Effective magnetic hyperthermia induced by mitochondria‐targeted nanoparticles modified with triphenylphosphonium‐containing phospholipid polymers

Abstract

Magnetic hyperthermia (MHT) is a promising cancer treatment because tumor tissue can be specifically damaged by utilizing the heat generated by nano‐heaters such as magnetite nanoparticles (MNPs) under an alternating magnetic field. MNPs are taken up by cancer cells, enabling intracellular MHT. Subcellular localization of MNPs can affect the efficiency of intracellular MHT. In this study, we attempted to improve the therapeutic efficacy of MHT by using mitochondria‐targeting MNPs. Mitochondria‐targeting MNPs were prepared by the modification of carboxyl phospholipid polymers containing triphenylphosphonium (TPP) moieties that accumulate in mitochondria. The mitochondrial localization of polymer‐modified MNPs was supported by transmission electron microscopy observations of murine colon cancer CT26 cells treated with polymer‐modified MNPs. In vitro and in vivo MHT using polymer‐modified MNPs revealed that the therapeutic effects were enhanced by introducing TPP. Our results indicate the validity of mitochondria targeting in enhancing the therapeutic outcome of MHT. These findings will pave the way for developing a new strategy for the surface design of MNPs and therapeutic strategies for MHT.

Mitochondria‐targeting magnetite nanoparticles for hyperthermia were prepared by modification of triphenylphosphonium‐containing phospholipid polymers. The therapeutic effects of magnetic hyperthermia were enhanced by mitochondrial targeting in vitro and in vivo in murine colon cancer.

Abbreviations

AMF

alternating magnetic field

ARGET ATRP

activators regenerated by electron transfer atom transfer radical polymerization

DLS

dynamic light scattering

EBiB

2‐bromoisobutyrate

FBS

fetal bovine serum

FT‐IR

Fourier transform infrared spectroscopy

MES

2‐methacryloyloxyethyl succinate

MHT

magnetic hyperthermia

MNPs

magnetite nanoparticles

MPC

2‐methacryloyloxyethyl phosphorylcholine

NIR

near‐infrared

PME

poly(MPC‐co‐MES)

PMET

poly(MPC‐co‐MES‐co‐VTPP)

PTT

photothermal therapy

TEM

transmission electron microscopy

TGA

thermogravimetric analysis

TPMA

tris[(2‐pyridyl)methyl]amine

VTPP

4‐vinylbenzyl(triphenyl)phosphonium chloride

1. INTRODUCTION

Hyperthermia is a cancer therapy that induces cell death by heating tumor tissue above 42.5°C. Tumor tissues are equipped with immature blood vessels, making it difficult to dissipate heat, and thus are more vulnerable to heat stress compared to normal tissues. The local heating of tumors can be achieved using nanoparticles supplied to the tumor tissues. Metal nanoparticles that exhibit magnetism or surface plasmon resonance can generate heat on irradiation with an alternating magnetic field (AMF) for magnetic hyperthermia (MHT) or near‐infrared (NIR) laser for photothermal therapy (PTT), respectively. ¹ , ² , ³ Nanoparticle‐mediated hyperthermia enables the specific heating of tumors and is expected to be a promising cancer treatment with minimal side effects.

Compared with PTT, MHT is applicable to deep‐seated tumors owing to the excellent tissue permeability of AMF. Among the diverse magnetic nanoparticles that generate heat upon AMF exposure, Fe₃O₄ magnetite nanoparticles (MNPs) are considered one of the most promising owing to their high stability and low toxicity. ⁴ , ⁵ , ⁶ MNPs can be taken up by cancer cells, and we previously developed intracellular hyperthermia using MNPs. ⁷ Intratumorally administered functional MNPs generate heat upon exposure to AMF, owing to Brownian and Néel relaxation ⁸ or hysteresis losses. ⁹ The therapeutic effects of MHT on cancer have been previously examined, and complete tumor regression has been observed in animal models with several types of tumors, including mouse mammary carcinoma, ¹⁰ mouse melanoma, ¹¹ and human prostate cancer in nude mice. ¹² Based on the results of a preclinical study, four patients with advanced metastatic melanoma in stages III and IV were enrolled in preliminary clinical trials ¹³ in which MNPs were injected directly into metastatic lymph nodes. Then, the patients were irradiated with AMF at 43°C for 30 min. While further clinical studies are needed to evaluate the therapeutic effects, novel strategies to enhance the antitumor effects of MHT are required to improve clinical outcomes.

Herein, we focus on the subcellular localization of nanoparticles. Controlling the subcellular localization of nanoparticles and heating thermally sensitive regions within cancer cells may improve the effects of intracellular hyperthermia. Mitochondria are susceptible to temperature elevation, and several studies have shown that mitochondria‐targeted hyperthermia improves the therapeutic effect of PTT with NIR. ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ , ¹⁸ , ¹⁹ , ²⁰ , ²¹ , ²² Thus, mitochondria‐targeting hyperthermia has gained increasing attention as a promising approach. Nonetheless, few studies have reported on the preparation of MNPs targeting mitochondria for MHT ²³ , ²⁴ and the therapeutic potency of solely mitochondria‐targeting MHT has yet to be elucidated.

The modification of biocompatible polymers has been investigated for the in vivo application of MNPs. For example, dextran, ²⁵ polyethylene glycol, ²⁶ and polyglycerol ²⁷ have been used to prepare biocompatible magnetic nanoparticles. In particular, 2‐methacryloyloxyethyl phosphorylcholine (MPC) polymers, which mimic the structure of phosphorylcholine that composes phospholipid bilayers, has been used to impart biocompatibility to various medical devices because of its high hydrophilicity and excellent biocompatibility. ²⁸ Modifying magnetic nanoparticles with MPC polymers improves dispersion stability, inhibits nonspecific adsorption of proteins, and reduces cytotoxicity. ²⁹ , ³⁰ , ³¹ , ³² , ³³ , ³⁴ , ³⁵ Despite the promising potential of MPC polymers, to the best of our knowledge there have been no reports on the application of magnetic nanoparticles modified with MPC polymers for MHT. Figure 1 shows a schematic of the present study. For the surface modification of nanoparticles, we used MPC, 2‐methacryloyloxyethyl succinate (MES), and 4‐vinylbenzyl(triphenyl)phosphonium chloride (VTPP) as monomers and synthesized poly(MPC‐co‐MES‐co‐VTPP) (PMET; Figure 1A) using activators regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP). The MPC units provide nanoparticles with dispersion stability in an aqueous solution and biocompatibility, whereas the MES units bind to the surface of MNPs via a carboxyl group. The VTPP units endow mitochondria‐targeting ability due to their lipophilic and cationic properties. ³⁶ Mitochondria‐targeting MNPs were prepared by the modification of PMET. Polymer‐modified MNPs were administered into a subcutaneously established tumor of CT26 mouse colon cancer and the therapeutic effect of MHT was evaluated. In this study, we developed mitochondria‐targeting MNPs and investigated their therapeutic efficiency (Figure 1B).

FIGURE 1.

(A) Chemical structure of carboxyl phospholipid polymer poly(MPC‐co‐MES‐co‐VTPP) (PMET). (B) Synthetic schemes of the polymer‐modified magnetite nanoparticles. (C) Schematic illustration of magnetic hyperthermia using mitochondria‐targeting magnetite nanoparticles. MES, 2‐methacryloyloxyethyl succinate; MPC, 2‐methacryloyloxyethyl phosphorylcholine; VTPP, 4‐vinylbenzyl(triphenyl)phosphonium chloride.

2. MATERIALS AND METHODS

2.1. Materials

MPC was purchased from NOF Corporation. MES, VTPP, CuBr₂, tris[(2‐pyridyl)methyl]amine (TPMA), and ethyl 2‐bromoisobutyrate (EBiB) were purchased from Sigma‐Aldrich. Ascorbic acid, FeCl₂·4H₂O, and FeCl₃·6H₂O were purchased from FUJIFILM Wako Pure Chemicals. All other reagents and solvents used in this study were commercially available reagents of ultrapure grade and were used without further purification.

2.2. Synthesis of PME and PMET

Poly(MPC‐co‐MES) (PME) and PMET were synthesized by ARGET ATRP using MPC, MES, and VTPP as monomers, CuBr₂ as the catalyst, TPMA as the ligand, ascorbic acid as the reducing agent, and EBiB as the initiator (see also Table S1 for the monomer feed ratio). The total concentration of the monomers ([monomer]) was set to 0.5 M. The ratio of ATRP reagents was determined to be [monomer]:[EBiB]:[ascorbic acid]:[TPMA]:[CuBr₂] = 100:1:1:0.1:0.01. The synthetic procedure for PMET was as follows. To a glass test‐tube, 1.92 g (6.5 mmol) of MPC, 0.691 g (3.0 mmol) of MES, and 0.207 g (0.50 mmol) of VTPP were added and dissolved in 20 mL of a mixed solvent of methanol/deionized water = 2/1 (v/v). To the monomer solution, 17.6 mg (0.10 mmol) of ascorbic acid, 2.94 mg (10 μmol) of TPMA, and 0.223 mg (1.0 μmol) of CuBr₂ were added, followed by purging with Ar gas for 15 min. Thereafter, 14.7 μL (0.10 mmol) of EBiB was added and the tube was shielded. Polymerization was carried out for 48 h at 40°C. For the synthesis of PME, polymerization was performed for 24 h. The reaction was terminated by exposing the polymer solution to air. The polymerization solution was re‐precipitated in a mixture of diethyl ether/chloroform = 9/1 (v/v). The resulting precipitate was dissolved in deionized water and dialyzed for over 2 days using a dialysis membrane (1 kDa). The polymer solutions were lyophilized to obtain the polymer in powder form.¹H NMR measurements were used to analyze the polymer structure in a mixed solvent of C₂D₅OD/D₂O = 2/1 (v/v).

2.3. Preparation of polymer‐modified MNPs

MNPs were synthesized using a coprecipitation method. Fe²⁺ and Fe³⁺ solutions were prepared by dissolving FeCl₂·4H₂O and FeCl₃·6H₂O in deionized water. Then, 5 mL of 0.2 M FeCl₂ aqueous solution, 10 mL of 0.2 M FeCl₃ aqueous solution, and 5 mL of deionized water were added to a three‐necked flask and mechanically stirred at 300 rpm. Next, Ar purging was performed for 30 min. Thereafter, 6 mL of ammonia water (28 wt%) was added while stirring, and the solution was heated at 80°C for 1 h, resulting in the formation of MNPs. For the modification of the polymers, the polymer aqueous solution was added to the magnetite solution to achieve a molar ratio of iron ions and MES units of 1:1 and stirred at 80°C for 1 h.

Next, the solution was collected and centrifuged (5,000 g for 15 min). The resulting precipitates were washed by centrifugation (10,000 g, 60 min) twice with deionized water to obtain polymer‐coated MNP dispersions. The iron concentrations of the dispersions were measured using the potassium thiocyanate method. ³⁷ If necessary, the magnetite solution was lyophilized to prepare the MNP powder. Transmission electron microscopy (TEM) observations of MNPs were performed by JEM‐2100plus (Jeol). Thermogravimetric analysis (TGA) was performed using DTG‐60AH (Shimadzu) under a nitrogen atmosphere while temperature was increased to 900°C at 10°C/min. Fourier transform infrared spectroscopy (FT‐IR) measurements were conducted using an FT/IR‐4100 (JASCO).

2.4. Evaluation of the heating property of MNPs under AMF exposure

To evaluate the heating property, 1.0 mL of 2.0 mg/mL MNPs dispersions in deionized water was introduced into a vial. The vial was placed in the center of a coil, and AMF was applied for 5 min using an AMF generator (HI‐HEATER 5010; Dai‐ichi High Frequency). AMF was applied at 350 kHz and 8 kW. A fiber‐optic thermometer was inserted into the dispersion to track the temperature.

2.5. Cell culture

Murine colon cancer CT26 cells (American Type Culture Collection) were maintained in 100 mm tissue culture dishes and cultured in RPMI1640 (FUJIFILM Wako Pure Chemical) medium containing 10% fetal bovine serum (FBS; Funakoshi) and 1% penicillin–streptomycin solution (Fujifilm Wako Pure Chemical) under 5% CO₂ at 37°C.

2.6. Effect of the administration of MNPs on the viability of CT26 cells

CT26 cells in 100 μL of RPMI medium were seeded at 3 × 10³ cells/well in 96‐well plates and incubated at 37°C for 24 h. After preculture, 10 μL of magnetite dispersion was added to each well to obtain final concentrations of 10, 100, and 1000 μg/mL, and incubated for 24 h. Thereafter, the cells were washed twice with 100 μL of Hanks' Balanced Salt Solution (Fujifilm Wako Pure Chemical), and 100 μL of RPMI1640 medium was added. Next, 10 μL of Cell Counting Kit 8 (Dojindo) was added and incubated for 2 h. Cell viability was calculated by measuring absorbance at 450 nm using a microplate reader (iMark Microplate Absorbance Reader; Bio‐Rad Laboratories). The absorbance of cells cultured without MNPs was used as a control. The absorbance of RPMI1640 medium without cells was used as a blank.

2.7. Evaluation of the cellular uptake of MNPs

CT26 cells (6 × 10⁵ cells) in 2 mL of RPMI1640 medium were seeded in wells of six‐well plates and incubated at 37°C for 24 h. After preculture, the medium was replaced with RPMI1640 containing 1000 μg/mL MNPs. The cells were incubated for 24 h, washed twice with 1 mL of Dulbecco's phosphate buffered saline (DPBS) (Fujifilm Wako Pure Chemical), and incubated with 200 μL of trypsin for 5 min. The cells were collected and suspended in 1 mL of RPMI1640. The suspensions were centrifuged (200 g, 5 min), to which 200 μL of 12 N HCl was added. Then, the cell suspension was maintained at 4°C for 24 h. Next, trichloroacetic acid (5%, 1 mL) was added and the cells were incubated at 4°C for 30 min, resulting in the precipitation of cellular proteins. After removing the precipitates by centrifugation (10,000 g, 20 min), the supernatant was collected, and the iron concentration was evaluated using the potassium thiocyanate method. ³⁷

2.8. In vitro hyperthermia

CT26 cells (1 × 10⁶ cells) in 10 mL of RPMI1640 medium were seeded in a 100‐mm tissue culture dish and incubated at 37°C for 24 h. After preculture, the medium was replaced with RPMI1640 containing 1000 μg/mL MNPs and incubated for 24 h. The cells were collected by trypsin treatment and suspended in 10 mL of RPMI1640. The number of cells was counted using the trypan blue dye‐exclusion method. The cells were centrifuged (200 g, 5 min), washed twice with DPBS, and resuspended in DPBS. For in vitro hyperthermia, 1 mL of cell suspension was transferred to a vial and irradiated with AMF for 5 min. The vial was placed in the center of a coil, and AMF was applied for 15 min using an AMF generator (HI‐HEATER 5010; Dai‐ichi High Frequency). AMF was applied at 350 kHz and 8 kW. A fiber‐optic thermometer was inserted into the vial to track the temperature of the suspension. After AMF irradiation, the cells were centrifuged (200 g, 5 min) and suspended in 1 mL of RPMI medium. After in vitro hyperthermia, the cells were seeded in a 100‐mm tissue culture dish and cultured at 37°C for 24 h. The medium was then removed and the cells were collected by trypsin treatment and suspended in RPMI1640. The number of cells was counted using the trypan blue dye‐exclusion method.

2.9. TEM observation of CT26 cells treated with MNPs

CT26 cells at 5 × 10³ cells in 300 μL of RPMI1640 medium were seeded in a Lovetec Chamber Slide (Thermo Fisher Scientific) and incubated at 37°C for 24 h. After the preculture, the medium was replaced with RPMI1640 containing 1000 μg/mL of the MNP and incubated for 24 h. Thereafter, the cells were washed twice with DPBS and treated with deionized water containing 2.5% glutaraldehyde and 0.1 M phosphate buffer (pH 7.4) overnight. The fixed cells were dehydrated using various percentages of ethanol and propylene oxide. The dehydrated samples were passed through propylene oxide/epoxy resin (1/1 and 1/3) mixtures and embedded in an epoxy resin. The embedded samples were baked at 60°C for 72 h. Sections were prepared using an ultramicrotome (EM UC6; Leica), which were then stained with uranyl acetate and lead citrate, followed by observation using TEM (H‐7650; Hitachi).

2.10. In vivo hyperthermia

All animal experiments were approved by the Ethics Committee for Animal Experiments of the School of Engineering, Nagoya University (G220016). CT26‐tumor‐bearing mice were established via a subcutaneous injection of 100 μL of CT26 cell suspension in DPBS (3 × 10⁶ cells/mL) into the left flank of 4‐week‐old female BALB/c mice (Japan SLC). MHT was performed on days 0, 1, and 2 (day 0 was set as the day when the tumor volume exceeded 50 mm³). For MHT, 4 mg of PME‐modified magnetite nanoparticles (PME‐mag) or PMET‐modified magnetite nanoparticles (PMET‐mag) was injected intratumorally. The mice were subsequently subjected to hyperthermia treatment under general anesthesia using isoflurane inhalation solution (Mylan). The magnetic field was created using a horizontal coil (inner diameter 70 mm, length 70 mm) with a transistor inverter (118 kHz, 5 kW). The mouse was placed in the center of a coil and AMF was applied for 30 min using an AMF generator (LTG‐150‐05; Dai‐ichi High Frequency). The temperature of the tumor surface was measured for 30 min using a fiber‐optic thermometer. The AMF output was regulated to maintain the tumor surface at 43°C. Tumor volume was monitored with calipers every 3 days and was calculated using the following equation:

$$V=\frac{l\times s^2}{2}$$

where V is tumor volume, l is the long diameter, and s is the short diameter.

Mice with tumor volumes exceeding 2000 mm³ or tumor ulceration were euthanized.

3. RESULTS AND DISCUSSION

3.1. Preparation and characterization of polymer‐modified MNPs

Carboxyl phospholipid polymers containing mitochondria‐targeting VTPP units (PMET, Figure 1) were synthesized using ARGET ATRP (Table S1). PME without VTPP units was also synthesized for comparison. Based on the ¹H NMR analysis (Figure S1), the synthesized polymers were confirmed to have similar unit ratios to the in‐feed monomer ratios.

MNPs were synthesized using a co‐precipitation method. Ammonia solution (28% in water) was added to an aqueous solution containing Fe²⁺ and Fe³⁺ with stirring at 80°C, resulting in the formation of black precipitates. The dispersion was maintained at 80°C for 1 h to allow crystal growth. The surface modification with the carboxyl phospholipid polymers (PME‐mag and PMET‐mag) was performed by adding the polymers to the suspensions and stirring at 80°C for 1 h. X‐ray diffraction measurements confirmed that the obtained nanoparticles consisted of magnetite (Figure S2), ³⁸ and the modification of the polymers did not affect the essential crystal structure. Based on the TEM observations, the average size of the MNPs was approximately 10–11 nm (Figure S3).

The chemical structures of the MNP surfaces were analyzed by FT‐IR (Figure S4A). Peaks at approximately 570 cm⁻¹ were attributed to the Fe–O stretching vibrations of magnetite. In the FT‐IR spectra of the polymer‐modified MNPs, characteristic peaks were detected at approximately 970, 1070, and 1710 cm⁻¹. These peaks were attributed to the stretching vibrations of N⁺ (CH₃)₃, P‐O‐C, and C=O. These functional groups were included in the MPC units, indicating the presence of PME or PMET on the MNPs. The amount of polymer on the MNPs was analyzed by TGA. Figure S4B shows the TGA curves of naked magnetite and polymer‐modified MNPs. Weight loss on temperature increase was observed in all the samples. For the polymer‐modified MNPs, the weight losses were higher than those of the unmodified MNPs, indicative of the thermal decomposition of the polymers on the surface. The amount of polymer that existed on the surface was estimated by calculating the differences in the weight losses at 200 and 800°C (Table S2), since weight loss at 200°C could be attributed mainly to the evaporation of residual water. A comparison between PME‐mag and PMET‐mag implied that introducing VTPP units resulted in a smaller modification efficiency. Collectively, the carboxyl phospholipid polymers were successfully modified on the surface of MNPs.

According to dynamic light scattering (DLS) measurements, the average hydrodynamic sizes of the naked MNPs, PME‐mag, and PMET‐mag were 76.0, 111, and 129 nm, respectively. (Figure 2A). Considering the particle size of MNPs determined by TEM (Figure S3), MNPs were assumed to have formed aggregates composed of multiple core nanoparticles. The hydrodynamic size of the MNPs increased after polymer modification, which can be attributed to the formation of a polymer layer on the surface of the MNPs. The ζ‐potential of PMET‐mag was positively shifted (Figure 2B) compared with that of the PME‐mag, suggesting that the cationic triphenylphosphonium moieties were on the surface of the polymer layer. Figure S5 depicts a time course of photographs of the MNP dispersion in PBS and FBS. Bare MNPs precipitated in PBS in approximately 10 min, whereas PME‐mag and PMET‐mag remained dispersed even after 6 h. In FBS, bare MNPs slightly precipitated after 7 days, whereas PME‐mag and PMET‐mag remained dispersed for 14 days. These results indicate that modification of MPC polymers improved the dispersion stability of MNPs.

FIGURE 2.

(A) Dynamic light scattering charts of naked magnetite nanoparticles, PME‐mag, and PMET‐mag. (B) ζ‐potential of an aqueous dispersion of naked magnetite nanoparticles, PME‐mag, and PMET‐mag. PME‐mag, PME‐modified magnetite nanoparticles; PMET‐mag, PMET‐modified magnetite nanoparticles.

Next, the performance of the polymer‐modified MNPs as nano‐heaters for MHT was assessed. The water‐dispersed MNPs were irradiated with AMF and the temperature was monitored. The temperature increase was slower for the polymer‐modified magnetite than for the unmodified MNPs, and there was no apparent difference in the effect of the polymer structure on the heating capacity (Figure S6). The heat generation of MNPs could be attributed to Néel relaxation and Brownian relaxation ⁸ because MNPs smaller than 20 nm exhibit superparamagnetic properties. ³⁹ , ⁴⁰ , ⁴¹ As a mechanism of lower heat generation capacity of the polymer‐modified magnetite, we speculated that the binding of carboxy groups to the MNPs caused spin canting at the surface layer of the nanoparticles, leading to a decrease in magnetization. ⁴² Moreover, the formation of polymer layers may reduce the mobility of MNPs, which could attenuate the heat generation based on Brownian relaxation. ⁸ It remains to be elucidated how the formation of polymer layers affects heat generation in tumor tissues and cancer cells because Brownian relaxation depends on the environment around the MNPs.

3.2. In vitro MHT with polymer‐modified MNPs

To examine the cytotoxicity of polymer‐modified MNPs without AMF exposure, various concentrations of polymer‐modified MNPs were added to CT26 cell culture. No obvious decrease in cell viability was observed at any concentration after 24 h of incubation (Figure S7), indicating that polymer‐modified MNPs were not cytotoxic in the concentration range <1000 μg/mL.

Based on these results, we investigated the in vitro cell killing activity of MHT with polymer‐modified MNPs at 1000 μg/mL. After 24 h of incubation with polymer‐modified MNPs, the amount of magnetite internalized into the CT26 cells was measured. As a result, the amount of magnetite incorporated into the cells in both PME‐mag and PMET‐mag was approximately 4% of the total amount added (Figure 3A). CT26 cells treated with MNPs were irradiated with AMF for 15 min. All the cell pellets reached 43°C in 5 min and maintained the temperature at 43°C by controlling the power of the AMF generator (Figure 3B). The viability of CT26 cells after MHT treatment with MNPs was reduced, and a more prominent decrease in cell viability was observed when cells were treated with PMET‐mag (Figure 3C). Given that there was no apparent difference in magnetite uptake (Figure 3A) and the heating temperature of the cells (Figure 3B), it is plausible to assume that the presence of mitochondria‐targeting VTPP units enhanced the effect of hyperthermia (Figure 3C). To examine the mitochondrial damage caused by MHT, the mitochondrial membrane potential was measured using JC‐1 dye, which emits green when monomeric but red when accumulated and aggregated in healthy mitochondria. The intensity of red fluorescence decreases when mitochondria are damaged and depolarized. Figure S8 depicts the results of the flow cytometric analysis of CT26 cells stained with JC‐1 dye after MHT. In the hyperthermia group, the percentage of cells with depolarized mitochondria was elevated. Moreover, CT26 cells treated with PMET‐mag exhibited an increased level of depolarized mitochondria compared with that of CT26 cells treated with PME‐mag. These findings suggest that the presence of TPP units increased mitochondrial damage caused by MHT.

FIGURE 3.

(A) Uptake of polymer‐modified magnetite nanoparticles by CT26 cells incubated in the presence of PME‐mag and PMET‐mag for 24 h. Error bars represent standard deviation (n = 3). (B) Temperature profiles of in vitro magnetic hyperthermia for CT26 cells. The power output of alternating magnetic field was adjusted to maintain at 43°C. Error bars represent standard deviation (n = 3). (C) Viability of CT26 cells after in vitro magnetic hyperthermia. Cell viability was normalized by nontreated cells. Error bars represent standard deviation (n = 3). PME‐mag, PME‐modified magnetite nanoparticles; PMET‐mag, PMET‐modified magnetite nanoparticles.

3.3. Subcellular localization of polymer‐modified MNPs

To investigate the subcellular localization of MNPs, CT26 cells incubated with PME‐mag or PMET‐mag were observed using TEM (Figure 4). Black MNPs were observed in both PME‐mag‐ and PMET‐mag‐treated cells (Figure 4A,B). MNPs were present in the aggregates, which is consistent with the particle distribution measured by DLS (Figure 2A). In general, nanoparticles are internalized by cells mainly via endocytosis. ⁴³ Focusing on intracellular MNPs, PME‐mag was observed in endosomes (Figure 4C), suggesting that PME‐mag was taken up via endocytosis. In contrast, PMET‐mag was present in the cytoplasm (Figure 4D), suggesting that PMET‐mag directly penetrated the plasma membrane or escaped from endosomes and migrated into the cytoplasm. It was presumed that PMET‐mag entered the cytoplasm due to the membrane‐disruptive nature of the VTPP units, ⁴⁴ which is supported by previous studies showing that cationic nanoparticles can undergo direct penetration and endosomal escape. ⁴⁵ , ⁴⁶ The mitochondria of the cells are shown in Figure 4E,F. For PME‐mag, no obvious accumulation of MNPs was observed in the mitochondria (Figure 4E). In contrast, PMET‐mag was observed in mitochondria (Figure 4F). These results indicate that the introduction of VTPP units confers mitochondrial localization properties to MNPs, and we successfully prepared mitochondria‐targeting MNPs.

FIGURE 4.

TEM images of CT26 cells incubated in the presence of PME‐mag or PMET‐mag for 24 h. The images of a whole CT26 cell treated with (A) PME‐mag and (B) PMET‐mag. The enraged images in cytosol near cell membrane of a CT26 cell treated with (C) PME‐mag and (D) PMET‐mag. The images near mitochondria in a CT26 cell treated with (E) PME‐mag and (F) PMET‐mag. M, mitochondria; PME‐mag, PME‐modified magnetite nanoparticles; PMET‐mag, PMET‐modified magnetite nanoparticles.

3.4. In vivo MHT with polymer‐modified MNPs

The in vivo therapeutic effects of mitochondria‐targeting MHT were evaluated. PME‐mag and PMET‐mag were intratumorally injected into CT26 tumor‐bearing mice. MNP administration and AMF irradiation were performed on days 0, 1, and 2 (Figure 5A). Tumor temperature during AMF irradiation was monitored, and the therapeutic temperature was set at 43°C by adjusting the power of the AMF generator. As shown in Figure 5B,C, the temperature profiles of PME‐mag and PMET‐mag in tumor were almost the same, suggesting that the intratumoral levels of nanoparticles were approximately the same. Figure 5D–F shows the therapeutic effects on tumor volume in mice treated with MHT. Tumors grew progressively in untreated mice (Figure 5D). Tumor growth was suppressed and two out of six tumors were cured in mice treated with MHT using PME‐mag (Figure 5E). For PMET‐mag, tumor growth was strongly suppressed for 30 days, and five out of six mice showed complete tumor regression. No obvious weight loss was observed in the MHT‐treated groups (Figure S9). These results indicate that mitochondria‐targeting MHT enhances therapeutic efficacy.

FIGURE 5.

In vivo magnetic hyperthermia. (A) Therapeutic protocol of magnetic hyperthermia for CT26‐tumor‐bearing mice. The mice were treated with magnetic hyperthermia when the tumor volume reached 50 mm³. The alternating magnetic fields were applied after the intratumor administration of magnetite nanoparticles on days 0, 1, and 2. The temperature profiles for the surface of the tumor during the magnetic hyperthermia with (B) PME‐mag and (C) PMET‐mag on day 2. Error bars represent standard deviation (n = 6). Tumor growth curves in (D) nontreated mice and mice treated with magnetic hyperthermia using (E) PME‐mag and (F) PMET‐mag. Each group included six mice. *Mice cured of tumor, n. H, hyperthermia; M, injection of magnetite nanoparticles; PME‐mag, PME‐modified magnetite nanoparticles; PMET‐mag, PMET‐modified magnetite nanoparticles.

Although the administration of PME‐mag and PMET‐mag exhibited no evident toxicity in vitro (Figure S7) and in vivo (Figure S9), indicating high biocompatibility, additional research on the toxicity of MNPs must be conducted before their clinical application. Analysis of blood biomarkers such as inflammatory cytokines would be among future studies. Furthermore, the pharmacokinetics of MNPs must be elucidated for the optimization of the therapeutic protocol.

This study aimed to prepare mitochondria‐targeting MNPs and investigate the therapeutic potency of MHT in the local heating of the mitochondria. The modification of MNPs achieved mitochondrial localization using carboxyl phospholipid polymers containing mitochondria‐targeting VTTP units. Moreover, mitochondria‐targeting MNPs enhanced therapeutic outcomes in vivo, as evidenced by MHT in CT26 tumor‐bearing mice. Our results indicate that the local heating of mitochondria can improve the efficiency of MHT. These findings provide insights that promote the development of a new strategy for the surface design of MNPs and therapeutic strategies for MHT.

AUTHOR CONTRIBUTIONS

Masahiro Kaneko and Akira Ito designed the study. Masahiro Kaneko, Hiroto Yamazaki, and Takahiro Ono carried out the polymer synthesis and prepared the polymer‐modified magnetite nanoparticles. Hiroto Yamazaki performed the in vitro and in vivo hyperthermia experiments. Takahiro Ono evaluated the mitochondrial membrane potential after in vitro hyperthermia. Masanobu Horie performed the TEM observation of CT26 cells. Masahiro Kaneko and Hiroto Yamazaki analyzed the data. Masahiro Kaneko, Hiroto Yamazaki, and Akira Ito prepared the figures. Masahiro Kaneko wrote the manuscript with input from all authors. All authors read and approved the final version of the manuscript.

FUNDING INFORMATION

This work was supported by JSPS KAKENHI 20H02538 (A.I.) and 21K14469 (M.K.), and the Uehara Memorial Foundation (M.K.).

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

ETHICS STATEMENT

Approval of the research protocol by an Institutional Reviewer Board: N/A.

Informed Consent: N/A.

Registry and the Registration No: N/A.

Animal Studies: Ethics Committee for Animal Experiments of the School of Engineering, Nagoya University (G220016).

Supporting information

APPENDIX S1

Kaneko M, Yamazaki H, Ono T, Horie M, Ito A. Effective magnetic hyperthermia induced by mitochondria‐targeted nanoparticles modified with triphenylphosphonium‐containing phospholipid polymers. Cancer Sci. 2023;114:3750‐3758. doi: 10.1111/cas.15895