorporating TiO 2 nanoparticles in vitro with HeLa cells, where decreased cell viability was observed in cells treated with PIC micelles by ultrasound irradiation compared to that in the untreated cells [24].

Results and Discussion
PMPC, developed by Ishihara et al., is water-soluble and has a high biocompatibility derived from the phosphorylcholine motif of the lipid bilayer [59,60]. To modify PMPC as a biocompatible polymer on TiO2 nanoparticles, a new water-soluble dithiobenzoatebased RAFT agent possessing a phosphonic acid group (RAFT-PO4H2) was synthesized, where the phosphonic acid group can form a stable linkage with TiO2 nanoparticles. 4-Cyano-4-(phenylcarbonothioylthio)pentanoic acid N-succinimidyl ester was reacted with O-phosphoryl ethanolamine in a mixture of dimethylsulfoxide/water. The product was purified via inverse silica gel chromatography. The purification of the target RAFT agent was confirmed by 1 H-NMR and 13 C-NMR spectroscopy (see in Figure 1 and Supplementary Materials Figure S1). From ultraviolet-visible (UV-Vis) spectral measurements, RAFT-PO4H2 has a maximum absorbance wavelength of 305 nm with a small peak at 480 nm (see in Supplementary Materials Figure S2), indicating that RAFT-PO4H2 has a phenylcarbonothioylthio group. RAFT-COOH has a low solubility in water (even under basic conditions); however, RAFT-PO4H2 is easily dissolved in water. The high water solubility of RAFT-PO4H2 may be attributed to its phosphonic acid and amide groups.

Results and Discussion
PMPC, developed by Ishihara et al., is water-soluble and has a high biocompatibility derived from the phosphorylcholine motif of the lipid bilayer [59,60]. To modify PMPC as a biocompatible polymer on TiO 2 nanoparticles, a new water-soluble dithiobenzoatebased RAFT agent possessing a phosphonic acid group (RAFT-PO 4 H 2 ) was synthesized, where the phosphonic acid group can form a stable linkage with TiO 2 nanoparticles. 4-Cyano-4-(phenylcarbonothioylthio)pentanoic acid N-succinimidyl ester was reacted with O-phosphoryl ethanolamine in a mixture of dimethylsulfoxide/water. The product was purified via inverse silica gel chromatography. The purification of the target RAFT agent was confirmed by 1 H-NMR and 13 C-NMR spectroscopy (see in Figure 1 and Supplementary Materials Figure S1). From ultraviolet-visible (UV-Vis) spectral measurements, RAFT-PO 4 H 2 has a maximum absorbance wavelength of 305 nm with a small peak at 480 nm (see in Supplementary Materials Figure S2), indicating that RAFT-PO 4 H 2 has a phenylcarbonothioylthio group. RAFT-COOH has a low solubility in water (even under basic conditions); however, RAFT-PO 4 H 2 is easily dissolved in water. The high water solubility of RAFT-PO 4 H 2 may be attributed to its phosphonic acid and amide groups.   The polymerization control capability of RAFT-PO 4 H 2 as a control agent was investigated via RAFT polymerization of MPC by comparing the performance of RAFT-COOH. In the experiments, the target molecular weights of PMPC were regulated by changing the feed molar ratio of MPC to RAFT-PO 4

H 2 , where the feed molar ratio [MPC]/[RAFT-PO 4 H 2 ]
was set to 10, 20, 40, and 80. Deionized water was selected as the solvent for polymerization. The monomer conversions in all polymerizations using RAFT-PO 4 H 2 were estimated to be approximately 99% by 1 H-NMR ( Figure 2). The number-average molecular weights (M n ) of PMPC prepared using RAFT-PO 4 H 2 (PMPC-PO 4 H 2 ) were evaluated to be approximately 5600 (M w /M n :1.10), 7600 (M w /M n :1.11), 12,000 (M w /M n :1.12), and 20,000 (M w /M n :1.15) by gel permeation chromatography (GPC) when the target molecular weights were set to 3400, 6300, 12,200, and 24,000, respectively ( Table 1). The difference between the experimental and theoretical M n values was caused by the difference in the excluded volume of the polymer chains between PMPC and PEG, which was used as a standard polymer for preparing a calibration curve for GPC measurements.  Table 1). These results indicate that PMPC-PO 4 H 2 and PMPC-COOH were successfully prepared with narrow molecular weight distributions using RAFT-PO 4 H 2 and RAFT-COOH, respectively. Furthermore, PMPC-PO 4 H 2 and PMPC-COOH showed absorbances at 491 nm and 488 nm, respectively, which were derived from the dithiobenzoate groups of the RAFT end groups (see in Supplementary Materials Figures S4 and S5).      For the preparation of PMPC-modified TiO 2 nanoparticles, we used the grafting-to approach to prepare PMPC-modified TiO 2 nanoparticles using PMPC-PO 4 H 2 and PMPC-COOH. In the modification step, PMPC-PO 4 H 2 was added to the aqueous dispersion of TiO 2 in the presence of polyoxyethylene (20) oleyl ether (Brij98), where the pH of the aqueous media was adjusted to 4.0, to form OH groups on the TiO 2 nanoparticles. Notably, the TiO 2 nanoparticles were coagulated while adjusting to pH 4.0 without Bij98, whereas the particles were stably dispersed upon the addition of Brij98; this phenomenon was caused by the steric repulsion derived from the adsorbed Brij98 on TiO 2 nanoparticles. The adsorption of Brij98 on TiO 2 nanoparticles was supported by zeta potential measurements, that is, the zeta potential of the original TiO 2 nanoparticles (without Brij98) at pH 4.0 was +41.5 mV, whereas the zeta potential of the TiO 2 nanoparticles decreased slightly to +34.6 mV upon addition of Brij98, indicating that Brij98 was slightly adsorbed on the TiO 2 nanoparticles. The Brij98-stabilized TiO 2 nanoparticle dispersion had a monomodal particle size distribution, with a size of 38 nm (PDI: 0.256) (Figure 4). After the modification of PMPC-PO 4 H 2 (M n : 5600) on the TiO 2 nanoparticles, a peak derived from the submicrometer-sized coagulate TiO 2 particles was observed with a peak derived from the non-coagulated TiO 2 particles (average particle size: 55 nm, PDI: 0.256), indicating that the steric repulsion between TiO 2 particles is not effective for maintaining the colloidal stability when using the short PMPC-PO 4 H 2 . When PMPC-PO 4 H 2 of 7600 in M n was used, a small coagulate peak was also observed [47.2 nm (PDI: 0.263) for 7600 in M n ]. However, the coagulated TiO 2 particles were not detected when using the other PMPC-PO 4 H 2 of 12,000 and 20,000 in M n [49.3 nm (PDI: 0.194) for 12,000 in M n , and 55.7 nm (PDI: 0.151) for 20,000 in M n ]. Furthermore, the zeta potential of PMPC-modified TiO 2 nanoparticles prepared with PMPC-PO 4 H 2 (M n : 20,000) decreased markedly to +4.8 mV, which indicates that the modification of PMPC on TiO 2 particles was successful. Moreover, the similar particle size distribution of PMPC-modified TiO 2 particles prepared with PMPC-PO 4 H 2 of 12,000, and 20,000 in M n was maintained even after 240 min in pure water and after 100 times dilution of these particles in pure water [48.2 nm (PDI: 0.203) for 12,000 in M n , and 53.3 nm (PDI: 0.172) for 20,000 in M n after 240 min] (see in Supplementary Materials Figure S6). Thus, colloidally stable PMPC-modified TiO 2 nanoparticles were successfully prepared using PMPC-PO 4 H 2 with sufficient molecular weight. A similar molecular weight effect on the TiO 2 particle size distribution was observed when using PMPC-COOH with different molecular weights. A notable coagulation of TiO 2 particles was detected in the particle size distribution of TiO 2 particles incubated with PMPC-COOH of 5100 in M n . However, when PMPC-COOH with a higher molecular weight was used, particle size distributions with small peaks derived from coagulated TiO 2 particles were observed. The zeta potential of PMPC-COOH (M n : 16,000) was approximately +17.9 mV, which is smaller than that of the original TiO 2 particles but is higher than that of the PMPC-PO 4 H 2 -modified TiO 2 particles. Furthermore, the average particle size of the PMPC-COOH-modified TiO 2 particles increased [84.0 nm (PDI: 0.236) for 16,000 in M n , and 102.0 nm (PDI: 0.240) for 9800 in M n ] just after 100 times dilution using pure water; a more marked increase in the particle size of PMPC-COOH-modified TiO 2 particles just after dilution was detected using lower molecular weight PMPC-COOH, although the particle size was maintained after 240 min (see in Supplementary Materials Figure S6). The difference in the colloidal stability of PMPC-PO 4 H 2 -and PMPC-COOH-modified TiO 2 particles during pure water dilution may be caused by the higher affinity of the PO 4 H 2 group to the TiO 2 particles than that of the COOH group. These results indicate that the colloidal stability of PMPC-PO 4 H 2 -modified TiO 2 particles is higher than that of PMPC-COOH-modified TiO 2 particles against pure water dilution. just after 100 times dilution using pure water; a more marked increase in the particle size of PMPC-COOH-modified TiO2 particles just after dilution was detected using lower molecular weight PMPC-COOH, although the particle size was maintained after 240 min (see in Supplementary Materials Figure S6). The difference in the colloidal stability of PMPC-PO4H2and PMPC-COOH-modified TiO2 particles during pure water dilution may be caused by the higher affinity of the PO4H2 group to the TiO2 particles than that of the COOH group. These results indicate that the colloidal stability of PMPC-PO4H2-modified TiO2 particles is higher than that of PMPC-COOH-modified TiO2 particles against pure water dilution. We further investigated the effect of the PMPC-PO4H2 concentration on the particle size of obtained PMPC-modified TiO2 particles using PMPC-PO4H2 (Mn: 7600) (see in Supplementary Materials Figure S7 and Table 2). The average particle size of PMPC-modified TiO2 particles increased with increasing concentration from 2.5 mg/mL (41.6 nm, PDI: 0.181) to 5.0 mg/mL (49.1 nm, PDI: 0.264). However, the average particle size was almost saturated above 5.0 mg/mL, i.e., 47.2 nm (PDI: 0.263) and 49.3 nm (PDI: 0.271), and a monodispersed distribution was maintained when the PMPC-PO4H2 concentration was set to 0, 2.5, 5.0, 10.0, and 20.0 mg/mL, respectively (see in Supplementary Materials Figure S7). We further investigated the effect of the PMPC-PO 4 H 2 concentration on the particle size of obtained PMPC-modified TiO 2 particles using PMPC-PO 4 H 2 (M n : 7600) (see in Supplementary Materials Figure S7 and Table 2). The average particle size of PMPC-  Figure S7). For application as a sonosensitizer, PMPC-modified TiO 2 particles with high colloidal stability under physiological conditions, including pH and ionic concentration, are required. Thus, the colloidal stability of PMPC-modified TiO 2 particles prepared using PMPC-PO 4 H 2 and PMPC-COOH was investigated in PBS (pH 7.4) using DLS. The particles of Brij98 stabilized TiO 2 particles immediately coagulated in PBS (Figure 5g). PMPC-modified TiO 2 particles prepared using PMPC-PO 4 H 2 (M n : 5600) showed higher stability than unmodified TiO 2 particles because the particles were not immediately coagulated in PBS. However, the particle size significantly increased with increasing incubation time, reaching 199 nm (PDI: 0.279) after 60 min of incubation. We found that the colloidal stability of the PMPC-modified TiO 2 particles increased with the increasing molecular weight of PMPC-PO 4 H 2 . The particle sizes of PMPC-modified TiO 2 particles after 60 min of incubation were 86.5 nm (PDI: 0.200), 77.4 nm (PDI: 0.188), and 56.7 nm (PDI: 0.159) when PMPC-PO 4 H 2 of 7600, 12,000, and 20,000 in M n was used, respectively. In particular, the PMPC 20,000 -modified TiO 2 particles were maintained at less than 100 nm even after 240 min of incubation. In contrast to PMPC-PO 4 H 2 , the particle size of the PMPC-modified TiO 2 nanoparticles increased immediately even when high-molecular-weight PMPC-COOH (9800 and 16,000) was used ( Figure 5). These results strongly indicate that the phosphonic acid groups of PMPC-PO 4 H 2 are necessary to obtain colloidally stable PMPC-modified TiO 2 nanoparticles under physiological conditions. Previously, to prepare self-assembled monolayers (SAMs) on TiO 2 substrates or to form modification layers of TiO 2 photocatalysts, various molecules possessing acidic functional groups (e.g., carboxylic acid and phosphonic acid) were widely used, where these acidic groups work as interaction sites for the TiO 2 surface [55][56][57][58]. Several groups have reported that phosphonic acids interact more strongly with TiO 2 surfaces compared to carboxylic acids [61]. Gao et al. reported that well-ordered SAMs were formed on TiO 2 surfaces with phosphonic acid compounds, whereas most carboxylic acid compounds were removed from the TiO 2 surface during the washing process [62]. Thus, it appears that PMPC-COOH may be desorbed from TiO 2 nanoparticles in the buffered aqueous solution, resulting in particle coagulation, whereas TiO 2 nanoparticles with high colloidal stability were obtained with PMPC-PO 4 H 2 and had a stronger interaction capability with TiO 2 .  Finally, we investigated the 1 O 2 generation capability of PMPC 20,000 -PO 4 H 2 -modified TiO 2 particles under sonication in PBS using singlet oxygen sensor green (SOSG) as a probe molecule; the fluorescence intensity derived from SOSG increases upon reaction with 1 O 2 . As shown in Figure 6, the fluorescence intensity of SOSG increased gradually with increasing sonication time and was significantly higher than that of the control sample in the absence of PMPC 20,000 -PO 4 H 2 -modified TiO 2 particles (buffer solution). Furthermore, the fluorescence intensity derived from 1 O 2 -reacted SOSG for PMPC 20,000 -PO 4 H 2 -modified TiO 2 particles was higher than that for PMPC 7600 -PO 4 H 2 -modified TiO 2 particles. These results indicate that the PMPC-PO 4 H 2 -modified TiO 2 particles with high colloidal stability in the buffer solution exhibited 1 O 2 generation ability under sonication conditions in aqueous media. Figure 5. Particle size distributions, average particle size, and PDI values of PMPC-modified TiO2 nanoparticles prepared with PMPC20,000-PO4H2 (a), PMPC12,000-PO4H2 (b), PMPC7600-PO4H2 (c), PMPC5600-PO4H2 (d), PMPC30,000-COOH (e), PMPC15,000-COOH (f), and Brij98-stabilized TiO2 nanoparticles (g) at different incubation times (0 min: purple, 30 min: blue, 60 min: green, 120 min: yellow, 180 min: orange, 240 min: pink) in PBS. Figure 6. Fluorescence spectra derived from singlet oxygen sensor green at various sonication times in PBS without (a) and with PMPC20,000-PO4H2-modified TiO2 nanoparticles (b). Fluorescent intensity

Conclusions
In this study, we successfully created PMPC-modified TiO 2 nanoparticles with high colloidal stability in PBS as novel sonosensitizers using PMPC-PO 4 H 2 . To prepare PMPC-PO 4 H 2 , a new water-soluble RAFT agent possessing a phosphonic acid group (RAFT-PO 4 H 2 ) was synthesized. Using RAFT-PO 4 H 2 , PMPC-PO 4 H 2 with a narrow molecular weight distribution was prepared. Further, the molecular weight of PMPC-PO 4 H 2 could be regulated by changing the molar ratio [MPC]/[ RAFT-PO 4 H 2 ]. The grafting-to approach using PMPC-PO 4 H 2 yielded PMPC-modified TiO 2 nanoparticles, and PMPC-PO 4 H 2 with a higher molecular weight yielded greater colloidal stability of the PMPC-modified TiO 2 nanoparticles. Moreover, the PMPC-PO 4 H 2 -modified TiO 2 nanoparticles had greater colloidal stability under physiological conditions than the PMPC-COOH-modified TiO 2 nanoparticles. Furthermore, the sonosensitizing effect of the PMPC-modified TiO 2 nanoparticles in assisting 1 O 2 generation in an aqueous medium was clarified. Utilizing the RAFT polymerization, the TiO 2 nanoparticles can be further functionalized. For example, the sonosensitizer can be further functionalized by 2nd block chain extension and/or RAFT chain end modification. We believe that the various functionalized biocompatible polymerfunctionalized TiO 2 nanoparticles will be developed for sonodynamic therapy.  ). NaCl, HCl, NaOH, and dimethyl sulfoxide (DMSO) were purchased from Nacalai Tesque (Kyoto, Japan). A dispersion of TiO 2 nanoparticles (STS-100) was purchased from Ishihara Sangyo Kaisha Ltd. (Osaka, Japan). Deionized water was obtained using a Millipore Milli-Q purification system. SOSG was purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Apparatus
UV-Vis spectral measurements were performed using a V-560 spectrophotometer (Jasco Ltd., Tokyo, Japan). Fluorescence spectral measurements were performed using an FP-8300 spectrophotometer (Jasco Ltd., Tokyo, Japan). 1 H-NMR spectra were measured using a 400-MHz Fourier transform (FT)-NMR apparatus (JNM-ECX400, FT-NMR system, JEOL Ltd., Tokyo, Japan). The particle size distribution and zeta potential of the obtained particles were measured using a ZETASIZER NANO-ZS instrument (Malvern, UK). Ultrasonication was performed using Sonitron2000 (NEPA GENE, Chiba, Japan). The numberand weight-average molecular weights (M n and M w , respectively) were analyzed by GPC at 40 • C using TSKgel G3000PW and TSKgel G4000PW (7.8 mm i.d. × 300 mm, Tosoh Corp.) with 20 mM phosphate buffer (pH 7.4) as the eluent, coupled with a refractive index detector (RI-2031 Plus, JASCO, Tokyo, Japan). A PEG standard (molecular weight range: 1080-107,000) was used to calibrate the molecular weight. Theoretical molecular weights were calculated using the following Equation (1). In Equation (1)

Synthesis of RAFT-PO 4 H 2
4-Cyano-4-(phenylcarbonothioylthio)pentanoic acid N-succinimidyl ester (546.6 mg, 1.45 mmol) was dissolved in DMSO (30 mL). O-phosphoryl ethanolamine (234.6 mg, 1.66 mmol) was dissolved in a carbonate buffer (pH 9, 15 mL). These solutions were mixed to facilitate a coupling reaction between these molecules at room temperature for 18 h in the dark. The product was purified via inverse silica gel chromatography using methanol and water. Methanol and water were removed by evaporation and freeze-drying, respectively, to yield a dry product. Yield: 82%.

PMPC-Modified TiO 2 Nanoparticles
TiO 2 nanoparticles were dispersed in deionized water by dissolving Brij98 (1.5 mL), and PMPC-PO 4 H 2 or PMPC-COOH aqueous solution (1.5 mL) was mixed with the dispersion of TiO 2 nanoparticles (final concentration:1 mg/mL TiO 2 , 0.5 mM Brij98, 10 mg/mL PMPC). After the pH was adjusted to 4.0, the mixture was incubated for 24 h at room temperature in the dark. The size distribution of the incubated particles was determined using DLS. The supernatant of the dispersion was separated by ultrafiltration (50,000 Da), and the UV-Vis spectra of the supernatant and the original PMPC aqueous solution were measured to evaluate the modification of PMPC-PO 4 H 2 or PMPC-COOH on TiO 2 nanoparticles.

Colloidal Stability of PMPC-Modified TiO 2 Nanoparticles in PBS
The dispersion of PMPC-modified TiO 2 nanoparticles (2.5 µL) was mixed with PBS (pH 7.4, 2.9975 mL). After several incubation periods, the particle size distributions were measured using DLS. Stabilized TiO 2 nanoparticles were used as a reference instead of PMPC-modified TiO 2 nanoparticles.

Data Availability Statement:
The data presented in this study are available in the article.