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Article

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

by
Yukiya Kitayama
1,2,
Aoi Katayama
2,
Zhicheng Shao
1 and
Atsushi Harada
1,2,*
1
Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 599-8531, Osaka, Japan
2
Department of Applied Chemistry, Graduate School of Engineering, Osaka Metropolitan University, 1-1 Gakuen-cho, Naka-ku, Sakai 599-8531, Osaka, Japan
*
Author to whom correspondence should be addressed.
Polymers 2023, 15(11), 2426; https://doi.org/10.3390/polym15112426
Submission received: 27 March 2023 / Revised: 15 May 2023 / Accepted: 19 May 2023 / Published: 23 May 2023
(This article belongs to the Special Issue Polymeric Drugs and Drug Delivery Systems)
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Abstract

:
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.

1. 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 (1O2) 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 TiO2 [9,10], porphyrin and its derivatives [11,12,13,14], BaTiO3 [15], and PtCu3 [16,17] have been developed to enhance ROS generation and therapeutic effects. TiO2 nanoparticles have the potential for targeted delivery to tumors through enhanced permeability and retentivity effects [18,19] owing to their nanometer size [5]. TiO2 nanoparticles have high chemical and physical stabilities; however, the colloidal stability of TiO2 nanoparticles under physiological conditions is poor. TiO2 nanoparticles have an isoelectric point at pH 6.2 when TiO2 is formed as an anatase crystal [20]. Thus, TiO2 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 TiO2 nanoparticles. An important requirement to ensure the efficacy of the sonodynamic therapy is the high colloidal stability of TiO2 nanoparticles under physiological conditions.
Several approaches have been reported to prepare TiO2 nanoparticles with high colloidal stability for SDT under physiological conditions. Poly(ethylene glycol) (PEG) modification of TiO2 nanoparticles is used to create highly stable TiO2 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 TiO2 nanoparticles have also been prepared for sonodynamic cancer therapy [10,22]. In our previous studies, polyion complex (PIC) micelles incorporating TiO2 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 TiO2 nanoparticles at neutral pH, where the micellar structure was stabilized via electrostatic and van der Waals interactions between the polyallylamine and TiO2 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 TiO2 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].
Recently, reversible deactivation radical polymerization (RDRP) has been developed as an emerging synthetic method to achieve precise molecular design and create various functional polymers via radical reaction [25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44]. Using RDRPs, the surface modification of inorganic nanoparticles can be easily achieved using grafting-to or grafting-from approaches [45,46,47,48,49,50]. Charpentier et al. reported the surface-initiated reversible addition–fragmentation chain transfer (RAFT) polymerization of methyl methacrylate from TiO2 nanoparticles modified with 4-cyano-4-(dodecyl-sulfanylthiocarbonyl) sulfanyl pentanoic acid [51]. Wang et al. reported the successful surface-initiated atom transfer radical polymerization (ATRP) of styrene from TiO2 nanoparticles using an initiator possessing a trimethoxysilane group [52]. Charpentier et al. reported water-dispersible poly(acrylic acid)-modified TiO2 nanoparticles by RAFT polymerization from TiO2 nanoparticles modified with 4-cyano-4-(dodecyl-sulfanylthiocarbonyl)sulfanyl pentanoic acid [53]. Haddleton et al. reported surface modification of 2-(dimethylamino)ethyl methacrylate and 2-(diethylamino)ethyl methacrylate on TiO2 nanoparticles using grafting-to or grafting-from approaches with Cu(0)-mediated living radical polymerization using a designed initiator containing catechol as a binding site to TiO2 [54]. However, to the best of our knowledge, the preparation of biocompatible polymer-modified TiO2 nanoparticles via RDRPs and their application as sonosensitizers has never been reported.
In this study, we report the fabrication of biocompatible poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC)-modified TiO2 nanoparticles with high colloidal stability under physiological conditions using RDRPs and investigate the sonosensitizing effect of TiO2 particles (Scheme 1). To achieve this goal, a RAFT agent containing a phosphonic acid group was newly designed; the phosphonic-acid-functionalized poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC-PO4H2) was synthesized via RAFT polymerization with the RAFT agent because the phosphonic acid groups can interact more strongly with the TiO2 surface compared to carboxylic acids [55,56,57,58]. We clarify that the colloidal stability of PMPC-PO4H2-modified TiO2 nanoparticles under physiological conditions was higher than that of poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) possessing carboxylic acid (PMPC-COOH)-modified TiO2 nanoparticles; PMPC-COOH was prepared using a commercially available RAFT agent [4-cyano-4-(phenylcarbonothioylthio)pentanoic acid: RAFT-COOH]. The effect of the molecular weight of PMPC-PO4H2 on the colloidal stability of the 2-methacryloyloxyethyl phosphorylcholine (MPC)-modified TiO2 nanoparticles was also investigated in detail. Finally, the sonosensitizing activity of the PMPC-modified TiO2 nanoparticles was investigated.

2. 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 dithiobenzoate-based 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 1H-NMR and 13C-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.
The polymerization control capability of RAFT-PO4H2 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-PO4H2, where the feed molar ratio [MPC]/[RAFT-PO4H2] 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-PO4H2 were estimated to be approximately 99% by 1H-NMR (Figure 2). The number-average molecular weights (Mn) of PMPC prepared using RAFT-PO4H2 (PMPC-PO4H2) were evaluated to be approximately 5600 (Mw/Mn:1.10), 7600 (Mw/Mn:1.11), 12,000 (Mw/Mn:1.12), and 20,000 (Mw/Mn: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 Mn 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. Methanol was selected as the solvent for RAFT polymerization of MPC with RAFT-COOH because of the low solubility of RAFT-COOH. The conversion of RAFT polymerization of MPC reached 99% at all feed molar ratios of [MPC]/[RAFT-COOH] (see in Supplementary Materials Figure S3). The Mn of PMPC prepared using RAFT-COOH (PMPC-COOH) was evaluated to be approximately 5100 (Mw/Mn:1.11), 6400 (Mw/Mn:1.11), 9800 (Mw/Mn:1.16), and 16,000 (Mw/Mn:1.21) when the target molecular weights were 3200, 6200, 12,100, and 24,000, respectively (Figure 3, Table 1). These results indicate that PMPC-PO4H2 and PMPC-COOH were successfully prepared with narrow molecular weight distributions using RAFT-PO4H2 and RAFT-COOH, respectively. Furthermore, PMPC-PO4H2 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 TiO2 nanoparticles, we used the grafting-to approach to prepare PMPC-modified TiO2 nanoparticles using PMPC-PO4H2 and PMPC-COOH. In the modification step, PMPC-PO4H2 was added to the aqueous dispersion of TiO2 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 TiO2 nanoparticles. Notably, the TiO2 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 TiO2 nanoparticles. The adsorption of Brij98 on TiO2 nanoparticles was supported by zeta potential measurements, that is, the zeta potential of the original TiO2 nanoparticles (without Brij98) at pH 4.0 was +41.5 mV, whereas the zeta potential of the TiO2 nanoparticles decreased slightly to +34.6 mV upon addition of Brij98, indicating that Brij98 was slightly adsorbed on the TiO2 nanoparticles. The Brij98-stabilized TiO2 nanoparticle dispersion had a monomodal particle size distribution, with a size of 38 nm (PDI: 0.256) (Figure 4). After the modification of PMPC-PO4H2 (Mn: 5600) on the TiO2 nanoparticles, a peak derived from the submicrometer-sized coagulate TiO2 particles was observed with a peak derived from the non-coagulated TiO2 particles (average particle size: 55 nm, PDI: 0.256), indicating that the steric repulsion between TiO2 particles is not effective for maintaining the colloidal stability when using the short PMPC-PO4H2. When PMPC-PO4H2 of 7600 in Mn was used, a small coagulate peak was also observed [47.2 nm (PDI: 0.263) for 7600 in Mn]. However, the coagulated TiO2 particles were not detected when using the other PMPC-PO4H2 of 12,000 and 20,000 in Mn [49.3 nm (PDI: 0.194) for 12,000 in Mn, and 55.7 nm (PDI: 0.151) for 20,000 in Mn]. Furthermore, the zeta potential of PMPC-modified TiO2 nanoparticles prepared with PMPC-PO4H2 (Mn: 20,000) decreased markedly to +4.8 mV, which indicates that the modification of PMPC on TiO2 particles was successful. Moreover, the similar particle size distribution of PMPC-modified TiO2 particles prepared with PMPC-PO4H2 of 12,000, and 20,000 in Mn 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 Mn, and 53.3 nm (PDI: 0.172) for 20,000 in Mn after 240 min] (see in Supplementary Materials Figure S6). Thus, colloidally stable PMPC-modified TiO2 nanoparticles were successfully prepared using PMPC-PO4H2 with sufficient molecular weight. A similar molecular weight effect on the TiO2 particle size distribution was observed when using PMPC-COOH with different molecular weights. A notable coagulation of TiO2 particles was detected in the particle size distribution of TiO2 particles incubated with PMPC-COOH of 5100 in Mn. However, when PMPC-COOH with a higher molecular weight was used, particle size distributions with small peaks derived from coagulated TiO2 particles were observed. The zeta potential of PMPC-COOH (Mn: 16,000) was approximately +17.9 mV, which is smaller than that of the original TiO2 particles but is higher than that of the PMPC-PO4H2-modified TiO2 particles. Furthermore, the average particle size of the PMPC-COOH-modified TiO2 particles increased [84.0 nm (PDI: 0.236) for 16,000 in Mn, and 102.0 nm (PDI: 0.240) for 9800 in Mn] 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-PO4H2- and 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).
For application as a sonosensitizer, PMPC-modified TiO2 particles with high colloidal stability under physiological conditions, including pH and ionic concentration, are required. Thus, the colloidal stability of PMPC-modified TiO2 particles prepared using PMPC-PO4H2 and PMPC-COOH was investigated in PBS (pH 7.4) using DLS. The particles of Brij98 stabilized TiO2 particles immediately coagulated in PBS (Figure 5g). PMPC-modified TiO2 particles prepared using PMPC-PO4H2 (Mn: 5600) showed higher stability than unmodified TiO2 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 TiO2 particles increased with the increasing molecular weight of PMPC-PO4H2. The particle sizes of PMPC-modified TiO2 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- PO4H2 of 7600, 12,000, and 20,000 in Mn was used, respectively. In particular, the PMPC20,000-modified TiO2 particles were maintained at less than 100 nm even after 240 min of incubation. In contrast to PMPC-PO4H2, the particle size of the PMPC-modified TiO2 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-PO4H2 are necessary to obtain colloidally stable PMPC-modified TiO2 nanoparticles under physiological conditions. Previously, to prepare self-assembled monolayers (SAMs) on TiO2 substrates or to form modification layers of TiO2 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 TiO2 surface [55,56,57,58]. Several groups have reported that phosphonic acids interact more strongly with TiO2 surfaces compared to carboxylic acids [61]. Gao et al. reported that well-ordered SAMs were formed on TiO2 surfaces with phosphonic acid compounds, whereas most carboxylic acid compounds were removed from the TiO2 surface during the washing process [62]. Thus, it appears that PMPC-COOH may be desorbed from TiO2 nanoparticles in the buffered aqueous solution, resulting in particle coagulation, whereas TiO2 nanoparticles with high colloidal stability were obtained with PMPC- PO4H2 and had a stronger interaction capability with TiO2.
Finally, we investigated the 1O2 generation capability of PMPC20,000-PO4H2-modified TiO2 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 1O2. 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 PMPC20,000-PO4H2-modified TiO2 particles (buffer solution). Furthermore, the fluorescence intensity derived from 1O2-reacted SOSG for PMPC20,000-PO4H2-modified TiO2 particles was higher than that for PMPC7600-PO4H2-modified TiO2 particles. These results indicate that the PMPC-PO4H2-modified TiO2 particles with high colloidal stability in the buffer solution exhibited 1O2 generation ability under sonication conditions in aqueous media.

3. Conclusions

In this study, we successfully created PMPC-modified TiO2 nanoparticles with high colloidal stability in PBS as novel sonosensitizers using PMPC-PO4H2. To prepare PMPC-PO4H2, a new water-soluble RAFT agent possessing a phosphonic acid group (RAFT-PO4H2) was synthesized. Using RAFT-PO4H2, PMPC-PO4H2 with a narrow molecular weight distribution was prepared. Further, the molecular weight of PMPC-PO4H2 could be regulated by changing the molar ratio [MPC]/[ RAFT-PO4H2]. The grafting-to approach using PMPC-PO4H2 yielded PMPC-modified TiO2 nanoparticles, and PMPC-PO4H2 with a higher molecular weight yielded greater colloidal stability of the PMPC-modified TiO2 nanoparticles. Moreover, the PMPC-PO4H2-modified TiO2 nanoparticles had greater colloidal stability under physiological conditions than the PMPC-COOH-modified TiO2 nanoparticles. Furthermore, the sonosensitizing effect of the PMPC-modified TiO2 nanoparticles in assisting 1O2 generation in an aqueous medium was clarified. Utilizing the RAFT polymerization, the TiO2 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 polymer-functionalized TiO2 nanoparticles will be developed for sonodynamic therapy.

4. Materials and Methods

4.1. Materials

4-Cyano-4-(phenylcarbonothioylthio)pentanoic acid N-succinimidyl ester, O-phosphoryl ethanolamine, and MPC were purchased from Sigma–Aldrich (St. Louis, MO, USA). 2,2’-Azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (VA-044) and Brij98 were purchased from Wako Pure Chemical Co., Ltd. (Osaka, Japan). NaCl, HCl, NaOH, and dimethyl sulfoxide (DMSO) were purchased from Nacalai Tesque (Kyoto, Japan). A dispersion of TiO2 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).

4.2. 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). 1H-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 number- and weight-average molecular weights (Mn and Mw, 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), Mn.Monomer and Mn.RAFT are molecular weights of monomer and RAFT agent, respectively, [Monomer] and [RAFT agent] were molar concentrations of monomer and RAFT agent, respectively.
M n . th = M n . Monomer × Monomer RAFT   agent × Conversion + M n . RAFT

4.3. Synthesis of RAFT- PO4H2

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%.

4.4. Synthesis of PMPC by RAFT Polymerization

MPC (1 mmol), RAFT-PO4H2 (10, 20, 40, 80 μmol), and VA-044 (2.5, 5.0, 10, 20 μmol) were dissolved in deionized water (3, 3, 5, 8 mL). The solution was added to a Schlenk flask. After several N2/degassing processes, the polymerization began with heating the solution at 40 °C for 24 h in the dark. After polymerization, the polymer (PMPC-PO4H2) was obtained by freeze-drying. To prepare PMPC-COOH, RAFT polymerization was performed under the same conditions as the prepolymer solution of methanol (3 mL) containing MPC (1 mmol), RAFT-COOH (10, 20, 40, and 80 μmol), and VA-044 (2.5, 5.0, 10 and 20 μmol).

4.5. PMPC-Modified TiO2 Nanoparticles

TiO2 nanoparticles were dispersed in deionized water by dissolving Brij98 (1.5 mL), and PMPC- PO4H2 or PMPC-COOH aqueous solution (1.5 mL) was mixed with the dispersion of TiO2 nanoparticles (final concentration:1 mg/mL TiO2, 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-PO4H2 or PMPC-COOH on TiO2 nanoparticles.

4.6. Colloidal Stability of PMPC-Modified TiO2 Nanoparticles in PBS

The dispersion of PMPC-modified TiO2 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 TiO2 nanoparticles were used as a reference instead of PMPC-modified TiO2 nanoparticles.

4.7. Sonosensitizing Effect of PMPC-Modified TiO2 Nanoparticles

The dispersion of PMPC-modified TiO2 nanoparticles (3 mL, TiO2 concentration: 45 μL/mL) was mixed with a methanol solution of SOSG (0.5 M, 6 μL). Thereafter, sonication (intensity: 0.5 W/cm2) was performed for 2, 4, 6, 8, and 10 min. The fluorescence intensity of SOSG was measured at excitation wavelength of 485 nm and an emission wavelength of 525 nm.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/polym15112426/s1, Figure S1: 13C NMR spectrum of RAFT-PO4H2; Figure S2: UV–Vis spectrum of RAFT-PO4H2; Figure S3: 1H NMR spectra of MPC and PMPC; Figure S4: UV–Vis spectra of PMPC-PO4H2; Figure S5: UV–Vis spectra of PMPC-COOH; Figure S6: Colloidal stability of PMPC-PO4H2-and PMPC-COOH-modified TiO2 particles; Figure S7: Particle size distributions of PMPC-PO4H2-modified TiO2 particles prepared with different concentrations of PMPC7600-PO4H2.

Author Contributions

Conceptualization, A.H.; methodology, A.H.; investigation, A.K. and Z.S.; writing—original draft preparation, Y.K.; writing—review and editing, A.H.; supervision, A.H.; All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by JSPS KAKENHI (Grant Number 21H03823).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in the article.

Acknowledgments

The authors thank Junya Emoto for technical support.

Conflicts of Interest

The authors declare no conflict of interest.

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Polymers 15 02426 sch001 550
Scheme 1. Schematic of preparation of PMPC-modified TiO2 nanoparticles as biocompatible sonosensitizer.
Scheme 1. Schematic of preparation of PMPC-modified TiO2 nanoparticles as biocompatible sonosensitizer.
Polymers 15 02426 sch001
Polymers 15 02426 g001 550
Figure 1. (a) Synthetic scheme of RAFT-PO4H2. (b) 1H NMR spectrum and corresponding chemical structure of RAFT-PO4H2.
Figure 1. (a) Synthetic scheme of RAFT-PO4H2. (b) 1H NMR spectrum and corresponding chemical structure of RAFT-PO4H2.
Polymers 15 02426 g001
Polymers 15 02426 g002 550
Figure 2. Top: Scheme of RAFT polymerization of MPC with RAFT-PO4H2. Bottom: 1H NMR spectra of MPC and polymer solutions obtained after RAFT polymerizations of MPC using RAFT-PO4H2 with different molar ratios of MPC and RAFT-PO4H2.
Figure 2. Top: Scheme of RAFT polymerization of MPC with RAFT-PO4H2. Bottom: 1H NMR spectra of MPC and polymer solutions obtained after RAFT polymerizations of MPC using RAFT-PO4H2 with different molar ratios of MPC and RAFT-PO4H2.
Polymers 15 02426 g002
Polymers 15 02426 g003 550
Figure 3. GPC charts of PMPC-PO4H2 (a) and PMPC-COOH (b) obtained after RAFT polymerizations of MPC using RAFT-PO4H2 and RAFT-COOH, respectively, with different target molecular weights.
Figure 3. GPC charts of PMPC-PO4H2 (a) and PMPC-COOH (b) obtained after RAFT polymerizations of MPC using RAFT-PO4H2 and RAFT-COOH, respectively, with different target molecular weights.
Polymers 15 02426 g003
Polymers 15 02426 g004 550
Figure 4. Particle size distributions of PMPC-PO4H2- (a) and PMPC-COOH- (b) modified TiO2 nanoparticles. PMPC-PO4H2 (a) and PMPC-COOH (b) possessing different molecular weights were obtained after RAFT polymerizations of MPC using RAFT-PO4H2 and RAFT-COOH, respectively.
Figure 4. Particle size distributions of PMPC-PO4H2- (a) and PMPC-COOH- (b) modified TiO2 nanoparticles. PMPC-PO4H2 (a) and PMPC-COOH (b) possessing different molecular weights were obtained after RAFT polymerizations of MPC using RAFT-PO4H2 and RAFT-COOH, respectively.
Polymers 15 02426 g004
Polymers 15 02426 g005 550
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 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.
Polymers 15 02426 g005
Polymers 15 02426 g006 550
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 change at 525 nm after 10 min sonication without and with PMPC7600-PO4H2-modified TiO2 nanoparticles, and with PMPC20,000-PO4H2-modified TiO2 nanoparticles. (c) Fluorescent intensity change at 525 nm after 10 min sonication without and with PMPC7600-PO4H2-modified TiO2 nanoparticles, and with PMPC20,000-PO4H2-modified TiO2 nano-particles.
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 change at 525 nm after 10 min sonication without and with PMPC7600-PO4H2-modified TiO2 nanoparticles, and with PMPC20,000-PO4H2-modified TiO2 nanoparticles. (c) Fluorescent intensity change at 525 nm after 10 min sonication without and with PMPC7600-PO4H2-modified TiO2 nanoparticles, and with PMPC20,000-PO4H2-modified TiO2 nano-particles.
Polymers 15 02426 g006
Table
Table 1. RAFT polymerization of MPC with RAFT-PO4H2 or RAFT-COOH a.
Table 1. RAFT polymerization of MPC with RAFT-PO4H2 or RAFT-COOH a.
RunRAFT Agent[MPC]0/[RAFT Agent]0/Conv. (%)MnMn,thMw/Mn
1 bRAFT-PO4H210/1>99560034001.10
2 bRAFT-PO4H220/1>99760063001.11
3 bRAFT-PO4H240/1>9912,00012,2001.12
4 bRAFT-PO4H280/1>9920,00024,0001.15
5 cRAFT-COOH10/1>99510032001.11
6 cRAFT-COOH20/1>99640062001.11
7 cRAFT-COOH40/1>99980012,1001.16
8 cRAFT-COOH80/1>9916,00024,0001.21
a: Polymerization time: 24 h; Polymerization temperature: 70 °C; Initiator: V-501; b: Solvent: deionized water; c: Solvent: methanol.
Table
Table 2. Average particle sizes and PDI of TiO2 nanoparticles before and after modification of PMPC-PO4H2. Different concentrations of PMPC-PO4H2 were used.
Table 2. Average particle sizes and PDI of TiO2 nanoparticles before and after modification of PMPC-PO4H2. Different concentrations of PMPC-PO4H2 were used.
SampleConcentration (mg/mL)Size (nm)PDI
Original-38.30.256
PMPC-PO4H2 a2.541.60.181
5.049.10.264
1047.20.263
2049.30.271
a: Number-average molecular weight: 7600.
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Kitayama, Y.; Katayama, A.; Shao, Z.; Harada, A. Biocompatible Polymer-Grafted TiO2 Nanoparticle Sonosensitizers Prepared Using Phosphonic Acid-Functionalized RAFT Agent. Polymers 2023, 15, 2426. https://doi.org/10.3390/polym15112426

AMA Style

Kitayama Y, Katayama A, Shao Z, Harada A. Biocompatible Polymer-Grafted TiO2 Nanoparticle Sonosensitizers Prepared Using Phosphonic Acid-Functionalized RAFT Agent. Polymers. 2023; 15(11):2426. https://doi.org/10.3390/polym15112426

Chicago/Turabian Style

Kitayama, Yukiya, Aoi Katayama, Zhicheng Shao, and Atsushi Harada. 2023. "Biocompatible Polymer-Grafted TiO2 Nanoparticle Sonosensitizers Prepared Using Phosphonic Acid-Functionalized RAFT Agent" Polymers 15, no. 11: 2426. https://doi.org/10.3390/polym15112426

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