Enhanced Photodynamic Anticancer Activities of Multifunctional Magnetic Nanoparticles (Fe3O4) Conjugated with Chlorin e6 and Folic Acid in Prostate and Breast Cancer Cells

Photodynamic therapy (PDT) is a promising alternative to conventional cancer treatment methods. Nonetheless, improvement of in vivo light penetration and cancer cell-targeting efficiency remain major challenges in clinical photodynamic therapy. This study aimed to develop multifunctional magnetic nanoparticles conjugated with a photosensitizer (PS) and cancer-targeting molecules via a simple surface modification process for PDT. To selectively target cancer cells and PDT functionality, core magnetic (Fe3O4) nanoparticles were covalently bound with chlorin e6 (Ce6) as a PS and folic acid (FA). When irradiated with a 660-nm long-wavelength light source, the Fe3O4-Ce6-FA nanoparticles with good biocompatibility exerted marked anticancer effects via apoptosis, as confirmed by analyzing the translocation of the plasma membrane, nuclear fragmentation, activities of caspase-3/7 in prostate (PC-3) and breast (MCF-7) cancer cells. Ce6, used herein as a PS, is thus more useful for PDT because of its ability to produce a high singlet oxygen quantum yield, which is owed to deep penetration by virtue of its long-wavelength absorption band; however, further in vivo studies are required to verify its biological effects for clinical applications.


Introduction
Cancer is a leading cause of mortality worldwide. Every year, an estimated 11 million individuals are diagnosed with cancer, and approximately 7 million individuals die of cancer according to the World Health Organization (WHO) [1]. Therefore, cancer currently ranks among the deadliest diseases, and advancements in medical technology have yielded various methods for cancer treatment over the last few decades [2]. Among them, traditional chemotherapy is limited by its severe toxicity, poor tumor-specific delivery, and the possibility of inducing multi-drug resistance [3][4][5]. However, in comparison with chemotherapy, photodynamic therapy (PDT) offers certain unique advantages including minimal invasiveness, fewer side effects, negligible chemotherapeutic resistance, and low systematic toxicity [6][7][8].
In PDT, photosensitizers (PS) are the key components that transfer photo-energy to the surrounding O2 molecules, generating reactive oxygen species (ROS), primarily singlet oxygen ( 1 O2), to eliminate proximal cancer cells [9][10][11][12]. According to a recent study, various PSs have developed, which absorb light over a broad range from ultraviolet (UV) to the near-infrared (NIR) range [13,14]. However, the absorption bands of many PSs are primarily in the UV-Vis region [15,16]. Furthermore, the PSs with the absorption band in the NIR range have low 1 O2 quantum yield owing to a low population of PSs in the triplet state [17,18]. Therefore, these PSs are often limited by their low 1 O2 quantum yields, and low depth of penetration resulting from a short excitation wavelength [15,16]. In addition, currently available PSs are mostly nonspecifically activated and have poor water solubility and stability and low accumulation at the target site, resulting in treatment-related toxicity, light-induced degradation of drug molecules, and other side effects on adjacent normal tissue and blood cells [6,[19][20][21]. To overcome these limitations, various inorganic and organic nanocarriers, including Fe3O4 nanoclusters, Au nanoparticles, graphene oxide, mesoporous silica nanoparticles, and polymer micelles, have been used to improve the stability and therapeutic outcomes of these PSs [22][23][24][25][26]. Nonetheless, the development of multifunctional nanoparticles with enhanced anticancer efficiency remains a major challenge in PDT.
Herein, to achieve enhanced photodynamic anticancer activity, we designed and fabricated a novel Fe3O4 nanoparticle conjugated with chlorin e6 (Ce6) and folic acid (FA) (Fe3O4-Ce6-FA) via simple surface modification. To enhance the PDT efficiency, magnetic core particles were conjugated with Ce6 and FA as PDT agents to increase the in vivo penetration depth of the light source and selectively eliminate cancer cells. In addition, we evaluated the efficiency of the Fe3O4-Ce6-FA nanoparticles for specific targeting and photodynamic anticancer activity in vitro. The Fe3O4-Ce6-FA nanoparticles developed herein could be a promising multifunctional nanoreagent for photodynamic tumor therapy and multifunctional drug delivery in the future.

Characterization of Multifunctional Fe3O4-Ce6-FA
Multifunctional 20-nm Fe3O4-Ce6-FA nanoparticles were fabricated via a simple surface modification via a wet chemical process as shown in Scheme 1. Ce6, having a long-wavelength absorption band and high singlet oxygen quantum yield, was conjugated with Fe ions on the surface of the Fe3O4 nanoparticles via esterification. Additionally, the multifunctional nanoparticles were functionalized with FA used as a targeting molecule to deliver these particles to the cancer cell membrane. Transmission electron microscopy (TEM), field emission scanning electron microscopy (FE-SEM), and X-ray diffraction (XRD) analysis were performed to confirm the appearance, size distribution, and crystallinity of the Fe3O4-Ce6-FA nanoparticles. As shown in Figure 1a,b, Fe3O4-Ce6-FA nanoparticles had a uniform spherical structure and a rough surface, measuring approximately 20 nm in diameter (Figure 1a inset). High-resolution TEM (HRTEM) images revealed regular parallel lattice fringes, indicating the high crystallinity of the Fe3O4-Ce6-FA nanoparticles (inset of Figure 1b). Transmission electron microscopy (TEM), field emission scanning electron microscopy (FE-SEM), and X-ray diffraction (XRD) analysis were performed to confirm the appearance, size distribution, and crystallinity of the Fe 3 O 4 -Ce6-FA nanoparticles. As shown in Figure 1a Figure 2a shows the magnetic hysteresis loops of pure Fe3O4 and Fe3O4-Ce6-FA nanoparticles at room temperature. As shown, both samples exhibited superparamagnetic behavior without obvious remnant magnetization and coercivity owing to their small magnetite nanocrystal composition. The saturation magnetization (Ms) of pure Fe3O4 was 80.5 emu/g. After coating with PS and FA, the Ms of Fe3O4-Ce6-FA nanoparticles decreased to 58.5 emu/g. The minor reduction in magnetization primarily resulted from the reduction in the density of Fe3O4 due to the presence of non-magnetic coating layers. However, the Fe3O4-Ce6-FA nanoparticles (20 nm) still showed strong magnetization, thereby suggesting their suitability for magnetic separation and magnetic resonance (MR) imaging. Figure 2b shows the photoluminescence (PL) and photoluminescence excitation (PLE) spectra of the pure Ce6 and the Fe3O4-Ce6-FA nanoparticles in THF. Ce6 displayed three main UV-Vis absorption peaks with an intense Soret band at 400 nm and two relatively weak Q-bands at 500 and  the strong bonding between Ce6 and the magnetite nanoparticle [27]. Upon excitation at 660 nm, free Ce6 exhibited two strong emission peaks at 672 and 707 nm. The Fe 3 O 4 -Ce6-FA nanoparticles also exhibited a red shift and broadening compared with free Ce6, concurrent with the phenomenon observed during absorption. 662 nm, respectively. After encapsulation of the Fe3O4 nanoparticles, a remarkable red shift and peak broadening in the UV-Vis spectrum of Fe3O4-Ce6-FA nanoparticles were observed, indicating the strong bonding between Ce6 and the magnetite nanoparticle [27]. Upon excitation at 660 nm, free Ce6 exhibited two strong emission peaks at 672 and 707 nm. The Fe3O4-Ce6-FA nanoparticles also exhibited a red shift and broadening compared with free Ce6, concurrent with the phenomenon observed during absorption. We used 1,3-diphenylisobenzofuran (DPBF), a specific 1 O2 probe, to quantify the 1 O2 generated from the Fe3O4-Ce6-FA nanoparticles by monitoring the absorbance of DPBF at 424 nm. Figure 3a exhibits the time-dependent UV-Vis absorption spectra of complexes of DPBF and Fe3O4-Ce6-FA in ethanol, which were irradiated with a red light-emitting diode (LED) light source. Upon excitation at 660 nm, the intensity of the absorbance peak of DPBF at 424 nm decreased gradually with the increase in irradiation time in the presence of the Fe3O4-Ce6-FA nanoparticles (Figure 3b). In the blank condition, no appreciable degradation of DPBF was observed after irradiation for 35 min. Near-complete photodegradation of DPBF in the presence of Fe3O4-Ce6-FA nanoparticles was observed within 35 min. This clearly indicates that the Fe3O4-Ce6-FA nanoparticles can effectively generate the 1 O2 ROS. Based on the reaction kinetics, which was well fitted into the equation In(C/C0) = −kobs × time (min), the apparent first-order rate constant, kobs, of DPBF photo-oxidation was 0.05094 min −1 for the Fe3O4-Ce6-FA nanoparticles ( Figure 3c).  662 nm, respectively. After encapsulation of the Fe3O4 nanoparticles, a remarkable red shift and peak broadening in the UV-Vis spectrum of Fe3O4-Ce6-FA nanoparticles were observed, indicating the strong bonding between Ce6 and the magnetite nanoparticle [27]. Upon excitation at 660 nm, free Ce6 exhibited two strong emission peaks at 672 and 707 nm. The Fe3O4-Ce6-FA nanoparticles also exhibited a red shift and broadening compared with free Ce6, concurrent with the phenomenon observed during absorption. We used 1,3-diphenylisobenzofuran (DPBF), a specific 1 O2 probe, to quantify the 1 O2 generated from the Fe3O4-Ce6-FA nanoparticles by monitoring the absorbance of DPBF at 424 nm. Figure 3a exhibits the time-dependent UV-Vis absorption spectra of complexes of DPBF and Fe3O4-Ce6-FA in ethanol, which were irradiated with a red light-emitting diode (LED) light source. Upon excitation at 660 nm, the intensity of the absorbance peak of DPBF at 424 nm decreased gradually with the increase in irradiation time in the presence of the Fe3O4-Ce6-FA nanoparticles (Figure 3b). In the blank condition, no appreciable degradation of DPBF was observed after irradiation for 35 min. Near-complete photodegradation of DPBF in the presence of Fe3O4-Ce6-FA nanoparticles was observed within 35 min. This clearly indicates that the Fe3O4-Ce6-FA nanoparticles can effectively generate the 1 O2 ROS. Based on the reaction kinetics, which was well fitted into the equation In(C/C0) = −kobs × time (min), the apparent first-order rate constant, kobs, of DPBF photo-oxidation was 0.05094 min −1 for the Fe3O4-Ce6-FA nanoparticles (Figure 3c).

In Vitro Cytotoxicity of Multifunctional Fe 3 O 4 -Ce6-FA Nanoparticles
For biomaterials to be used for biomedical applications, a basic biocompatibility assay is necessary to evaluate their cytotoxicity. Therefore, the in vitro cytotoxicity of Fe 3 O 4 -Ce6-FA was evaluated in normal fibroblast (L-929), breast cancer (MCF-7), and prostate cancer (PC-3) cell lines, as described previously [28][29][30][31][32][33]. Twofold-diluted concentrations of Fe 3 O 4 -Ce6-FA from 100 to 6.25 µg/mL were tested, and non-treated cells constituted the control. As shown in Figure 4a, the cell viabilities of all cells exceeded 95%, indicating that Fe 3 O 4 -Ce6-FA displayed no cytotoxicity in all cells, suggesting that Fe 3 O 4 -Ce6-FA nanoparticles may have biomedical applications with excellent biocompatibility.

In Vitro Cytotoxicity of Multifunctional Fe3O4-Ce6-FA Nanoparticles
For biomaterials to be used for biomedical applications, a basic biocompatibility assay is necessary to evaluate their cytotoxicity. Therefore, the in vitro cytotoxicity of Fe3O4-Ce6-FA was evaluated in normal fibroblast (L-929), breast cancer (MCF-7), and prostate cancer (PC-3) cell lines, as described previously [28][29][30][31][32][33]. Twofold-diluted concentrations of Fe3O4-Ce6-FA from 100 to 6.25 μg/mL were tested, and non-treated cells constituted the control. As shown in Figure 4a, the cell viabilities of all cells exceeded 95%, indicating that Fe3O4-Ce6-FA displayed no cytotoxicity in all cells, suggesting that Fe3O4-Ce6-FA nanoparticles may have biomedical applications with excellent biocompatibility.

In Vitro Photodynamic Anticancer Activity of Fe3O4-Ce6-FA Nanoparticles
To confirm the photo-killing ability of Fe3O4-Ce6-FA nanoparticles, PC-3 and MCF-7 cell lines were exposed to LED irradiation for 10 min after incubation with various concentrations of Fe3O4-Ce6-FA nanoparticles for 2 h. As shown in Figure 4b, cell viabilities of the two cell lines were significantly decreased with an increase in nanoparticle concentration, and the cell viability of PC-3

In Vitro Photodynamic Anticancer Activity of Fe 3 O 4 -Ce6-FA Nanoparticles
To confirm the photo-killing ability of Fe 3 O 4 -Ce6-FA nanoparticles, PC-3 and MCF-7 cell lines were exposed to LED irradiation for 10 min after incubation with various concentrations of Fe 3 O 4 -Ce6-FA nanoparticles for 2 h. As shown in Figure 4b, cell viabilities of the two cell lines were significantly decreased with an increase in nanoparticle concentration, and the cell viability of PC-3 cells was even more drastically decreased compared to that of MCF-7 cells, even at the lowest concentration of 6.25 µg/mL. This indicated that the photo-killing efficacy of Fe 3 O 4 -Ce6-FA nanoparticles was concentration-dependent. Moreover, Fe 3 O 4 -Ce6-FA nanoparticles are more effective than Fe 3 O 4 conjugated with hematoporphyrins (HPs) and FA, as reported previously [30,31]. The PS (Ce6) used herein is more applicable for PDT owing to its attributes, which include a high singlet oxygen quantum yield and long-wavelength absorption band, resulting in deeper light penetration in vivo compared with HP-conjugated nanoparticles. In other words, the photodynamic anticancer efficacy of Fe 3 O 4 -Ce6-FA nanoparticles was closely associated with singlet oxygen quantum yield and the concentration of the Fe 3 O 4 -Ce6-FA nanoparticles.
Considering the photodynamic anticancer activity of Fe 3 O 4 -Ce6-FA nanoparticles, the mechanisms underlying cancer cell death were evaluated via analysis of the translocation of the plasma membrane, using an Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit, nuclear fragmentation using a fluorescent dye (Hoechst 33342), and enzyme activities of caspase-3/7 using a CellEvent Caspase-3/7 Green Detection reagent. First, PC-3 and MCF-7 cells were stained with Annexin V-FITC reagent post-irradiation after incubation with Fe 3 O 4 -Ce6-FA nanoparticles for 2 h to confirm phosphatidylserine translocation from the intracellular to the extracellular leaflet of the plasma membrane, which is a hallmark of the early stage of apoptotic cell death. Figure 4c shows the images of live and apoptotic cells stained with Annexin V-FITC, post-irradiation. Both cell types (MCF-7 and PC-3) in the Fe 3 O 4 -Ce6-FA nanoparticle-treated groups showed green fluorescence, whereas control cells did not. These results indicate that PDT following treatment with Fe 3 O 4 -Ce6-FA nanoparticles induced cancer cell death via apoptosis.
Additionally, nuclear fragmentation of cancer cells, which is also a hallmark of apoptotic cell death, was confirmed via staining with Hoechst 33342 dye. As shown in Figure 4d, the nuclei of both cell types treated with Fe 3 O 4 -Ce6-FA nanoparticles were more condensed than those of control cells, and most nuclei of PC-3 cells rapidly changed to granular apoptotic nuclear bodies. However, no changes were detected in the control cells of both cell lines. These results also indicated that irradiation after treatment with Fe 3 O 4 -Ce6-FA nanoparticles enhanced apoptotic cell death.
Finally, caspase-3/7 activity, which essentially contributes to apoptotic cell death, were also evaluated using a fluorogenic substrate highly specific for activated caspase-3 and -7. As shown in Figure 4d

Preparation of Fe 3 O 4 -Ce6-FA Nanoparticles
Multifunctional Fe 3 O 4 -Ce6-FA nanocomposites were synthesized in accordance with a previously reported procedure with minor modifications [28]. In brief, FeCl 3 ·6H 2 O (0.54 g) and NaAc·3H 2 O (1.5 g) in 20 mL ethylene glycol (EG) and diethylene glycol (DEG) (1:19) were added in a 200 mL round-bottom flask, and the mixture was vigorously stirred for 30 min. Thereafter, the yellowish homogeneous solution formed was sealed in a teflon-lined stainless steel autoclave. The autoclave was heated to and maintained at 200 • C for 10 h and cooled to ambient temperature. The black precipitate was harvested via magnetic decantation, washed with deionized water and absolute alcohol several times, and then dried in a vacuum oven at 60 • C for 12 h. The photoactive and targeting functionalities of the Fe 3 O 4 nanoparticles were achieved using a wet chemical process similar to our previous method [29]. Briefly, 20 mg of precipitated Fe 3 O 4 nanoparticles with 20 nm size were mixed with a solution of Ce6/EtOH (final concentration, 10 −4 M). The Ce6 molecules are easily conjugated to the surface of magnetite nanoparticles owing to the three terminal carboxyl groups, which initiate covalent bonding. Furthermore, the Ce6 molecules have a high singlet oxygen quantum yield of 0.77 in solution [34]. This value of singlet oxygen quantum yield is higher than that of the other photosensitizers such as hematoporphyrin (0.51) [35] and protoporphyrin (0.63) [36]. The solution was vigorously agitated for 24 h at room temperature. After the reaction was completed, the product was washed several times with EtOH. To facilitate targeting functionality, the FA molecules were conjugated to Ce6-bonded Fe 3

Physical Characterization of Multifunctional Fe 3 O 4 -Ce6-FA Particles
TEM (JEM-2100F, JEOL, Tokyo, Japan) and FE-SEM (SU-70, Hitachi, Tokyo, Japan) were performed to study the morphology of the multifunctional nanoparticles. The crystallographic structure of the composite particles was investigated via XRD (X'Pert Pro MPD, PANalytical, Almelo, Netherlands), using Cu Kα radiation. A vibrating sample magnetometer (VSM; Lakeshore 7300, Lake Shore Cryotronics, Westerville, OH, USA) was used to obtain magnetization versus magnetic field loop up to H = 10 kOe at room temperature. Steady-state absorption and PL and PLE spectra were measured using a UV-Vis spectrophotometer (U-2800, Hitachi, Tokyo, Japan) and spectrofluorometer (F-4500, Hitachi, Tokyo, Japan), respectively.

Photodynamic Anticancer Activity of Multifunctional FCF Nanoparticles
Anticancer activity of the FCF nanoparticles was also assessed in MCF-7 and PC-3 cells on the basis of cell viability determined using CCK-8 after irradiation, as described previously [33][34][35][36]. Each cell type was plated in a 24-well plate at the same concentration and incubated as described in 3.3. Thereafter, the cells were further incubated with various concentrations (0, 6.25, 12.5, 25, 50, and 100 µg/mL) of FCF nanoparticles for 2 h in the dark, followed by three washes with DPBS, replenishment of the media, and irradiation with a red light-emitting diode (LED; FD-332R-N1, Fedy Technology Co., Shenzhen, China). The LEDs were driven using a constant current buck driver (LED-2800, TMC Co., Gunpo, Korea) and light intensity was regulated via pulse width modulation (CB210, Comfile Technology, Seoul, Korea). LED irradiation was applied at a maximum wavelength of 660 nm at 20 mW/cm 2 . After irradiation for 30 min, cancer cells were further incubated for 24 h, and cell viability was determined via a CCK-8 assay, as described in Section 3.3.
To evaluate the mechanisms underlying cancer cell death by Fe 3 O 4 -Ce6-FA after irradiation, both cancer cell types pre-cultured for 24 h were further incubated with 12.5 µg/mL of FCF after replenishing media in the dark. After 2 h of incubation, each cell type was irradiated with LED light at the same power for 10 min, as described previously in this section, and further incubated for 6 h to induce cell death. Thereafter, the plasma membranes and nuclei of both cell types were stained with an Annexin V-FITC apoptosis detection reagent (Komabiotech Inc., Seoul, Korea), Hoechst 33342 dye (Invitrogen, Molecular Probes, Eugene, OR, USA), and a CellEvent Caspase-3/7 Green Detection reagent (Invitrogen) in accordance with the manufacturers' instructions. After staining each sample, fluorescence microscopic images were acquired using an automated live cell imager (Lionheart FX; BioTek Instruments, Inc., VT, USA).

Conclusions
In summary, we synthesized Fe 3 O 4 -Ce6-FA nanoparticles for FA receptor-targeted PDT. PS and FA were covalently bound to the surface of the magnetite nanoparticles. The prepared multifunctional Fe 3 O 4 -Ce6-FA nanocomposites had high water solubility and good biocompatibility without any cytotoxicity. Moreover, Fe 3 O 4 -Ce6-FA exhibited more effective anticancer activity via apoptosis in prostate (PC-3) and breast cancer (MCF-7) cell lines in comparison with Fe 3 O 4 conjugated with HPs. Thus, the PS (Ce6) used in this study is more useful for PDT applications owing to its ability to produce a high singlet oxygen quantum yield and deep penetration owing to its long-wavelength absorption band. However, further in vivo studies are required to verify its biological effects for clinical applications, although we believe that our study makes a significant contribution to PDT because it supports the use of chlorin e6 as a PS in multifunctional nanomaterials for effective PTD.

Conflicts of Interest:
The authors declare no conflict of interest.