Preparation of a thermo-responsive drug carrier consisting of a biocompatible triblock copolymer and fullerene

Abstract

A triblock copolymer (PEG-b-PUEM-b-PMPC; EUM) comprising poly(ethylene glycol) (PEG), thermo-responsive poly(2-ureidoethyl methacrylate) (PUEM), and poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC) blocks was synthesized via controlled radical polymerization. PEG and PMPC blocks exhibit hydrophilicity and biocompatibility. The PUEM block exhibits an upper critical solution temperature (UCST). PMPC can dissolve hydrophobic fullerenes in water to form a complex by grinding PMPC and fullerene powders. Fullerene-C₇₀ (C₇₀) and EUM were ground in a mortar and phosphate-buffered saline (PBS) was added to synthesize a water-soluble complex (C₇₀/EUM). C₇₀/EUM has a core–shell-corona structure, whose core is a complex of C₇₀ and PMPC, the shell is PUEM, and corona is PEG. The maximum C₇₀ concentration dissolved in PBS was 0.313 g L⁻¹ at an EUM concentration of 2 g L⁻¹. The C₇₀/EUM hydrodynamic radius (R_h) was 34 nm in PBS at 10 °C, which increased due to the PUEM block's UCST phase transition with increasing temperature, and R_h attained a constant value of 38 nm above 36 °C. An anticancer drug, doxorubicin, was encapsulated in the PUEM shell by hydrophobic interactions in C₇₀/EUM at room temperature, which can be released by heating. The generation of singlet oxygen (¹O₂) from C₇₀/EUM upon visible-light irradiation was confirmed using the singlet oxygen sensor green indicator. Water-soluble C₇₀/EUM may be used as a carrier that releases encapsulated drugs when heated and as a photosensitizer for photodynamic therapy.

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Introduction

Fullerenes such as C₆₀ and C₇₀ and their derivatives have been reported to function as enzyme inhibitors,^1–3 antivirals,^4–6 DNA scission agents,^7–9 and radical quenchers.¹⁰ Furthermore, fullerenes such as C₆₀ and C₇₀ are expected to be used as photosensitizers for photodynamic therapy (PDT) since they generate singlet oxygen with high efficiency upon visible-light irradiation.^11–13 However, their biological applications are limited by their low water solubility.¹⁴ Various fullerene derivatives with improved water solubility have been reported.^15–17 Fullerenes have been solubilized, forming a complex between fullerenes and host molecules such as cyclodextrins,^18,19 calixarenes,²⁰ micelles,^21,22 liposomes,^23,24 and poly(N-vinylpyrrolidone) (PVP).^25–27 Particularly, PVP can solubilize fullerenes with high concentrations to form a water-soluble complex due to charge-transfer interactions. Yamakoshi et al.²⁸ reported that C₆₀ and C₇₀ can be solubilized in water at 0.4 and 0.2 g L⁻¹, respectively, using 50 g L⁻¹ PVP.

Poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC) is water-soluble and biocompatible because the pendant phosphorylcholine group has the same chemical structure as the polar group in phospholipids from biological membranes.^29–31 The phosphorylcholine group is an electrically neutral zwitterionic structure. PMPC shows no biological reactions with biomolecules because it does not interact with plasma proteins.^32–34 We have reported that a water-soluble complex (C₇₀/PMPC) can be synthesized by mixing C₇₀ and PMPC powders and grinding them.³⁵ 0.49 g L⁻¹ C₇₀ can be solubilized in water using 1 g L⁻¹ PMPC.

The photosensitizer should be accumulated around the affected region for effective PDT with low side effects. There are tens to hundreds of nanometer-sized pores that are not observed in normal tissue at the vascular endothelium of cancer tissue. Particles of several nanometers in size are immediately excreted from the kidneys, and those larger than 400 nm are rapidly eliminated by macrophages. However, tens to hundreds of nanometers of particles accumulate in cancer tissue due to enhanced permeation and retention (EPR) effects.^36–38 The photosensitizer accumulated around the cancer tissue damages only cancer through the singlet oxygen generated upon light irradiation. The light should be irradiated to the photosensitizer inside the body for PDT. PDT uses ordinary visible or near-infrared light; however, it is difficult for it to penetrate deep inside the body.³⁹ Thus, it should be combined with other PDT treatments for treating deep cancers inside the body. The photosensitizer C₇₀ in a water-soluble C₇₀/PMPC complex can generate singlet oxygen upon visible-light irradiation. Therefore, the controlled release function of anticancer drugs upon heating was added to the C₇₀/PMPC complex. We focused on poly(2-ureidoethyl methacrylate) (PUEM), which exhibits an upper critical solution temperature (UCST).⁴⁰ The PUEM's pendant ureido groups combine with interpolymer chains below the UCST due to hydrogen bonds, and PUEM cannot dissolve in water. Alternatively, PUEM can dissolve in water due to an increase in molecular motion to break the hydrogen bonds above the UCST. UCST type polymers are applied for controlled release for drug delivery systems.⁴¹ Deng et al.⁴² synthesized a diblock copolymer (PEG-b-P(NAGA-co-AN)) composed of a hydrophilic poly(ethylene glycol) (PEG) and a thermo-responsive random copolymer (P(NAGA-co-AN)) of N-acryloyl glycolinamide and acrylonitrile. P(NAGA-co-AN) exhibits UCST due to hydrogen bonding interactions. PEG-b-P(NAGA-co-AN) forms core–shell micelles to associate with the P(NAGA-co-AN) blocks below the UCST. Hydrophobic drugs can be encapsulated in the core and released by heating.

In this study, we have synthesized a triblock copolymer (EUM; PEG₄₅-b-PUEM₁₀₁-b-PMPC₉₉) consisting of PEG for colloid stabilization in water, PUEM exhibiting UCST, and PMPC solubilizing C₇₀ in water to synthesize a thermoresponsive drug carrier containing C₇₀ photosensitizer. EUM was synthesized via reversible addition–fragmentation chain transfer (RAFT)-controlled radical polymerization using a PEG-macro chain transfer agent (CTA). The water-soluble complex (C₇₀/EUM) was synthesized by crushing powders of EUM and C₇₀ together in a mortar and then adding water. The C₇₀/EUM complex formed a core–shell-corona micelle with a C₇₀/PMPC core, thermoresponsive PUEM shell, and hydrophilic PEG corona (Fig. 1). C₇₀/EUM can encapsulate guest molecules such as hydrophobic drugs in the PUEM shell because the PUEM block becomes hydrophobic below the UCST. Water-soluble PEG exhibits biocompatibility.^43–45 C₇₀/EUM covered with biocompatible PEG coronas is expected to accumulate in the affected region due to the EPR effect in the body. Heating above the UCST of the PUEM shell can release the encapsulated hydrophobic drugs in C₇₀/EUM. C₇₀/EUM is thought to have two functions: singlet oxygen generation upon visible-light irradiation and a drug carrier that can release the drug when heated.

Fig. 1 (a) The chemical structure of PEG₄₅-b-PUEM₁₀₁-b-PMPC₉₉ (EUM) and a conceptual illustration of C₇₀/EUM micelle formation in water, (b) its thermo-responsive behavior, and (c) its light-responsive behavior upon visible-light irradiation.

Experimental

Materials

2-(Methacryloyloxy)ethyl phosphorylcholine (MPC) was purchased from NOF Corp. (Tokyo, Japan) and used after recrystallization from acetonitrile.³¹ Poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC, degree of polymerization (DP) = 100, number-average molecular weight (M_n) = 2.98 × 10⁴ g mol⁻¹, molecular weight distribution (M_w/M_n) = 1.16) was synthesized via RAFT polymerization using 4-cyanopentanoic acid dithiobenzoate as a CTA. The CTA, α-methyltrithiocarbonate-S-phenylacetic acid (MTPA), was synthesized according to the literature.⁴⁶ 4,4′-Azobis(4-cyanovaleric acid) dihydrochloride (V-501, 95%), N,N′-dicyclohexylcarbodiimide (DCC, 95%), imidazole (98%), potassium cyanate (KOCN, 95%), fullerene-C₇₀ (C₇₀, 99%), and doxorubicin (DOX) hydrochloride were purchased from Wako Pure Chemical (Osaka, Japan). 4-Dimethylaminopyridine (DMAP, 99%), poly(ethylene glycol) monomethyl ether (PEG₄₅, M_n = 2000, DP = 45), 2-aminoethyl methacrylate hydrochloride (AEM, 90%), phosphate-buffered saline (PBS), and deuterium oxide (D₂O) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Singlet oxygen sensor green (SOSG) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Ion-exchanged water was used as pure water.

Synthesis of PEG₄₅-MTPA

PEG₄₅ (22.4 g, 11.2 mmol), MTPA (3.47 g, 13.4 mmol), and DMAP (40.0 mg, 0.327 mmol) were dissolved in dichloromethane (100 mL). A dichloromethane solution (100 mL) of DCC (5.66 g, 27.4 mmol) was added dropwise to the solution for 30 min under an Ar atmosphere. After refluxing at 40 °C for 16 h, purification was performed on a silica-gel column using chloroform as the eluent. The solvent was evaporated. The residue was further dissolved in pure water, and PEG₄₅-MTPA was obtained by a freeze-drying technique (6.48 g, 25.1%). According to ¹H NMR, the number-average degree of polymerization (DP(NMR)) and number-average molecular weight (M_n(NMR)) determined from ¹H NMR were 45 and 2.24 × 10³ g mol⁻¹, respectively (Fig. S1a, ESI†). According to gel-permeation chromatography (GPC) using tetrahydrofuran (THF) as an eluent, the number-average molecular weight M_n(GPC) and molecular weight distribution (M_w/M_n) were 4.40 × 10³ g mol⁻¹ and 1.03, respectively (Fig. S2a, ESI†).

Synthesis of PEG₄₅-b-PAEM₁₀₁-b-PMPC₉₉ (EAM)

The triblock copolymer (EAM; PEG₄₅-b-PAEM₁₀₁-b-PMPC₉₉) synthesis using PEG₄₅-MTPA as a macro CTA was performed in one pot as follows: AEM (2.09 g, 12.6 mmol), PEG₄₅-MTPA (274 mg, 0.121 mmol), and V-501 (13.9 mg, 0.0496 mmol) were dissolved in imidazole buffer ([imidazole] = 1.00 M, pH = 6.00, 12.1 mL) with a molar ratio of [AEM]/[PEG₄₅-MTPA]/[V-501] = 104/1/0.4. The solution was heated at 70 °C for 6 h under an Ar atmosphere. The conversion (PEG₄₅-b-PAEM₁₀₁) after polymerization obtained using ¹H NMR was 97.5%. DP(NMR) and M_n(NMR) obtained using ¹H NMR were 101 and 1.90 × 10⁴ g mol⁻¹, respectively (Fig. S1b, ESI†). The M_n(GPC) and M_w/M_n determined by GPC measurement using acetate buffer as an eluent were 4.69 × 10⁴ g mol⁻¹ and 1.25, respectively (Fig. S2b, ESI†).

MPC (3.59 g, 12.2 mmol) and V-501 (14.1 mg, 0.0503 mmol) were dissolved in imidazole buffer (12.1 mL). The MPC solution was degassed with Ar and was added to the polymerization solution of PEG₄₅-b-PAEM₁₀₁ with a molar ratio of [MPC]/[PEG₄₅-b-PAEM₁₀₁]/[V-501] = 100/1/0.416. The solution was heated at 70 °C for 16 h under an Ar atmosphere. The polymer conversion after polymerization (PEG₄₅-b-PAEM₁₀₁-b-PMPC₉₉) obtained from ¹H NMR was 100%. Dialysis was performed overnight against pure water, and PEG₄₅-b-PAEM₁₀₁-b-PMPC₉₉ (EAM) was obtained via freeze-drying (5.51 g, 92.6%). DP(NMR) and M_n(NMR) obtained from ¹H NMR were 99 and 4.80 × 10⁴ g mol⁻¹, respectively (Fig. S1c, ESI†). The M_n(GPC) and M_w/M_n determined using GPC were 5.29 × 10⁴ g mol⁻¹ and 1.61, respectively (Fig. S2b, ESI†).

Synthesis of PEG₄₅-b-PUEM₁₀₁-b-PMPC₉₉ (EUM)

Ureido groups were introduced in the pendant amino groups in the PAEM block in EAM as follows: EAM (4.00 g, 0.0828 mmol) and KOCN (808 mg, 9.94 mmol) were dissolved in imidazole buffer (16.0 mL) with molar ratio of [EAM]/[KOCN] = 1/120. The solution was heated at 50 °C for 24 h. PEG₄₅-b-PUEM₁₀₁-b-PMPC₉₉ (EUM) was obtained via freeze-drying (3.30 g, 70.6%) after dialysis against pure water for three days. The conversion from the amine to ureido groups estimated using ¹H NMR was 100% (Fig. S3, ESI†). The M_n(GPC) and M_w/M_n obtained using GPC were 8.91 × 10⁴ g mol⁻¹ and 1.64, respectively (Fig. S2b, ESI†).

Preparation of the C₇₀/EUM Complex

C₇₀ (6.0 mg, 7.14 × 10⁻³ mmol) and EUM (20 mg, 4.14 × 10⁻⁴ mmol, M_n(theo) = 4.83 × 10⁴ g mol⁻¹, M_w/M_n = 1.64) powders were ground in a mortar for 30 min. PBS (10 mL) was further added to adjust the polymer concentration (C_p) = 2 g L⁻¹ to synthesize a water-soluble complex (C₇₀/EUM). Undissolved C₇₀ was eliminated by centrifugation at 6000 rpm for 30 min and then filtered through a 0.2 μm pore-sized filter. The C₇₀/PMPC complex, which comprises C₇₀ and PMPC, was synthesized in the same way as the C₇₀/EUM complex for comparison.

Measurements

¹H NMR spectra were obtained using a Bruker (Massachusetts, United States) DRX-500 and JEOL (Tokyo, Japan) ECZ400. GPC measurements using THF as the eluent were performed using a Shodex (Tokyo, Japan) DS-4 pump and a Shodex (Tokyo, Japan) RI-101 refractive index (RI) detector equipped with a Shodex (Tokyo, Japan) 10.0 μm bead size GF-7M column (exclusion limit ∼ 10⁷) working at 40 °C, with a flow rate of 1.0 mL min⁻¹. The M_n(GPC) and M_w/M_n were calibrated using standard poly(styrene) samples. GPC measurements using acetate buffer as the eluent were performed using a JASCO (Tokyo, Japan) PU-8020 pump and a JASCO (Tokyo, Japan) RI-2031 Plus RI detector equipped with a Shodex (Tokyo, Japan) 10.0 μm bead size Ohpak SB-804 HQ column (exclusion limit of approximately 10⁷) working at 40 °C, with a flow rate of 0.6 mL min⁻¹. A 0.5 M acetic acid aqueous solution containing 0.3 M sodium sulfate was used as the eluent. The M_n(GPC) and M_w/M_n were calibrated using standard poly(2-vinylpyridine) samples. Ultraviolet-visible (UV-vis) absorption spectra and percent transmittance (%T) at a wavelength of 700 nm were measured at varying temperatures using a JASCO (Tokyo, Japan) V-630BIO UV-vis spectrophotometer. Dynamic light scattering (DLS) measurements were performed with a Malvern (Worcestershire, UK) Zetasizer Nano ZS using a He–Ne laser (4 mW) with a wavelength of 632.8 nm as the light source. The hydrodynamic radius (R_h) and light scattering intensity (LSI) were obtained. Static light scattering (SLS) measurement was performed using an Otsuka Electronics Photal (Osaka, Japan) DLS-7000 HL light scattering spectrometer. A He–Ne laser (632.8 nm, 10.0 mW) was used as the light source. The weight-average molecular weight (M_w(SLS)) and radius of gyration (R_g) were estimated from the Zimm plots. The RI increment (dn/dC_p) values at 633 nm were determined using an Otsuka Electronics Photal (Osaka, Japan) DRM-3000 differential refractometer. Transmission electron microscopy (TEM) observations were performed using a JEOL (Tokyo, Japan) JEM-2100 microscope at an acceleration voltage of 160 kV. The TEM sample was prepared by placing one drop of aqueous solution on a copper grid coated with Formvar thin films. Excess water was blotted using filter paper. The samples were stained with sodium phosphotungstate and dried under vacuum conditions. Fluorescence measurement was performed using a Hitachi F-2500 (Tokyo, Japan). Visible light at 590 nm was irradiated using OptCode (Tokyo, Japan) EX-590 with a power consumption of 3 W.

Characterization of C₇₀/EUM

The complex solution was diluted 100-fold and the UV-vis absorption spectrum was measured at 25 °C. The complex solution's absorbance before dilution was calculated by multiplying the absorbance of the complex solution after dilution by 100. The concentration of C₇₀ ([C₇₀]_s) was calculated from its molar extinction coefficient in water at 384 nm (40.4 L g⁻¹ cm⁻¹)²⁸ and the absorbance.

Encapsulation and controlled release of DOX

DOX encapsulation in C₇₀/EUM was confirmed via fluorescence measurements. DOX (0.02 g L⁻¹) was dissolved in a C₇₀/EUM (C_p = 2 g L⁻¹, [C₇₀]_s = 0.313 g L⁻¹) PBS solution and stirred at 25 °C for 3 days to synthesize DOX-encapsulating C₇₀/EUM (DOX@C₇₀/EUM). Dialysis was performed against a substantial excess of PBS at 25 °C for 3 days with a polycarbonate dialysis membrane (Harvard MFP, Massachusetts, USA) with a pore size of 10 nm to remove the DOX that could not be encapsulated. It was confirmed that the free DOX in the C₇₀/EUM solution was eliminated by comparing the fluorescence intensity inside the dialysis membrane of the DOX@C₇₀/EUM solution with the DOX-only blank solution. The concentration of DOX was calculated from the fluorescence intensity to estimate the amount of DOX in C₇₀/EUM. The release of DOX from DOX@C₇₀/EUM by heating was confirmed by fluorescence. The DOX@C₇₀/EUM ([DOX] = 0.0099 g L⁻¹) and DOX (0.01 g L⁻¹) blank solutions were dialyzed against PBS using a polycarbonate dialysis membrane at 25 °C and 50 °C. The fluorescence intensity of DOX emitted to the outside of the dialysis membrane was used to estimate the release rate.

Generation of singlet oxygen from C₇₀/EUM upon visible-light irradiation

SOSG was used to confirm ¹O₂ generation. The fluorescence intensity increases when SOSG is oxidized with ¹O₂ (Scheme S1, ESI†). The C₇₀/EUM PBS solutions at C_p = 2 g L⁻¹ and [C₇₀]_s = 0.313 g L⁻¹ were diluted 10-fold with PBS, and SOSG was added to make it 2 μM. The solution was irradiated upon 590 nm visible light using an OptCode (Tokyo, Japan) EX-590 LED light, and the fluorescence spectra of SOSG were obtained. A quartz cell with a 1 cm optical path length was used. The SOSG fluorescence was measured at an excitation wavelength of 420 nm with excitation and emission slight widths at 20 nm.

Results and discussion

Preparation of PEG₄₅-b-PAEM₁₀₁-b-PMPC₉₉ (EUM)

A macro CTA, PEG₄₅-MTPA, was synthesized via condensation reaction of mono-methoxy PEG₄₅ having an OH group at the chain end with MTPA having a carboxylic acid (Scheme S2, ESI†). DP(NMR) of PEG₄₅-MTPA estimated from the integrated intensity ratio of the peaks attributed to PEG methylene protons at 3.4 ppm and the MTPA methyl proton at 2.7 ppm was 45 (Fig. S1a, ESI†). AEM and MPC were continuously polymerized in one pot via RAFT polymerization using PEG₄₅-MTPA. The PAEM block's DP(NMR) estimated from the integrated intensity ratio of the peaks attributed to PEG methylene proton at 3.6 ppm to the pendant methylene protons in the PAEM block at 3.3 ppm was 101 (Fig. S1b, ESI†). The PMPC block's DP(NMR) estimated from the integrated intensity ratio of the peaks of the pendant methylene protons in the PAEM block at 3.3 ppm to the pendant methyl protons in the PMPC block at 3.1 ppm was 99 (Fig. S1c, ESI†). GPC measurements were performed for PEG₄₅-MTPA using THF as the eluent (Fig. S2a, ESI†), and for PEG₄₅-b-PAEM₁₀₁, EAM, and EUM using aqueous acetic acid solution (Fig. S2b, ESI†). M_w/M_n of PEG₄₅-MTPA, PEG₄₅-b-PAEM₁₀₁, EAM, and EUM were 1.03, 1.25, 1.61, and 1.64, respectively. Theoretical DP (DP(theo)) and number-average molecular weight (M_n(theo)) values were calculated from the following equations

M_n(theo) = DP(theo) × M_m + M_CTA (2)

where x is the monomer's percent conversion, [M]₀ and [CTA]₀ are the initial concentrations of the monomer and CTA, respectively, and M_m and M_CTA are the molecular weights of the monomer and CTA, respectively. Table 1 summarizes the results.

Table 1 Molecular characteristics of the synthesized polymers

Polymers	M n (theoretical) × 10^−4 (g mol^−1)	DP (theoretical)	M n (NMR) × 10^−4 (g mol^−1)	DP (NMR)	M n (GPC) × 10^−4 (g mol^−1)	M w/Mn
a DP of PEG. b DP of PAEM. c DP of PMPC. d DP of PUEM. e The signals were overlapped.
PEG45-MTPA	—	—	0.224	45^a	0.44	1.03
PEG45-b-PAEM101	1.90	101^b	1.90	101^b	4.69	1.25
PEG45-b-PAEM101-b-PMPC99	4.82	100^c	4.80	99^c	5.29	1.61
EUM	5.24	101^d	—^e	—^e	8.91	1.64

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The ¹H NMR spectra for EAM and EUM were measured in D₂O at 25 °C and 80 °C (Fig. S3, ESI†). The peak of pendant methylene protons adjacent to the primary amine in the PAEM block at 3.3 ppm (g) disappeared in the ¹H NMR for EUM. This suggests that the pendant primary amine groups in the PAEM block were replaced with 100% ureido groups in the PUEM block. Although the peak intensity of pendant methylene protons adjacent to the ureido in the PUEM block in EUM at 3.4 ppm (i) was barely observed at 25 °C (Fig. S3b, ESI†), the peak intensity at 3.4 ppm (i) increased at 80 °C (Fig. S3c, ESI†). The NMR peak becomes broad when the proton mobility decreases due to a decrease in the spin–spin relaxation time.⁴⁷ Therefore, this result suggests that the PUEM block's motion in EUM was restricted in D₂O at 25 °C.

The triblock copolymer, EUM, which contains the thermo-responsive PUEM block bearing pendant ureido groups exhibits UCST behavior in water. The polymer concentration (C_p) dependence on the thermo-responsive behavior of EUM was investigated. EUM was dissolved in PBS at various C_p values. The percent transmittance (%T) at 700 nm was measured as a function of temperature at the cooling process (Fig. 2a). The phase transition temperatures (T_p) at C_p = 1.0, 2.0, and 5.0 g L⁻¹ were 43 °C, 50 °C, and 65 °C, respectively. T_p increased linearly with C_p (Fig. 2b). This phenomenon suggests that the frequency of collisions between polymer chains increased with increasing C_p to easily form hydrogen bonds between the pendant ureido groups in the PUEM blocks.

Fig. 2 (a) Percent transmittance (%T) of EUM in PBS at various C_p = 1.0 (), 2.0 (), and 5.0 g L⁻¹ () as a function of temperature with the cooling process, and (b) phase transition temperature (T_p) of EUM in PBS as a function of C_p.

Preparation of C₇₀/EUM

The maximum C₇₀ concentration ([C₇₀]_s) to be solubilized by the formation of a water-soluble C₇₀/EUM complex was determined. EUM and various amounts of C₇₀ ([C₇₀]_f) powders were ground and mixed in a mortar for 30 min. PBS was added to the mixture to synthesize C₇₀/EUM aqueous solution with C_p = 2.0 g L⁻¹. A water-soluble C₇₀/PMPC complex was prepared using C₇₀ and PMPC powders by the same method for comparison. From the UV-vis absorption spectra of C₇₀/EUM (Fig. 3) and C₇₀/PMPC (Fig. S4, ESI†), maximum absorptions are observed at 250 and 384 nm for C₇₀, suggesting that C₇₀ could be solubilized in PBS. The absorbance of C₇₀ increased for C₇₀/EUM and C₇₀/PMPC with an increase in [C₇₀]_f. The [C₇₀]_s values were calculated using the molar extinction coefficient of C₇₀ in water at 384 nm (40.4 L g⁻¹ cm⁻¹)²⁸ and the absorbance of the complex aqueous solution. The maximum [C₇₀]_s values using EUM and PMPC were 0.31 g L⁻¹ (Fig. 3b) and 0.83 g L⁻¹ (Fig. S4b, ESI†), respectively. The maximum EUM [C₇₀]_s was lower than that of PMPC at C_p = 2 g L⁻¹ because the EUM's PEG and PUEM blocks cannot contribute to solubilizing C₇₀. C₇₀ powder was mixed with PEG₄₅ or PUEM₁₀₀ powders at [C₇₀]_f = 0.10 g L⁻¹; however, C₇₀ could not be solubilized in PBS (Fig. S5, ESI†). This observation suggests that the C₇₀/EUM core was formed from the C₇₀ and PMPC block. The maximum [C₇₀]_s (0.313 g L⁻¹) obtained using EUM with C_p = 2 g L⁻¹ was higher than that ([C₇₀]_s = 0.2 g L⁻¹) obtained using PVP (MW = 4.00 × 10⁴ g mol⁻¹) with C_p = 50 g L⁻¹.²⁸ All subsequent experiments were conducted using C₇₀/EUM with [C₇₀]_s = 0.313 g L⁻¹.

Fig. 3 (a) UV-vis absorption spectra of C₇₀/EUM at C_p = 2 g L⁻¹ and (b) the amount of solubilized C₇₀ in PBS ([C₇₀]_s) as a function of the amount of C₇₀ ([C₇₀]_f) used.

Characterization of C₇₀/EUM

¹H NMR spectra for C₇₀/EUM were obtained in D₂O at 25 °C and 80 °C (Fig. 4). Although the NMR signal of the pendant methylene protons adjacent to the ureido group in the PUEM block was observed at 3.4 ppm at 80 °C, the signal disappeared at 25 °C. This observation suggests that 80 °C was higher than the phase transition temperature (T_p) of the PUEM block in C₇₀/EUM. Alternatively, the NMR signal for the PUEM shells in C₇₀/EUM became broad at 25 °C due to the decreasing mobility caused by the hydrophobic interactions of the PUEM shells below T_p.

Fig. 4 ¹H NMR spectra for C₇₀/EUM in D₂O at (a) 25 °C and (b) at 80 °C.

SLS measurements were performed in PBS to characterize the association state of the complex (Fig. S6 and Table 2, ESI†). The theoretical expanded polymer chain lengths calculated from the DP values of EUM (245) and PMPC (100) were 61.3 and 25.0 nm, respectively, based on the C–C bond chain length. The R_g values of C₇₀/EUM and C₇₀/PMPC were 58.4 and 40.0 nm, respectively. The R_g value of C₇₀/EUM was similar to the theoretical expanded polymer chain length of EUM. This result suggests that C₇₀/EUM formed the core–shell-corona micelle structure without intermicellar aggregation. The R_g value of C₇₀/PMPC was larger than the theoretical chain length of 25.0 nm. This suggests that C₇₀/PMPC cannot form a clear core–shell structure. The aggregate's shape is a rigid sphere when the R_g/R_h value is 0.8, a random coil when it is 1, and a rod when it is larger than 2.⁴⁸ The R_g/R_h values of C₇₀/EUM and C₇₀/PMPC were 1.60 and 0.885, respectively. Therefore, C₇₀/EUM's shape was almost spherical, and C₇₀/PMPC formed aggregates similar to a rigid sphere. The inside of the C₇₀/PMPC complex was tightly packed because it does not have the shell and corona layers. Therefore, the R_g/R_h value of C₇₀/PMPC was similar to that of a rigid sphere.

Table 2 DLS and SLS data for the C₇₀/EUM and C₇₀/PMPC complexes in PBS at 25 °C

Samples	r	M w(SLS)^b × 10^−6 (g mol^−1)	R g (nm)	R h (nm)	R g/Rh	d (g cm^−3)	N agg	N C70	dn/dCp^h (mL g^−1)
a Molar ratio of C70 and polymer ([C70]/[polymer]). b Weight-average molecular weight estimated from SLS. c Radius of gyration estimated from SLS. d Hydrodynamic radius estimated from DLS. e Density of complex calculated from eqn (4). f Aggregation number of the polymer chains calculated from eqn (3). g Number of C70 in one complex calculated from r and Nagg. h Refractive index increment.
C70/EUM	9.8	6.67	58.4	36.3	1.60	0.0133	70.9	691	0.068
C70/PMPC	15	2.32	40.0	45.2	0.885	0.0144	46.7	686	0.780

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The aggregation number (N_agg), which is the number of polymer chains forming one aggregate, can be calculated from the following equation

where M_wpoly and M_wC70 are the weight-average molecular weights of the polymer and C₇₀, respectively, and r is the molar ratio of C₇₀ to the polymer. N_agg of C₇₀/EUM was calculated to be 70.9. The number of C₇₀ (N_C70) contained in one C₇₀/EUM complex was calculated to be 691 from r and N_agg. A single EUM polymer chain solubilized about 10 C₇₀ molecules based on the N_agg and N_C70 of C₇₀/EUM. Similarly, a single PMPC chain can solubilize 15 C₇₀ molecules in the case of C₇₀/PMPC. PMPC can solubilize a greater amount of C₇₀ than EUM in water because the PEG and PUEM blocks in EUM cannot contribute to C₇₀ solubilization. The density (d) of the complex was calculated from the following formulawhere N_A represents Avogadro's number. The d values of C₇₀/EUM and C₇₀/PMPC were 0.0133 and 0.0144 g/cm³, respectively. The d value of C₇₀/EUM was lower than that of C₇₀/PMPC, because C₇₀/EUM contains the PUEM shell and PEG corona layers, which are well-hydrated.

TEM observation confirmed the spherical shape of the C₇₀/EUM complex (Fig. 5). The average R_TEM obtained from TEM was 33.5 nm, which was lower than the R_h = 36.3 nm calculated by DLS at 25 °C due to shrinkage of the TEM sample during the drying process.

Fig. 5 TEM image of C₇₀/EUM.

Thermo responsive behavior of C₇₀/EUM

R _h and LSI were investigated as a function of temperature to confirm the swelling of C₇₀/EUM in PBS due to the PUEM shell's UCST behavior upon heating (Fig. 6). T_p was defined as the temperature at which R_h became constant during a heating process. Below 36 °C, R_h increased with temperature. However, R_h became constant above 36 °C at about 38 nm. Therefore, C₇₀/EUM's T_p was determined to be 36 °C. The T_p of EUM without C₇₀ was 50 °C (Fig. 2); however, the T_p of C₇₀/EUM decreased to 36 °C. The PUEM block in C₇₀/EUM is anchored to the hydrophobic core containing C₇₀. The T_p of C₇₀/EUM was decreased because the PUEM polymer chain was affected by the core's hydrophobicity. LSI is sensitive to the formation of large aggregates due to intermicellar aggregations. No intermicellar aggregation of C₇₀/EUM occurred because the LSI was constant regardless of temperature. The R_h values were repeatedly measured at 25 °C and 70 °C to confirm the reversibility of swelling and shrinking of C₇₀/EUM by temperature (Fig. S7, ESI†). The R_h values at 25 °C and 70 °C were ca. 36.5 and ca. 38.2 nm, respectively, for five temperature cycles. Thus, the C₇₀/EUM complex phase transition is reversible.

Fig. 6 Hydrodynamic radius (R_h, ) and light scattering intensity (LSI, ) of C₇₀/EUM at C_p = 2 g L⁻¹ and [C₇₀]_s = 0.313 g L⁻¹ in PBS as a function of temperature.

Generation of singlet oxygen from C₇₀/EUM upon visible-light irradiation

The C₇₀/EUM solution with C_p = 2 g L⁻¹ and [C₇₀]_s = 0.313 g L⁻¹ was diluted 10-fold with PBS, and SOSG was added to make it 2 μM. The solution was irradiated with light at 590 nm and SOSG's fluorescence spectra were measured for the irradiation time, t (Fig. S8, ESI†). The ratios (I/I₀) of SOSG's maximum fluorescence intensity (I) at t to that before irradiation (I₀) were plotted against t (Fig. 7). SOSG is oxidized by ¹O₂ and the fluorescence intensity at 500–550 nm increased. The I/I₀ ratios were almost constant regardless of light irradiation when the light was irradiated towards SOSG PBS solution and SOSG PBS solution with EUM. Alternatively, when the SOSG PBS solution with the C₇₀/EUM complex containing C₇₀ was irradiated, I/I₀ of SOSG increased with the irradiation time. This observation indicates that C₇₀ in C₇₀/EUM's core can generate ¹O₂ under light irradiation. There is almost no error for the samples that cannot generate ¹O₂. The plots of C₇₀/EUM contains up to 1.5% error.

Fig. 7 Changes in SOSG fluorescence intensity (2 μM) with C₇₀/EUM at [C₇₀]_s = 0.03 g L⁻¹ (), without C₇₀ (), and without EUM and C₇₀ () as a function of irradiation time at 25 °C; I and I₀ were the fluorescence intensities at the maximum wavelength (525 nm) after and before 590 nm light irradiation.

Encapsulation and controlled release of doxorubicin (DOX)

The following method was used to encapsulate a guest molecule, DOX, in C₇₀/EUM: C₇₀/EUM and DOX were stirred in PBS for 3 days at room temperature, and any DOX that could not be encapsulated was removed by dialysis. The removal of free DOX from DOX@C₇₀/EUM solution was confirmed by comparing the fluorescence intensity inside the dialysis membrane of the DOX@C₇₀/EUM solution with that of the DOX-only solution (Fig. S9, ESI†). The fluorescence intensity of DOX@C₇₀/EUM was almost constant after 24 h dialysis, whereas that of the DOX-only solution decreased. These results show that DOX can be encapsulated in C₇₀/EUM. From eqn (5) and (6), DOX encapsulation efficiency (EE) and DOX encapsulation content (EC) were estimated after 72 h dialysis. EE is the ratio of the encapsulated DOX concentration ([DOX]) to the used DOX concentration ([DOX]₀). [DOX] can be estimated from the fluorescence intensity. EC is the ratio of [DOX] to C_p. EE(%) and EC(%) were calculated using the following equations:EE(%) and EC(%) for DOX@C₇₀/EUM were 49.5% and 0.495%, respectively.

The controlled release of DOX from DOX@C₇₀/EUM with increasing temperature was evaluated. The DOX@C₇₀/EUM solutions were dialyzed against PBS at 25 °C and 50 °C. The dialysis membrane with a 10 nm pore size was used. DOX can permeate the membrane, whereas large-sized C₇₀/EUM cannot. DOX fluorescence intensity released to the outside of the membrane was investigated with respect to the dialysis time (Fig. 8a). The DOX-only solution was studied using the same method for comparison. DOX release amounts at 50 °C and 25 °C after 200 min were 90% and 70%, respectively, for the DOX-only solution. The significant increase at 50 °C was attributed to an increase in DOX thermal motion due to high temperature. The amounts of DOX released from DOX@C₇₀/EUM at 50 °C and 25 °C after 200 min were 20% and 5.0%, respectively. The plots contain up to 1.2% error. At 50 °C the release amount of DOX@C₇₀/EUM solution was lower than that of the DOX-only solution. This suggests that not only is DOX encapsulated in the PUEM shell in C₇₀/EUM due to hydrophobic interactions but it also interacts with the hydrophobic core containing hydrophobic C₇₀. Assuming that DOX release follows the first-order mechanism,^49,50 the release rate constant (k) can be calculated from the following equation:

ln((I₀ − I)/I₀) = −kt (7)

where I₀ indicates the initial fluorescence intensity of DOX before dialysis, I indicates the fluorescence intensity outside of the membrane at t, and t is the dialysis time. The slope of the plots in Fig. 8b shows that k can be calculated. The k values for the comparative DOX-only solution at 25 °C and 50 °C were 0.264 s⁻¹ and 0.481 s⁻¹, respectively. Therefore, the release rate increased by 1.82 times at 50 °C compared to 25 °C due to the increase in DOX thermal motion. The k values for the DOX@C₇₀/EUM solution at 25 °C and 50 °C were 0.0180 s⁻¹ and 0.0792 s⁻¹, respectively. The rate of release rose by 4.40 times when the temperature was raised to 50 °C from 25 °C. The increase in k due to heating in the presence of C₇₀/EUM was larger than in the absence of C₇₀/EUM. This suggests that DOX release could be controlled by increasing the temperature rather than just a simple change in thermal motion. The affinity between DOX and the PUEM block of C₇₀/EUM was changed drastically at 25 °C and 50 °C due to the UCST behavior of the PUEM block. At 50 °C the affinity of DOX with the PUEM block was low. Therefore, DOX can be released from C₇₀/EUM at 50 °C.

Fig. 8 (a) DOX release from DOX@C₇₀/EUM at 25 °C () and 50 °C () and a blank solution without C₇₀/EUM at 25 °C () and 50 °C () in PBS. (b) −ln((I₀ − I)/I₀) was plotted against dialysis time; I is the fluorescence intensity and I₀ is the initial intensity.

The effect of DOX encapsulation and release on C₇₀/EUM size was evaluated using DLS (Fig. S10, ESI†). The R_h value of C₇₀/EUM without DOX was 36.3 nm. The R_h value of DOX@C₇₀/EUM was 39.8 nm. The R_h value of C₇₀/EUM decreased to 32.7 nm after DOX release. The LSIs of DOX@C₇₀/EUM before and after the release of DOX were 2160 and 1830 kcps, respectively. From these results, the influence of DOX encapsulation and release on the size and structure of the complex was minimal.

Conclusions

A thermo-responsive triblock copolymer, EUM, was synthesized via controlled radical polymerization. EUM can dissolve C₇₀ in PBS to form a C₇₀/EUM complex. The maximum solubilized C₇₀ concentration in PBS by EUM was 0.313 g L⁻¹, which was superior to a previously reported solubilization method using PVP. The T_p value of C₇₀/EUM in PBS was 36 °C according to DLS measurements. C₇₀/EUM can generate ¹O₂ by visible-light irradiation at 590 nm in PBS. C₇₀/EUM can encapsulate the anticancer agent, DOX, at 25 °C by hydrophobic interactions between the PUEM block in EUM and DOX. The encapsulated DOX can be released when heated. C₇₀/EUM may be expected to function as both a thermo-responsive drug carrier and a photosensitizer for PDT.

Author contributions

Conceptualization, S. Y.; data curation, K. K. and S. Y.; formal analysis, K. K. and S. Y.; investigation, K. K., K. I., and S. Y.; methodology, K. I. and S. Y.; project administration, S. Y.; supervision, S. Y.; visualization, K. K.; writing – original draft, K. K. and S. Y.; writing – review and editing, K. K. K. I. and S. Y. All authors have read and agreed to the published version of the manuscript.

Conflicts of interest

There are no conflicts of interest to declare.

Acknowledgements

This research was supported by KAKENHI grants (21H02005, 21K19931, 21H05027, 21H05535) from the Japan Society for the Promotion of Science (JSPS), JSPS Bilateral Joint Research Projects (JPJSBP120203509). and the Cooperative Research Program of “Network Joint Research Center for Materials and Devices (20214044).” The author would like to thank Enago for the English language editing.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/d1tb02183d

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