Journal of Photochemistry and Photobiology B: Biology 146 (2015) 34–43
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Enhanced photodynamic therapy efficacy of methylene blue-loaded calcium phosphate nanoparticles Da-Young Seong, Young-Jin Kim ⇑ Department of Biomedical Engineering, Catholic University of Daegu, Gyeongsan 712-702, Republic of Korea
a r t i c l e
i n f o
Article history: Received 19 August 2014 Received in revised form 17 February 2015 Accepted 19 February 2015 Available online 9 March 2015
a b s t r a c t Although methylene blue (MB) is the most inexpensive photosensitizer with promising applications in the photodynamic therapy (PDT) for its high quantum yield of singlet oxygen generation, the clinical use of MB has been limited by its rapid enzymatic reduction in the biological environment. To enhance PDT efficacy of MB by preventing the enzymatic reduction, we have developed a new mineralization method to produce highly biocompatible MB-loaded calcium phosphate (CaP-MB) nanoparticles in the presence of polymer templates. The resulting CaP-MB nanoparticles exhibited spherical shape with a size of under 50 nm. Fourier transform infrared (FT-IR) and zeta-potential analyses confirmed the insertion of MB into the CaP-MB nanoparticles. The encapsulation of MB in CaP nanoparticles could effectively protect MB from the enzymatic reduction. In addition, the CaP-MB nanoparticles exhibited a good biocompatibility in the dark condition and significantly enhanced PDT efficacy due to apoptotic cell death against human breast cancer cells as compared with free MB, implying that CaP-MB nanoparticle system might be potentially applicable in PDT. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Photodynamic therapy (PDT) has emerged as an important treatment modality for a variety of cancers, cardiovascular and ophthalmic diseases [1,2]. This modality involves the delivery of light-sensitive molecules called photosensitizers to target sites followed by irradiation with appropriate wavelength of light. Upon irradiation, the activated photosensitizers transfer their excess energy to the surrounding oxygen to form reactive oxygen species (ROS) such as singlet oxygen (1O2) or free radicals, which will cause irreversible damage of diseased cells and tissues. PDT is also expected to be a potential method of overcoming multidrug resistance (MDR) because cytotoxicity mechanism of photosensitizers onto cancer cells is different from that of other chemotherapy agents [3]. However, the clinical use of many photosensitizers has been hampered by their significant side effects including nonspecific damage to normal tissues due to low accumulation selectivity to specific cells or tissues, environmental degradation and hydrophobicity [4]. To solve these problems, various biocompatible nanocarrier systems for delivering photosensitizers such as liposomes, polymeric micelles and nanoparticles have been investigated [5– 7]. These nanocarriers have promoted uptake of photosensitizers into target sites and reduction of nonspecific damage to normal ⇑ Corresponding author. Tel.: +82 53 850 2512; fax: +82 53 359 6750. E-mail address: [email protected] (Y.-J. Kim). http://dx.doi.org/10.1016/j.jphotobiol.2015.02.022 1011-1344/Ó 2015 Elsevier B.V. All rights reserved.
tissues caused by free photosensitizers [8]. In addition, nanocarrier systems have offered stable aqueous dispersion of photosensitizers by surface modification and protected photosensitizers from environmental degradation [9]. Despite many advantages of the nanocarrier systems, the use of liposomes and micellar systems was limited because of their lower drug loading capacity and severe side effects like anaphylactic shocks [1,10]. Methylene blue (MB) is a phenothiazinium photosensitizer that has been employed in a variety of applications including PDT [9,11]. The high quantum yield of 1O2 generation by MB in the excitation of the therapeutic window (600–900 nm) makes MB a reasonable candidate for PDT. However, the clinical use of MB has been hindered by its propensity for rapid chemical alteration when systemically applied. MB is usually converted by accepting electrons from nicotinamide adenine dinucleotide (NADH)/nicotinamide adenine dinucleotide phosphate (NADPH) in the biological environment and the formed colorless leucomethylene blue has negligible photodynamic activity [12]. The presence of a transmembrane thiazine dye reductase at the cell surface is commonly recognized as the initial factor for MB reduction [13]. After cellular uptake of MB, the reduction also can be catalyzed by NADH/NADPH dehydrogenases. In this respect, the major obstacle for the use of MB in PDT applications is the difficulty in preparing pharmaceutical formulations that enable their facile administration. Therefore, biocompatible nanoparticles have received attention as an adequate means of encapsulating and delivering MB for PDT [9,11].
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Calcium phosphate (CaP) materials have been widely used in biomedical applications as useful drug carriers due to their excellent biocompatibility and bioactivity [14,15]. Among them, photosensitizers-incorporated CaP nanoparticles, which were synthesized in the presence of ionic polymers such as poly(ethylene imine) and poly(styrene sulfonic acid), exhibited a good phototoxicity against murine macrophages and bacteria [15]. In addition, CaP is absorbable in specific cellular environments (endosome/lysosome) as non-toxic ionic species [16]. Thus, the employment of controlled mineralization technology using self-assembled polymer templates would lead to the successful development of biocompatible and biodegradable nanocarriers of photosensitizers. The major purpose of this study was to enhance PDT efficacy of MB by increasing biostability such as the prevention of environmental degradation and enzymatic reduction under the biological conditions. For this reason, a new mineralization method was explored to produce highly biocompatible MB-loaded CaP (CaP-MB) nanoparticles in the presence of alginic acid sodium salt (alginate) and poly(ethylene glycol)–block–poly(propylene glycol)–block– poly(ethylene glycol) (PEG–PPG–PEG) triblock copolymer (Pluronic F-68) as polymer templates (Fig. 1). The prepared MBloaded CaP nanoparticles were systematically examined by considering their morphologies, chemical structures and particle size. The enzymatic reduction and yield of singlet oxygen generation were thoroughly investigated. Furthermore, the cellular internalization behavior and phototoxicity onto human breast cancer cell line (MCF-7) were evaluated via fluorescence microscopy and MTT assay.
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2. Materials and methods 2.1. Materials Methylene blue (MB), poly(ethylene glycol)–block–poly(propylene glycol)–block–poly(ethylene glycol) (PEG–PPG–PEG triblock copolymer, Pluronic F-68, Mn = 8400), alginic acid sodium salt (alginate), calcium nitrate tetrahydrate (Ca(NO3)24H2O), ammonium phosphate dibasic ((NH4)2HPO4), ammonium hydroxide solution (NH4OH), b-nicotinamide adenine dinucleotide, reduced dipotassium salt (NADH), diaphorase from C. kluyveri, paraformaldehyde and 9,10-dimethylanthracene (DMA) were purchased from Sigma–Aldrich (USA) and used without further purification. Human breast cancer cell line (MCF-7) was received from the Korean Cell Line Bank (KCLB, Korea). RPMI-1640, fetal bovine serum (FBS) and penicillin–streptomycin were obtained from Gibco BRL (USA). Slowfade gold antifade reagent and Live/Dead Viability/ Cytotoxicity assay kit were purchased from Molecular probes (USA). Annexin V-fluorescent isothiocyanate (FITC) fluorescence microscopy kit was from BD Biosciences (USA) and DePsipher kit was from Trevigen (USA). Human cytochrome c Quantikine ELISA kit was purchased from R&D Systems (USA). Other reagents and solvents were commercially available and were used as received.
2.2. Synthesis of MB-loaded CaP nanoparticles A synthesis of MB-loaded CaP (CaP-MB) nanoparticles is as follows. 1 w/v% MB solution was first added dropwise to 70 mL of
Fig. 1. Schematic images of methylene blue-loaded calcium phosphate (CaP-MB) nanoparticles for photodynamic therapy. (a) Synthetic scheme for CaP-MB nanoparticles and (b) schematic illustration of photoactivity mechanism for CaP-MB nanoparticles. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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2 w/v% Pluronic F-68 solution. To this solution, 10 mL of 0.2 w/v% alginate solution was added dropwise for preparing self-assembled polymer micelle nanotemplate solution containing drug. Then, 15 mL of 25 mM Ca(NO3)24H2O and 10 mL of 25 mM (NH4)2HPO4 solution were added dropwise in turn and pH was adjusted to 10 by the addition of NH4OH. The final concentration of MB in the reaction solutions was 0 (CaP-0MB), 1 (CaP-1MB), 5 (CaP-5MB) or 10 wt% (CaP-10MB) based on the weight of polymers and CaP precursors (Ca(NO3)24H2O and (NH4)2HPO4). The mixture was stirred at 40 °C under air to induce the nucleation and growth of CaP nanoparticles in the polymer micelle nanotemplates. After 24 h, the resultant CaP-MB nanoparticles were isolated by tubular membrane dialysis in distilled water (DW) for 12 h, followed by lypophilization in vacuo. 2.3. Characterization of nanoparticles The morphologies of CaP-MB nanoparticles were observed by transmission electron microscopy (TEM, H-7600, Hitachi, Japan). The average diameter of nanoparticles was determined by analyzing the TEM images with image analyzing software (Image-Pro Plus, Media Cybernetics Inc., USA). The zeta-potential of nanoparticles was determined using a Zetasizer Nano ZS (Malvern Instruments, UK). UV–visible spectra were recorded on a Hitachi U-2900 spectrometer (Japan) at 25 °C. Fourier transform infrared (FT-IR) spectra of the samples were obtained with an ALPHA spectrometer (Brucker Optics, USA) in the wavenumber range of 400–4000 cm–1. X-ray diffraction (XRD) measurements were carried out to characterize the crystalline phase of CaP-MB nanoparticles with a Panalytical X-ray diffractometer X’Pert Pro (Netherlands) with Cu Ka radiation at 40 kV/30 mA. The diffractograms were scanned in a 2h range of 20–60° at a rate of 2°/min.
2.6. Cell culture and incubation conditions For all of the experiments, human breast cancer cell line (MCF7) was used. MCF-7 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. Cells were incubated at 37 °C in humidified 5% CO2 atmosphere. When the cells reached 80% confluence, they were harvested by 0.25% trypsin–EDTA and seeded into a new tissue culture plate for subculture. The CaP-MB nanoparticles and the free MB were dispersed in serum-free medium. Untreated cells irradiated or kept in the dark were used as a reference standard. 2.7. Cellular uptake test To verify the cellular uptake of the free MB and the CaP-MB nanoparticles, a cell lysis test was utilized [18]. MCF-7 cells (1 106 cells/well) were seeded into 6 well plates in 2 mL of culture medium to estimate the concentration of cellular internalized MB using lysis buffer. After 24 h incubation, the cells were treated with the free MB or the CaP-5MB for 1, 2, 4, 8, 16 and 24 h at 37 °C. The MB concentration of CaP-5MB was adjusted to 10 lg/mL for comparison with 10 lg/mL of free MB. Then the cells were washed twice with DPBS, and treated with protein extraction solution (RIPA buffer) for cell lysis. The lysis solution was divided into the supernatant and the debris by centrifugation (12,000 rpm, 15 min). For the evaluation of cellular uptake amount of the free MB and the CaP-5MB, the absorbance of the supernatant at 653 nm was measured with a UV–visible spectrophotometer. Meanwhile, at 2 h after incubation with 30 lg/mL of CaP-0MB and CaP-5MB nanoparticles, the cells were rinsed with DPBS and made into TEM specimens, and observed via a TEM (H-7600, Hitachi, Japan) for visualizing the intracellular uptake of nanoparticles into cancer cells.
2.4. Enzymatic reduction test of MB To investigate the protection of CaP nanoparticles for the loaded MB against reduction by diaphorase, the enzymatic reduction test of MB in the nanoparticles has been conducted according to previously published procedures [9]. Solutions of 1.8 mg/mL CaP-5MB (1 mg/mL of MB) and 1 mg/mL free MB were prepared in Dulbecco’s phosphate buffered saline (DPBS, pH 7.4), respectively. Then, 0.45 lM of NADH and 50 lg diaphorase were sequentially added into 1 mL of CaP-5MB solution. The decrease in MB absorbance at 663 nm due to the reduction of the entrapped MB was measured at room temperature with incubation time up to half an hour, using a UV–visible spectrometer. The free MB solution was treated in the same way, and its absorption spectra were recorded for comparison. 2.5. Singlet oxygen detection Determination of singlet oxygen generation has been extensively reported by an indirect method using a chemical probe [17]. In this study, the generation of singlet oxygen from CaP-5MB under different conditions was detected using DMA as the singlet oxygen probe. CaP-5MB (0.3 mg/mL) was dispersed in N,N-dimethylformamide (DMF) or DPBS (pH 7.4) and then added to DMA stock solution to give a final concentration of 20 lM DMA. Samples containing CaP-5MB and DMA were irradiated at a light intensity of 50 mW/cm2 using a 670 nm laser source (LVI Technologies, Korea) for 1 min. The decrease in fluorescence intensity of DMA (excitation, 360 nm; emission, 430 nm) as a result of the photosensitization reaction was monitored with a Perkin-Elmer LS55 spectrofluorophotometer (USA). The singlet oxygen generation from free MB was also determined for comparison.
2.8. Cell phototoxicity assay To determine cell viability under dark condition, MCF-7 cells (1 104 cells/well) were seeded onto 96 well plates and incubated for 24 h at 37 °C. After cell stabilization, the culture medium was replaced with 200 lL of culture medium containing free MB or CaP-5MB (0–50 lg/mL of MB), followed by incubation for 4 h. The cells were then washed twice with serum-free medium and cell viability was evaluated by the MTT assay after 24 h. To determine in vitro phototoxicity after laser irradiation, MCF-7 cells (1 104 cells/well) were seeded onto 96 well plates and incubated for 24 h at 37 °C. Then, these cells were treated with free MB or CaP-5MB (0–50 lg/mL of MB). After 4 h incubation, the cells were washed twice with serum-free medium and irradiated with a 670 nm diode laser (100 mW/cm2) for 0–9 min onto 96 well plates. The cell viability of irradiated cells was evaluated by the MTT assay after 24 h incubation. Qualitative cell viability assay was performed by using the Live/ Dead Viability/Cytotoxicity assay kit. The kit contains calcein AM and ethidium homodimer-1 (EthD-1), which identifies live versus dead cells on the basis of membrane integrity and esterase activity. Calcein AM stains live cells green, whereas EthD-1 stains dead cells red [19]. MCF-7 cells (1 104 cells/well) were seeded onto 8 well chamber slide and incubated for 24 h at 37 °C. Then, these cells were treated with CaP-5MB (30 lg/mL of MB). After 4 h incubation, the cellular layers on the sample surface were rinsed twice with DPBS and irradiated with a 670 nm diode laser (100 mW/cm2) for 5 min, which were then treated for 10 min at 37 °C with 1 lM of calcein AM and 2 lM of EthD-1 to determine cell viability. Cells were finally observed using an inverted fluorescence microscope (Eclipse TS100, FITC-G2A filters, Nikon, Japan) equipped with a
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cooled CCD camera (DS-U2, Nikon, Japan) and with NIS-Elements Imaging Software. 2.9. Apoptotic analysis Cellular apoptosis was visualized using an Annexin V-FITC fluorescence microscopy kit. MCF-7 cells (1 104 cells/well) were seeded onto 8 well chamber slide for 24 h before being treated with drugs. The cells were incubated with CaP-5MB (30 lg/mL of MB) for 4 h, which were then rinsed twice with DPBS and illuminated with a 670 nm diode laser (100 mW/cm2) for 5 min. The cells were stained with l mL of Annexin V-FITC (10 wt%) for 15 min at room temperature. After staining, the cells were washed twice with DPBS and then fixed using 4% paraformaldehyde in DPBS for 30 min. A cover slip was mounted on a microscope slide with a drop of anti-fade mounting solution to reduce fluorescence photo-bleaching. Cells were observed with an inverted fluorescence microscope using a filter sect for FITC. 2.10. Assay for intracellular mitochondrial membrane potential Mitochondrial membrane potential in MCF-7 cells was analyzed using the Trevigen’s DePsipher kit. DePsipher is the fluorescent lipophilic cation 5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolcarbocyanine iodide which is used as a mitochondrial marker and detects early apoptosis in cells. The cells (1 104 cells/well) were seeded in 8 well chamber slide for 24 h and then incubated with CaP-5MB (30 lg/mL of MB) for 4 h, which were then rinsed twice with DPBS and illuminated with a 670 nm diode laser (100 mW/cm2) for 5 min. After irradiating, the cells were incubated with DePsipher solution at the concentration 5 lg/mL for 20 min at 37 °C, then washed with reaction buffer containing stabilizer, placed on a glass slide and covered with a glass cover slip. The stained cells were observed using an inverted fluorescence microscope equipped with FITC and G2A filters. 2.11. Determination of cytochrome c extrusion Cytochrome c was detected using the quantitative Quantikine ELISA kit (R&D systems). MCF-7 cells (1 106 cells/well) were seeded onto 6 well plates and allowed to adhere overnight. CaP0MB and CaP-5MB nanoparticles were added into the culture plates and incubated 2 h, respectively. After the incubation, the cells were washed twice with DPBS and irradiated with a 670 nm diode laser (100 mW/cm2) for 5 min. Then, the cells were harvested by centrifugation and suspended in lysis buffer. The cytosolic fraction was isolated by centrifugation for 15 min and used for cytochrome c assay. After reacting with cytochrome c conjugate and substrate, the absorbance was measured at 450 nm for calculating the amount of cytochrome c released from mitochondria to the cytosol. 3. Results and discussion 3.1. Morphology and physicochemical properties of MB-loaded CaP nanoparticles CaP nanoparticles are well known as carriers for the transport of genes and drugs into cells [20,21]. CaP is superior to other inorganic species such as silica in terms of biocompatibility because CaP is naturally found as the main mineral component in bone [14]. MB is a highly water-soluble near IR fluorescent dye. It was reported that the MB-doped silica nanoparticles showed weak fluorescence and suffered from dye leaching [22]. In the present study, the MB-loaded CaP (CaP-MB) nanoparticles were prepared by rapid
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precipitation from water in the presence of polymers which stabilized the nanoparticles as shown in Fig. 1(a). The resulting nanoparticles were named as CaP-0MB, CaP-1MB, CaP-5MB and CaP-10MB with the weight feed ratio of MB to polymers and CaP precursors in the reaction solutions (Table 1). Fig. 2 shows the morphological structure of CaP-MB nanoparticles. All of the resulting nanoparticles exhibited spherical shape with a size of under 50 nm and the size of nanoparticles was hardly affected by the content of MB. The average diameter of nanoparticles was 34.6 ± 4.2 nm for CaP-0MB, 33.2 ± 2.9 nm for CaP-1MB, 35.5 ± 4.6 nm for CaP-5MB and 41.5 ± 3.5 nm for CaP-10MB as shown in Table 1. The existence of alginate can provide a lot of binding sites giving rise to the accumulation of MB molecules and Ca2+ ions due to the strong ionic interaction and increasing the supersaturation near the negative positions. In addition, the formation of a specific stereo-chemical arrangement and the charge distribution of reactive groups in alginate–MB and alginate–Ca2+ complexes can proceed [23]. Then, alginate–Ca2+ complexes can strongly interact with the surface of PO3 ions to 4 nucleate the CaP-MB nanoparticles. As a result, the initial nucleation is preferentially triggered at the positions of carboxyl groups, and the particle size is related to the nucleation and growth. As photosensitizer, MB was chosen in this study because of its high singlet oxygen quantum yield and absorption/emission wavelength in near IR region with relatively efficient tissue penetration [9]. MB was readily loaded by the simple precipitation method in the presence of polymer templates. The amount of MB absorbed on the particles was determined by UV–visible spectroscopy. The loading efficiency of MB was 56.2% at 5 wt% feed ratio, which decreased to 38.8% at 10 wt% feed ratio (Table 1). Thus, we used CaP-5MB for further studies. FT-IR analysis was carried out for identifying the functional groups present in the CaP-MB nanoparticles, which in turn provided information about the constitution and phase composition of the products. As shown in Fig. 3, all of the samples synthesized in the absence and presence of MB exhibited characteristic absorption bands for the vibrational modes of PO3 4 appeared at around 1090, 1027, 960, 597 and 565 cm1, and the broad band centered at 3340 cm1 associated with OH of alginate and self-associated Pluronic F-68 [23,24]. In addition, the absorption bands for the asymmetric stretching mode of COO ion were observed at 1607 cm1, which was probably ascribed to alginate. Furthermore, the bands observed at 1420 and 879 cm1 are 3 attributed to the substitution of CO3 2 ions in the place of PO4 ions and thus can confirm the substitution of CO3 in CaP structure. 2 These CO3 ions were formed by the reaction of CO2 present in 2 the atmosphere with OH ions of reaction medium. In the case of CaP-5MB, new absorption bands were found at 1490 and 1333 cm1 assigned to aromatic nitro compound [25]. To confirm the incorporation of MB into the nanoparticles, the change of zeta-potential was also determined using 0.1 w/v% (1 mg/mL concentration) solutions of all samples, resulting that negative values of zeta-potential reduced by adding positively charged MB as shown in Table 1. These data clearly explain the insertion of MB into the CaP-MB nanoparticles. Crystallographic analysis was performed using XRD to elucidate the change in crystal structure of the CaP-MB nanoparticles. The CaP-0MB nanoparticles showed the peaks attributed to the hydroxyapatite (HA) crystalline phase at around 25.9°, 32.1°, 39.5°, 46.8°, 49.7° and 53.2°, which reflected characteristic of the (0 0 2), (2 1 1), (2 0 2), (2 2 2), (2 1 3) and (0 0 4) planes (Fig. 4) [23]. For the CaP-5MB nanoparticles, the intensity and position of the peaks arising from HA hardly changed, suggesting that the addition of MB did not affect the crystalline phases of HA. Additionally all the peaks were broad diffraction peaks indicating a poorly crystallized HA phase. This is owing to the complex formation of HA with
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Table 1 Characteristics of CaP-MB nanoparticles.
a b c d
Sample
MB feed ratio (wt%)a
Loading content (wt%)b
Loading efficiency (%)
Mean diameter of nanoparticles (nm)c
Zeta-potential (mV)d
CaP-0MB CaP-1MB CaP-5MB CaP-10MB
– 1 5 10
– 0.1 ± 0.02 2.8 ± 0.16 3.9 ± 0.09
– 10.0 ± 2.0 56.2 ± 3.2 38.8 ± 0.9
34.6 ± 4.2 33.2 ± 2.9 35.5 ± 4.6 41.5 ± 3.5
21.4 ± 1.8 18.6 ± 0.9 15.0 ± 1.1 13.6 ± 0.6
Weight feed ratio of MB to polymers and CaP precursors. Weight content of MB in CaP-MB nanoparticles, determined by UV–visible spectrophotometer. Determined by analyzing the TEM images with image analyzing software. Measured using a Zetasizer Nano ZS at 1 mg/mL concentration in PBS (pH 7.4).
Fig. 2. TEM micrographs of CaP-MB nanoparticles synthesized with different content of MB: (a) 0 wt% (CaP-0MB), (b) 1 wt% (CaP-1MB), (c) 5 wt% (CaP-5MB) and (d) 10 wt% (CaP-10MB).
amorphous polymer. Moreover, the isomorphous substitution of 3– PO3– 4 by CO2 derived from the absorption of CO2 in the air during preparation process of the CaP-MB nanoparticles affected the decrease of crystallinity. 3.2. Prevention of enzymatic reduction of MB by encapsulation MB in the biological environment is usually reduced by the enzyme to form colorless leucomethylene blue which has negligible photodynamic activity [12]. To verify the hypothesis that the encapsulation of MB in CaP nanoparticles could prevent the reduction of MB from the enzyme, free MB and CaP-5MB were tested in DPBS (pH 7.4) with a diaphorase from Clostridium kluyveri and the cofactor NADH. The reduction behaviors of free MB and CaP-5MB were confirmed by monitoring the decrease of absorption at 663 nm. Fig. 5 exhibits the result of enzyme reduction test. In
contrast to the approximately 100% decrease of absorption intensity in free MB solution, CaP-5MB showed minimal decrease over half an hour. These results suggest that the encapsulated MB stayed mostly intact after the treatment, while free MB in solution was rapidly diminished by the enzyme. Therefore, CaP-5MB is appropriate for PDT in biological system to prevent the reduction of MB by the enzyme. 3.3. Singlet oxygen generation of CaP-MB nanoparticles To prove the self-quenching effect of CaP-MB nanoparticles, the generation of singlet oxygen by CaP-5MB in DMF and DPBS (pH 7.4) was determined using DMA as the singlet oxygen trap. CaP5MB produced a sharp decline in DMA fluorescent intensity, meaning the rapid generation of singlet oxygen upon laser irradiation when CaP-5MB was dispersed in DMF (Fig. 6). However, the
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Fig. 3. FT-IR spectra of (a) CaP-0MB and (b) CaP-5MB nanoparticles.
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Fig. 6. Fluorescence intensity change of DMA due to generation of singlet oxygen by CaP-5MB nanoparticles in DMF and DPBS.
under aqueous condition [26]. That is, during circulation in the blood (pH = 7.4), photodynamic activity of CaP-5MB may be suppressed by a self-quenching effect between MB molecules similar to the fluorescence resonance energy transfer (FRET) effect [27]. As the nanoparticles penetrate tissue and internalize in cancer cells, photodynamic activity may be recovered due to loss of the FRET effect by acidic dissolution of CaP-5MB in the cellular compartment such as lysosomes (pH = 4.5) as shown in Fig. 1(b) [28]. The self-quenching effect of the MB-loaded CaP nanoparticles is advantageous for improving the therapeutic efficiency and lowering the side effects of PDT. 3.4. Cellular uptake and in vitro phototoxicity of CaP-MB nanoparticles
Fig. 4. X-ray diffraction patterns of (a) CaP-0MB and (b) CaP-5MB nanoparticles.
PDT is a less invasive technique for cancer therapy. CaP nanoparticles offer benefit of appropriate size for the delivery of MB to malignant tissues via a passive targeting mechanism. To investigate the cytotoxicity of CaP-MB nanoparticles, the intracellular uptake of free MB and CaP-5MB into cancer cells was first assessed using a cell lysis test with RIPA buffer and UV–visible spectroscopy observation. The samples were incubated with human breast cancer cells, MCF-7 cells, for 1, 2, 4, 8, 16 and 24 h. CaP-5MB exhibited initial fast cellular uptake within 4 h and subsequent slow increase up to 24 h as shown in Fig. 7. Furthermore, the cellular uptake amount of CaP-5MB was conspicuously higher than that of free MB. Nanoparticle delivery
Fig. 5. Absorption change of free MB and CaP-5MB in the presence of 0.45 lM NADH and 50 lg diaphorase as function of time at 663 nm.
generation of singlet oxygen was significantly decreased in DPBS because of the aggregation of MB, showing the quenching effect. On the other hand, free MB showed considerable generation of singlet oxygen even in DPBS. These results imply that CaP-5MB could not be activated into the triplet state by mutual energy transfer, resulting in a decline of the ability to generate singlet oxygen
Fig. 7. Cellular uptake of free MB and CaP-5MB as a function of incubation time.
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system can increase their cell adhesion and cellular uptake of encapsulated drug molecules, compared to free drugs [29]. In addition, the ultrastructure of MCF-7 cells was observed by TEM for confirming the intracellular uptake of nanoparticles. The thin section TEM images clearly showed the incorporation of CaP-0MB and CaP-5MB nanoparticles into MCF-7 cells after 2 h incubation (Fig. 8), meaning that CaP nanoparticle could be used as a vehicle for the transportation of drugs. This high cellular uptake of CaP5MB may be closely related to the enhanced phototoxicity on the cancer cells. The photosensitizer should not exhibit cytotoxicity in the absence of light, whereas it generates singlet oxygen for therapy by the irradiation with an appropriated wavelength [2]. Accordingly, the in vitro phototoxicity of free MB and CaP-5MB on MCF-7 cells was compared after treatment with or without laser irradiation in order to estimate the PDT efficacy, which was assessed by MTT assay (Fig. 9). The data indicated no dark-toxicity of CaP-5MB in the range from 5 lg/mL to 50 lg/mL in comparison with control and cell viability was more than 92%. However, CaP5MB showed significantly enhanced phototoxicity after laser irradiation, which might be due to the heightened cellular uptake. 15 lg/mL of CaP-5MB caused approximately 60% cell death, demonstrating an obvious photodynamic activity. In the case of free MB, the cytotoxicity on MCF-7 cells was very high despite without light treatment. These results suggest that the encapsulation of MB in CaP nanoparticles can improve the PDT efficacy of MB on human cells, and extremely lessen side effects. Cancer cell viability after CaP-5MB treatment and laser irradiation was further confirmed by a fluorescence staining study
with calcein AM (green fluorescence) and EthD-1 (red fluorescence) to distinguish the live and dead cells. As shown in Fig. 10, MCF-7 cells appeared green fluorescence before laser irradiation, indicating live cells. However, MCF-7 cells treated with CaP-5MB and subsequent laser irradiation appeared red fluorescence due to the cell death. This is in good agreement with the MTT assay result of CaP-5MB. 3.5. Cell apoptosis induction by CaP-MB nanoparticles Apoptotic cells lose the asymmetry of membrane phospholipid, which leaves phosphatidylserine on the outer leaflet of the plasma membrane [30]. One of the tools for assaying apoptosis is to measure the phosphatidylserine externalization by binding of Annexin V. In this point of view, to reveal the phototoxicity mechanism of CaP-5MB on MCF-7 cells, an Annexin V study was carried out. From the fluorescence microscopy images, the early apoptotic cell death in MCF-7 cells was clearly observed after the CaP-5MB treatment and laser irradiation (Fig. 11). The apoptotic cells were stained green because of Annexin V-FITC binding at the outer membrane. Mitochondria are the major cellular component involved in the intrinsic pathway of apoptosis. In particular, permeabilization of the mitochondrial membrane is a critical event in the process leading to apoptotic cell death [31]. To obtain further support for the induction of apoptosis by the CaP-5MB treatment and laser irradiation in MCF-7 cells, permeabilization of the mitochondrial membrane and dissipation of the transmembrane potential were investigated by assessing incorporation of a lipophilic cationic
Fig. 8. The thin section TEM images of MCF-7 cells incubated with (a) CaP-0MB and (c) CaP-5MB nanoparticles for 2 h. (b) and (d) are the magnified images of the areas in (a) and (c) marked by circle. Arrows denote the CaP-0MB and CaP-5MB nanoparticles.
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Fig. 9. In vitro phototoxicity and dark-toxicity of different concentration of (a) free MB and (b) CaP-5MB on MCF-7 cells with or without laser irradiation.
Fig. 10. Live/Dead fluorescence microscopy images of MCF-7 cells stained with calcein-AM (green) and EthD-1 (red) in the presence of CaP-5MB (a) before irradiation and (b) after irradiation using a 670 nm diode laser (100 mW/cm2) for 5 min. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 11. Annexin V-FITC fluorescence microscopy images of MCF-7 cells treated with CaP-5MB (a) before irradiation and (b) after irradiation using a 670 nm diode laser (100 mW/cm2) for 5 min.
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Fig. 12. Microscopic observations on the effect of CaP-5MB-induced disruption of mitochondrial membrane potential in MCF-7 cells (a) before irradiation and (b) after irradiation using a 670 nm diode laser. The cells were stained with Depsipher and observed using an inverted fluorescence microscope at absorbance of 530 nm and 590 nm.
fluorochrome (DePsipher) into mitochondria. The marker entered and aggregated in the undisrupted mitochondria of untreated cells, in which it generated red fluorescence as shown in Fig. 12(a). The CaP-5MB treatment and laser irradiation provoked the disruption of mitochondrial membrane potential, resulting in the cytosol accumulation of the marker in monomeric form and the generation of green fluorescence (Fig. 12(b)). This is an advantageous result since apoptosis is an ordered process during an organism’s life cycle and takes place without the drastic effect of inflammation as observed for necrosis. Furthermore, the amount of released cytochrome c was carefully checked to certify the apoptotic cell death of MCF-7 cells by the CaP-5MB treatment and laser irradiation. Mitochondria also play a central role in the control of apoptosis by releasing the apoptogenic proteins such as apoptosis-inducing factor and cytochrome c into the cytosol [32]. Among them, cytochrome c release is a key step in activation of caspase cascade for initiation of apoptosis. As shown in Fig. 13, the amount of released cytochrome c remarkably increased by the CaP-5MB treatment and laser irradiation, which
might be due to a partial leakage of mitochondrial membrane triggered by the cytotoxicity of CaP-5MB. This result is in good agreement with previous result showing that PDT caused cytochrome c release from the mitochondria into the cytosol prior to caspase activation [32].
4. Conclusions Currently, the engineered nanoparticles have received attention as a possible means of encapsulating and delivering photosensitizers. Such nanostructured materials have enhanced photosensitizers loading and intracellular uptake of photosensitizers due to protecting the photosensitizers from environmental degradation. In the present study, a novel and simple reaction for the preparation of MB-loaded CaP (CaP-MB) nanoparticles was successfully developed by rapid precipitation from water in the presence of polymers which stabilized the nanoparticles. The MB content affected the zeta-potential of CaP-MB nanoparticles, but not the size of nanoparticles. The encapsulation of MB in CaP nanoparticles could effectively protect MB from the enzymatic reduction. Although the MB was encapsulated in CaP nanoparticles, it could be sufficiently excited by laser irradiation to generate singlet oxygen. The resulting CaP-5MB nanoparticles exhibited significant phototoxicity ascribed to apoptotic cell death, while their darktoxicity was negligible in the range of 5–50 lg/mL. In addition, the CaP-5MB treatment and laser irradiation gave rise to the disruption of mitochondrial membrane potential and the increased cytochrome c release. Based on these results, the CaP-MB nanoparticles may contribute to the development of a new generation of photosensitizer carriers for enhanced PDT treatment of cancers.
Acknowledgements
Fig. 13. Effect of CaP-0MB and CaP-5MB on cytochrome c release in MCF-7 cells with or without laser irradiation.
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2013R1A1A2006665).
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Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Enhanced photodynamic therapy efficacy of methylene blue-loaded calcium phosphate nanoparticles Da-Young Seong, Young-Jin Kim ⇑ Department of Biomedical Engineering, Catholic University of Daegu, Gyeongsan 712-702, Republic of Korea
a r t i c l e
i n f o
Article history: Received 19 August 2014 Received in revised form 17 February 2015 Accepted 19 February 2015 Available online 9 March 2015
a b s t r a c t Although methylene blue (MB) is the most inexpensive photosensitizer with promising applications in the photodynamic therapy (PDT) for its high quantum yield of singlet oxygen generation, the clinical use of MB has been limited by its rapid enzymatic reduction in the biological environment. To enhance PDT efficacy of MB by preventing the enzymatic reduction, we have developed a new mineralization method to produce highly biocompatible MB-loaded calcium phosphate (CaP-MB) nanoparticles in the presence of polymer templates. The resulting CaP-MB nanoparticles exhibited spherical shape with a size of under 50 nm. Fourier transform infrared (FT-IR) and zeta-potential analyses confirmed the insertion of MB into the CaP-MB nanoparticles. The encapsulation of MB in CaP nanoparticles could effectively protect MB from the enzymatic reduction. In addition, the CaP-MB nanoparticles exhibited a good biocompatibility in the dark condition and significantly enhanced PDT efficacy due to apoptotic cell death against human breast cancer cells as compared with free MB, implying that CaP-MB nanoparticle system might be potentially applicable in PDT. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Photodynamic therapy (PDT) has emerged as an important treatment modality for a variety of cancers, cardiovascular and ophthalmic diseases [1,2]. This modality involves the delivery of light-sensitive molecules called photosensitizers to target sites followed by irradiation with appropriate wavelength of light. Upon irradiation, the activated photosensitizers transfer their excess energy to the surrounding oxygen to form reactive oxygen species (ROS) such as singlet oxygen (1O2) or free radicals, which will cause irreversible damage of diseased cells and tissues. PDT is also expected to be a potential method of overcoming multidrug resistance (MDR) because cytotoxicity mechanism of photosensitizers onto cancer cells is different from that of other chemotherapy agents [3]. However, the clinical use of many photosensitizers has been hampered by their significant side effects including nonspecific damage to normal tissues due to low accumulation selectivity to specific cells or tissues, environmental degradation and hydrophobicity [4]. To solve these problems, various biocompatible nanocarrier systems for delivering photosensitizers such as liposomes, polymeric micelles and nanoparticles have been investigated [5– 7]. These nanocarriers have promoted uptake of photosensitizers into target sites and reduction of nonspecific damage to normal ⇑ Corresponding author. Tel.: +82 53 850 2512; fax: +82 53 359 6750. E-mail address: [email protected] (Y.-J. Kim). http://dx.doi.org/10.1016/j.jphotobiol.2015.02.022 1011-1344/Ó 2015 Elsevier B.V. All rights reserved.
tissues caused by free photosensitizers [8]. In addition, nanocarrier systems have offered stable aqueous dispersion of photosensitizers by surface modification and protected photosensitizers from environmental degradation [9]. Despite many advantages of the nanocarrier systems, the use of liposomes and micellar systems was limited because of their lower drug loading capacity and severe side effects like anaphylactic shocks [1,10]. Methylene blue (MB) is a phenothiazinium photosensitizer that has been employed in a variety of applications including PDT [9,11]. The high quantum yield of 1O2 generation by MB in the excitation of the therapeutic window (600–900 nm) makes MB a reasonable candidate for PDT. However, the clinical use of MB has been hindered by its propensity for rapid chemical alteration when systemically applied. MB is usually converted by accepting electrons from nicotinamide adenine dinucleotide (NADH)/nicotinamide adenine dinucleotide phosphate (NADPH) in the biological environment and the formed colorless leucomethylene blue has negligible photodynamic activity [12]. The presence of a transmembrane thiazine dye reductase at the cell surface is commonly recognized as the initial factor for MB reduction [13]. After cellular uptake of MB, the reduction also can be catalyzed by NADH/NADPH dehydrogenases. In this respect, the major obstacle for the use of MB in PDT applications is the difficulty in preparing pharmaceutical formulations that enable their facile administration. Therefore, biocompatible nanoparticles have received attention as an adequate means of encapsulating and delivering MB for PDT [9,11].
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Calcium phosphate (CaP) materials have been widely used in biomedical applications as useful drug carriers due to their excellent biocompatibility and bioactivity [14,15]. Among them, photosensitizers-incorporated CaP nanoparticles, which were synthesized in the presence of ionic polymers such as poly(ethylene imine) and poly(styrene sulfonic acid), exhibited a good phototoxicity against murine macrophages and bacteria [15]. In addition, CaP is absorbable in specific cellular environments (endosome/lysosome) as non-toxic ionic species [16]. Thus, the employment of controlled mineralization technology using self-assembled polymer templates would lead to the successful development of biocompatible and biodegradable nanocarriers of photosensitizers. The major purpose of this study was to enhance PDT efficacy of MB by increasing biostability such as the prevention of environmental degradation and enzymatic reduction under the biological conditions. For this reason, a new mineralization method was explored to produce highly biocompatible MB-loaded CaP (CaP-MB) nanoparticles in the presence of alginic acid sodium salt (alginate) and poly(ethylene glycol)–block–poly(propylene glycol)–block– poly(ethylene glycol) (PEG–PPG–PEG) triblock copolymer (Pluronic F-68) as polymer templates (Fig. 1). The prepared MBloaded CaP nanoparticles were systematically examined by considering their morphologies, chemical structures and particle size. The enzymatic reduction and yield of singlet oxygen generation were thoroughly investigated. Furthermore, the cellular internalization behavior and phototoxicity onto human breast cancer cell line (MCF-7) were evaluated via fluorescence microscopy and MTT assay.
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2. Materials and methods 2.1. Materials Methylene blue (MB), poly(ethylene glycol)–block–poly(propylene glycol)–block–poly(ethylene glycol) (PEG–PPG–PEG triblock copolymer, Pluronic F-68, Mn = 8400), alginic acid sodium salt (alginate), calcium nitrate tetrahydrate (Ca(NO3)24H2O), ammonium phosphate dibasic ((NH4)2HPO4), ammonium hydroxide solution (NH4OH), b-nicotinamide adenine dinucleotide, reduced dipotassium salt (NADH), diaphorase from C. kluyveri, paraformaldehyde and 9,10-dimethylanthracene (DMA) were purchased from Sigma–Aldrich (USA) and used without further purification. Human breast cancer cell line (MCF-7) was received from the Korean Cell Line Bank (KCLB, Korea). RPMI-1640, fetal bovine serum (FBS) and penicillin–streptomycin were obtained from Gibco BRL (USA). Slowfade gold antifade reagent and Live/Dead Viability/ Cytotoxicity assay kit were purchased from Molecular probes (USA). Annexin V-fluorescent isothiocyanate (FITC) fluorescence microscopy kit was from BD Biosciences (USA) and DePsipher kit was from Trevigen (USA). Human cytochrome c Quantikine ELISA kit was purchased from R&D Systems (USA). Other reagents and solvents were commercially available and were used as received.
2.2. Synthesis of MB-loaded CaP nanoparticles A synthesis of MB-loaded CaP (CaP-MB) nanoparticles is as follows. 1 w/v% MB solution was first added dropwise to 70 mL of
Fig. 1. Schematic images of methylene blue-loaded calcium phosphate (CaP-MB) nanoparticles for photodynamic therapy. (a) Synthetic scheme for CaP-MB nanoparticles and (b) schematic illustration of photoactivity mechanism for CaP-MB nanoparticles. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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2 w/v% Pluronic F-68 solution. To this solution, 10 mL of 0.2 w/v% alginate solution was added dropwise for preparing self-assembled polymer micelle nanotemplate solution containing drug. Then, 15 mL of 25 mM Ca(NO3)24H2O and 10 mL of 25 mM (NH4)2HPO4 solution were added dropwise in turn and pH was adjusted to 10 by the addition of NH4OH. The final concentration of MB in the reaction solutions was 0 (CaP-0MB), 1 (CaP-1MB), 5 (CaP-5MB) or 10 wt% (CaP-10MB) based on the weight of polymers and CaP precursors (Ca(NO3)24H2O and (NH4)2HPO4). The mixture was stirred at 40 °C under air to induce the nucleation and growth of CaP nanoparticles in the polymer micelle nanotemplates. After 24 h, the resultant CaP-MB nanoparticles were isolated by tubular membrane dialysis in distilled water (DW) for 12 h, followed by lypophilization in vacuo. 2.3. Characterization of nanoparticles The morphologies of CaP-MB nanoparticles were observed by transmission electron microscopy (TEM, H-7600, Hitachi, Japan). The average diameter of nanoparticles was determined by analyzing the TEM images with image analyzing software (Image-Pro Plus, Media Cybernetics Inc., USA). The zeta-potential of nanoparticles was determined using a Zetasizer Nano ZS (Malvern Instruments, UK). UV–visible spectra were recorded on a Hitachi U-2900 spectrometer (Japan) at 25 °C. Fourier transform infrared (FT-IR) spectra of the samples were obtained with an ALPHA spectrometer (Brucker Optics, USA) in the wavenumber range of 400–4000 cm–1. X-ray diffraction (XRD) measurements were carried out to characterize the crystalline phase of CaP-MB nanoparticles with a Panalytical X-ray diffractometer X’Pert Pro (Netherlands) with Cu Ka radiation at 40 kV/30 mA. The diffractograms were scanned in a 2h range of 20–60° at a rate of 2°/min.
2.6. Cell culture and incubation conditions For all of the experiments, human breast cancer cell line (MCF7) was used. MCF-7 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. Cells were incubated at 37 °C in humidified 5% CO2 atmosphere. When the cells reached 80% confluence, they were harvested by 0.25% trypsin–EDTA and seeded into a new tissue culture plate for subculture. The CaP-MB nanoparticles and the free MB were dispersed in serum-free medium. Untreated cells irradiated or kept in the dark were used as a reference standard. 2.7. Cellular uptake test To verify the cellular uptake of the free MB and the CaP-MB nanoparticles, a cell lysis test was utilized [18]. MCF-7 cells (1 106 cells/well) were seeded into 6 well plates in 2 mL of culture medium to estimate the concentration of cellular internalized MB using lysis buffer. After 24 h incubation, the cells were treated with the free MB or the CaP-5MB for 1, 2, 4, 8, 16 and 24 h at 37 °C. The MB concentration of CaP-5MB was adjusted to 10 lg/mL for comparison with 10 lg/mL of free MB. Then the cells were washed twice with DPBS, and treated with protein extraction solution (RIPA buffer) for cell lysis. The lysis solution was divided into the supernatant and the debris by centrifugation (12,000 rpm, 15 min). For the evaluation of cellular uptake amount of the free MB and the CaP-5MB, the absorbance of the supernatant at 653 nm was measured with a UV–visible spectrophotometer. Meanwhile, at 2 h after incubation with 30 lg/mL of CaP-0MB and CaP-5MB nanoparticles, the cells were rinsed with DPBS and made into TEM specimens, and observed via a TEM (H-7600, Hitachi, Japan) for visualizing the intracellular uptake of nanoparticles into cancer cells.
2.4. Enzymatic reduction test of MB To investigate the protection of CaP nanoparticles for the loaded MB against reduction by diaphorase, the enzymatic reduction test of MB in the nanoparticles has been conducted according to previously published procedures [9]. Solutions of 1.8 mg/mL CaP-5MB (1 mg/mL of MB) and 1 mg/mL free MB were prepared in Dulbecco’s phosphate buffered saline (DPBS, pH 7.4), respectively. Then, 0.45 lM of NADH and 50 lg diaphorase were sequentially added into 1 mL of CaP-5MB solution. The decrease in MB absorbance at 663 nm due to the reduction of the entrapped MB was measured at room temperature with incubation time up to half an hour, using a UV–visible spectrometer. The free MB solution was treated in the same way, and its absorption spectra were recorded for comparison. 2.5. Singlet oxygen detection Determination of singlet oxygen generation has been extensively reported by an indirect method using a chemical probe [17]. In this study, the generation of singlet oxygen from CaP-5MB under different conditions was detected using DMA as the singlet oxygen probe. CaP-5MB (0.3 mg/mL) was dispersed in N,N-dimethylformamide (DMF) or DPBS (pH 7.4) and then added to DMA stock solution to give a final concentration of 20 lM DMA. Samples containing CaP-5MB and DMA were irradiated at a light intensity of 50 mW/cm2 using a 670 nm laser source (LVI Technologies, Korea) for 1 min. The decrease in fluorescence intensity of DMA (excitation, 360 nm; emission, 430 nm) as a result of the photosensitization reaction was monitored with a Perkin-Elmer LS55 spectrofluorophotometer (USA). The singlet oxygen generation from free MB was also determined for comparison.
2.8. Cell phototoxicity assay To determine cell viability under dark condition, MCF-7 cells (1 104 cells/well) were seeded onto 96 well plates and incubated for 24 h at 37 °C. After cell stabilization, the culture medium was replaced with 200 lL of culture medium containing free MB or CaP-5MB (0–50 lg/mL of MB), followed by incubation for 4 h. The cells were then washed twice with serum-free medium and cell viability was evaluated by the MTT assay after 24 h. To determine in vitro phototoxicity after laser irradiation, MCF-7 cells (1 104 cells/well) were seeded onto 96 well plates and incubated for 24 h at 37 °C. Then, these cells were treated with free MB or CaP-5MB (0–50 lg/mL of MB). After 4 h incubation, the cells were washed twice with serum-free medium and irradiated with a 670 nm diode laser (100 mW/cm2) for 0–9 min onto 96 well plates. The cell viability of irradiated cells was evaluated by the MTT assay after 24 h incubation. Qualitative cell viability assay was performed by using the Live/ Dead Viability/Cytotoxicity assay kit. The kit contains calcein AM and ethidium homodimer-1 (EthD-1), which identifies live versus dead cells on the basis of membrane integrity and esterase activity. Calcein AM stains live cells green, whereas EthD-1 stains dead cells red [19]. MCF-7 cells (1 104 cells/well) were seeded onto 8 well chamber slide and incubated for 24 h at 37 °C. Then, these cells were treated with CaP-5MB (30 lg/mL of MB). After 4 h incubation, the cellular layers on the sample surface were rinsed twice with DPBS and irradiated with a 670 nm diode laser (100 mW/cm2) for 5 min, which were then treated for 10 min at 37 °C with 1 lM of calcein AM and 2 lM of EthD-1 to determine cell viability. Cells were finally observed using an inverted fluorescence microscope (Eclipse TS100, FITC-G2A filters, Nikon, Japan) equipped with a
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cooled CCD camera (DS-U2, Nikon, Japan) and with NIS-Elements Imaging Software. 2.9. Apoptotic analysis Cellular apoptosis was visualized using an Annexin V-FITC fluorescence microscopy kit. MCF-7 cells (1 104 cells/well) were seeded onto 8 well chamber slide for 24 h before being treated with drugs. The cells were incubated with CaP-5MB (30 lg/mL of MB) for 4 h, which were then rinsed twice with DPBS and illuminated with a 670 nm diode laser (100 mW/cm2) for 5 min. The cells were stained with l mL of Annexin V-FITC (10 wt%) for 15 min at room temperature. After staining, the cells were washed twice with DPBS and then fixed using 4% paraformaldehyde in DPBS for 30 min. A cover slip was mounted on a microscope slide with a drop of anti-fade mounting solution to reduce fluorescence photo-bleaching. Cells were observed with an inverted fluorescence microscope using a filter sect for FITC. 2.10. Assay for intracellular mitochondrial membrane potential Mitochondrial membrane potential in MCF-7 cells was analyzed using the Trevigen’s DePsipher kit. DePsipher is the fluorescent lipophilic cation 5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolcarbocyanine iodide which is used as a mitochondrial marker and detects early apoptosis in cells. The cells (1 104 cells/well) were seeded in 8 well chamber slide for 24 h and then incubated with CaP-5MB (30 lg/mL of MB) for 4 h, which were then rinsed twice with DPBS and illuminated with a 670 nm diode laser (100 mW/cm2) for 5 min. After irradiating, the cells were incubated with DePsipher solution at the concentration 5 lg/mL for 20 min at 37 °C, then washed with reaction buffer containing stabilizer, placed on a glass slide and covered with a glass cover slip. The stained cells were observed using an inverted fluorescence microscope equipped with FITC and G2A filters. 2.11. Determination of cytochrome c extrusion Cytochrome c was detected using the quantitative Quantikine ELISA kit (R&D systems). MCF-7 cells (1 106 cells/well) were seeded onto 6 well plates and allowed to adhere overnight. CaP0MB and CaP-5MB nanoparticles were added into the culture plates and incubated 2 h, respectively. After the incubation, the cells were washed twice with DPBS and irradiated with a 670 nm diode laser (100 mW/cm2) for 5 min. Then, the cells were harvested by centrifugation and suspended in lysis buffer. The cytosolic fraction was isolated by centrifugation for 15 min and used for cytochrome c assay. After reacting with cytochrome c conjugate and substrate, the absorbance was measured at 450 nm for calculating the amount of cytochrome c released from mitochondria to the cytosol. 3. Results and discussion 3.1. Morphology and physicochemical properties of MB-loaded CaP nanoparticles CaP nanoparticles are well known as carriers for the transport of genes and drugs into cells [20,21]. CaP is superior to other inorganic species such as silica in terms of biocompatibility because CaP is naturally found as the main mineral component in bone [14]. MB is a highly water-soluble near IR fluorescent dye. It was reported that the MB-doped silica nanoparticles showed weak fluorescence and suffered from dye leaching [22]. In the present study, the MB-loaded CaP (CaP-MB) nanoparticles were prepared by rapid
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precipitation from water in the presence of polymers which stabilized the nanoparticles as shown in Fig. 1(a). The resulting nanoparticles were named as CaP-0MB, CaP-1MB, CaP-5MB and CaP-10MB with the weight feed ratio of MB to polymers and CaP precursors in the reaction solutions (Table 1). Fig. 2 shows the morphological structure of CaP-MB nanoparticles. All of the resulting nanoparticles exhibited spherical shape with a size of under 50 nm and the size of nanoparticles was hardly affected by the content of MB. The average diameter of nanoparticles was 34.6 ± 4.2 nm for CaP-0MB, 33.2 ± 2.9 nm for CaP-1MB, 35.5 ± 4.6 nm for CaP-5MB and 41.5 ± 3.5 nm for CaP-10MB as shown in Table 1. The existence of alginate can provide a lot of binding sites giving rise to the accumulation of MB molecules and Ca2+ ions due to the strong ionic interaction and increasing the supersaturation near the negative positions. In addition, the formation of a specific stereo-chemical arrangement and the charge distribution of reactive groups in alginate–MB and alginate–Ca2+ complexes can proceed [23]. Then, alginate–Ca2+ complexes can strongly interact with the surface of PO3 ions to 4 nucleate the CaP-MB nanoparticles. As a result, the initial nucleation is preferentially triggered at the positions of carboxyl groups, and the particle size is related to the nucleation and growth. As photosensitizer, MB was chosen in this study because of its high singlet oxygen quantum yield and absorption/emission wavelength in near IR region with relatively efficient tissue penetration [9]. MB was readily loaded by the simple precipitation method in the presence of polymer templates. The amount of MB absorbed on the particles was determined by UV–visible spectroscopy. The loading efficiency of MB was 56.2% at 5 wt% feed ratio, which decreased to 38.8% at 10 wt% feed ratio (Table 1). Thus, we used CaP-5MB for further studies. FT-IR analysis was carried out for identifying the functional groups present in the CaP-MB nanoparticles, which in turn provided information about the constitution and phase composition of the products. As shown in Fig. 3, all of the samples synthesized in the absence and presence of MB exhibited characteristic absorption bands for the vibrational modes of PO3 4 appeared at around 1090, 1027, 960, 597 and 565 cm1, and the broad band centered at 3340 cm1 associated with OH of alginate and self-associated Pluronic F-68 [23,24]. In addition, the absorption bands for the asymmetric stretching mode of COO ion were observed at 1607 cm1, which was probably ascribed to alginate. Furthermore, the bands observed at 1420 and 879 cm1 are 3 attributed to the substitution of CO3 2 ions in the place of PO4 ions and thus can confirm the substitution of CO3 in CaP structure. 2 These CO3 ions were formed by the reaction of CO2 present in 2 the atmosphere with OH ions of reaction medium. In the case of CaP-5MB, new absorption bands were found at 1490 and 1333 cm1 assigned to aromatic nitro compound [25]. To confirm the incorporation of MB into the nanoparticles, the change of zeta-potential was also determined using 0.1 w/v% (1 mg/mL concentration) solutions of all samples, resulting that negative values of zeta-potential reduced by adding positively charged MB as shown in Table 1. These data clearly explain the insertion of MB into the CaP-MB nanoparticles. Crystallographic analysis was performed using XRD to elucidate the change in crystal structure of the CaP-MB nanoparticles. The CaP-0MB nanoparticles showed the peaks attributed to the hydroxyapatite (HA) crystalline phase at around 25.9°, 32.1°, 39.5°, 46.8°, 49.7° and 53.2°, which reflected characteristic of the (0 0 2), (2 1 1), (2 0 2), (2 2 2), (2 1 3) and (0 0 4) planes (Fig. 4) [23]. For the CaP-5MB nanoparticles, the intensity and position of the peaks arising from HA hardly changed, suggesting that the addition of MB did not affect the crystalline phases of HA. Additionally all the peaks were broad diffraction peaks indicating a poorly crystallized HA phase. This is owing to the complex formation of HA with
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Table 1 Characteristics of CaP-MB nanoparticles.
a b c d
Sample
MB feed ratio (wt%)a
Loading content (wt%)b
Loading efficiency (%)
Mean diameter of nanoparticles (nm)c
Zeta-potential (mV)d
CaP-0MB CaP-1MB CaP-5MB CaP-10MB
– 1 5 10
– 0.1 ± 0.02 2.8 ± 0.16 3.9 ± 0.09
– 10.0 ± 2.0 56.2 ± 3.2 38.8 ± 0.9
34.6 ± 4.2 33.2 ± 2.9 35.5 ± 4.6 41.5 ± 3.5
21.4 ± 1.8 18.6 ± 0.9 15.0 ± 1.1 13.6 ± 0.6
Weight feed ratio of MB to polymers and CaP precursors. Weight content of MB in CaP-MB nanoparticles, determined by UV–visible spectrophotometer. Determined by analyzing the TEM images with image analyzing software. Measured using a Zetasizer Nano ZS at 1 mg/mL concentration in PBS (pH 7.4).
Fig. 2. TEM micrographs of CaP-MB nanoparticles synthesized with different content of MB: (a) 0 wt% (CaP-0MB), (b) 1 wt% (CaP-1MB), (c) 5 wt% (CaP-5MB) and (d) 10 wt% (CaP-10MB).
amorphous polymer. Moreover, the isomorphous substitution of 3– PO3– 4 by CO2 derived from the absorption of CO2 in the air during preparation process of the CaP-MB nanoparticles affected the decrease of crystallinity. 3.2. Prevention of enzymatic reduction of MB by encapsulation MB in the biological environment is usually reduced by the enzyme to form colorless leucomethylene blue which has negligible photodynamic activity [12]. To verify the hypothesis that the encapsulation of MB in CaP nanoparticles could prevent the reduction of MB from the enzyme, free MB and CaP-5MB were tested in DPBS (pH 7.4) with a diaphorase from Clostridium kluyveri and the cofactor NADH. The reduction behaviors of free MB and CaP-5MB were confirmed by monitoring the decrease of absorption at 663 nm. Fig. 5 exhibits the result of enzyme reduction test. In
contrast to the approximately 100% decrease of absorption intensity in free MB solution, CaP-5MB showed minimal decrease over half an hour. These results suggest that the encapsulated MB stayed mostly intact after the treatment, while free MB in solution was rapidly diminished by the enzyme. Therefore, CaP-5MB is appropriate for PDT in biological system to prevent the reduction of MB by the enzyme. 3.3. Singlet oxygen generation of CaP-MB nanoparticles To prove the self-quenching effect of CaP-MB nanoparticles, the generation of singlet oxygen by CaP-5MB in DMF and DPBS (pH 7.4) was determined using DMA as the singlet oxygen trap. CaP5MB produced a sharp decline in DMA fluorescent intensity, meaning the rapid generation of singlet oxygen upon laser irradiation when CaP-5MB was dispersed in DMF (Fig. 6). However, the
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Fig. 3. FT-IR spectra of (a) CaP-0MB and (b) CaP-5MB nanoparticles.
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Fig. 6. Fluorescence intensity change of DMA due to generation of singlet oxygen by CaP-5MB nanoparticles in DMF and DPBS.
under aqueous condition [26]. That is, during circulation in the blood (pH = 7.4), photodynamic activity of CaP-5MB may be suppressed by a self-quenching effect between MB molecules similar to the fluorescence resonance energy transfer (FRET) effect [27]. As the nanoparticles penetrate tissue and internalize in cancer cells, photodynamic activity may be recovered due to loss of the FRET effect by acidic dissolution of CaP-5MB in the cellular compartment such as lysosomes (pH = 4.5) as shown in Fig. 1(b) [28]. The self-quenching effect of the MB-loaded CaP nanoparticles is advantageous for improving the therapeutic efficiency and lowering the side effects of PDT. 3.4. Cellular uptake and in vitro phototoxicity of CaP-MB nanoparticles
Fig. 4. X-ray diffraction patterns of (a) CaP-0MB and (b) CaP-5MB nanoparticles.
PDT is a less invasive technique for cancer therapy. CaP nanoparticles offer benefit of appropriate size for the delivery of MB to malignant tissues via a passive targeting mechanism. To investigate the cytotoxicity of CaP-MB nanoparticles, the intracellular uptake of free MB and CaP-5MB into cancer cells was first assessed using a cell lysis test with RIPA buffer and UV–visible spectroscopy observation. The samples were incubated with human breast cancer cells, MCF-7 cells, for 1, 2, 4, 8, 16 and 24 h. CaP-5MB exhibited initial fast cellular uptake within 4 h and subsequent slow increase up to 24 h as shown in Fig. 7. Furthermore, the cellular uptake amount of CaP-5MB was conspicuously higher than that of free MB. Nanoparticle delivery
Fig. 5. Absorption change of free MB and CaP-5MB in the presence of 0.45 lM NADH and 50 lg diaphorase as function of time at 663 nm.
generation of singlet oxygen was significantly decreased in DPBS because of the aggregation of MB, showing the quenching effect. On the other hand, free MB showed considerable generation of singlet oxygen even in DPBS. These results imply that CaP-5MB could not be activated into the triplet state by mutual energy transfer, resulting in a decline of the ability to generate singlet oxygen
Fig. 7. Cellular uptake of free MB and CaP-5MB as a function of incubation time.
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system can increase their cell adhesion and cellular uptake of encapsulated drug molecules, compared to free drugs [29]. In addition, the ultrastructure of MCF-7 cells was observed by TEM for confirming the intracellular uptake of nanoparticles. The thin section TEM images clearly showed the incorporation of CaP-0MB and CaP-5MB nanoparticles into MCF-7 cells after 2 h incubation (Fig. 8), meaning that CaP nanoparticle could be used as a vehicle for the transportation of drugs. This high cellular uptake of CaP5MB may be closely related to the enhanced phototoxicity on the cancer cells. The photosensitizer should not exhibit cytotoxicity in the absence of light, whereas it generates singlet oxygen for therapy by the irradiation with an appropriated wavelength [2]. Accordingly, the in vitro phototoxicity of free MB and CaP-5MB on MCF-7 cells was compared after treatment with or without laser irradiation in order to estimate the PDT efficacy, which was assessed by MTT assay (Fig. 9). The data indicated no dark-toxicity of CaP-5MB in the range from 5 lg/mL to 50 lg/mL in comparison with control and cell viability was more than 92%. However, CaP5MB showed significantly enhanced phototoxicity after laser irradiation, which might be due to the heightened cellular uptake. 15 lg/mL of CaP-5MB caused approximately 60% cell death, demonstrating an obvious photodynamic activity. In the case of free MB, the cytotoxicity on MCF-7 cells was very high despite without light treatment. These results suggest that the encapsulation of MB in CaP nanoparticles can improve the PDT efficacy of MB on human cells, and extremely lessen side effects. Cancer cell viability after CaP-5MB treatment and laser irradiation was further confirmed by a fluorescence staining study
with calcein AM (green fluorescence) and EthD-1 (red fluorescence) to distinguish the live and dead cells. As shown in Fig. 10, MCF-7 cells appeared green fluorescence before laser irradiation, indicating live cells. However, MCF-7 cells treated with CaP-5MB and subsequent laser irradiation appeared red fluorescence due to the cell death. This is in good agreement with the MTT assay result of CaP-5MB. 3.5. Cell apoptosis induction by CaP-MB nanoparticles Apoptotic cells lose the asymmetry of membrane phospholipid, which leaves phosphatidylserine on the outer leaflet of the plasma membrane [30]. One of the tools for assaying apoptosis is to measure the phosphatidylserine externalization by binding of Annexin V. In this point of view, to reveal the phototoxicity mechanism of CaP-5MB on MCF-7 cells, an Annexin V study was carried out. From the fluorescence microscopy images, the early apoptotic cell death in MCF-7 cells was clearly observed after the CaP-5MB treatment and laser irradiation (Fig. 11). The apoptotic cells were stained green because of Annexin V-FITC binding at the outer membrane. Mitochondria are the major cellular component involved in the intrinsic pathway of apoptosis. In particular, permeabilization of the mitochondrial membrane is a critical event in the process leading to apoptotic cell death [31]. To obtain further support for the induction of apoptosis by the CaP-5MB treatment and laser irradiation in MCF-7 cells, permeabilization of the mitochondrial membrane and dissipation of the transmembrane potential were investigated by assessing incorporation of a lipophilic cationic
Fig. 8. The thin section TEM images of MCF-7 cells incubated with (a) CaP-0MB and (c) CaP-5MB nanoparticles for 2 h. (b) and (d) are the magnified images of the areas in (a) and (c) marked by circle. Arrows denote the CaP-0MB and CaP-5MB nanoparticles.
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Fig. 9. In vitro phototoxicity and dark-toxicity of different concentration of (a) free MB and (b) CaP-5MB on MCF-7 cells with or without laser irradiation.
Fig. 10. Live/Dead fluorescence microscopy images of MCF-7 cells stained with calcein-AM (green) and EthD-1 (red) in the presence of CaP-5MB (a) before irradiation and (b) after irradiation using a 670 nm diode laser (100 mW/cm2) for 5 min. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 11. Annexin V-FITC fluorescence microscopy images of MCF-7 cells treated with CaP-5MB (a) before irradiation and (b) after irradiation using a 670 nm diode laser (100 mW/cm2) for 5 min.
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Fig. 12. Microscopic observations on the effect of CaP-5MB-induced disruption of mitochondrial membrane potential in MCF-7 cells (a) before irradiation and (b) after irradiation using a 670 nm diode laser. The cells were stained with Depsipher and observed using an inverted fluorescence microscope at absorbance of 530 nm and 590 nm.
fluorochrome (DePsipher) into mitochondria. The marker entered and aggregated in the undisrupted mitochondria of untreated cells, in which it generated red fluorescence as shown in Fig. 12(a). The CaP-5MB treatment and laser irradiation provoked the disruption of mitochondrial membrane potential, resulting in the cytosol accumulation of the marker in monomeric form and the generation of green fluorescence (Fig. 12(b)). This is an advantageous result since apoptosis is an ordered process during an organism’s life cycle and takes place without the drastic effect of inflammation as observed for necrosis. Furthermore, the amount of released cytochrome c was carefully checked to certify the apoptotic cell death of MCF-7 cells by the CaP-5MB treatment and laser irradiation. Mitochondria also play a central role in the control of apoptosis by releasing the apoptogenic proteins such as apoptosis-inducing factor and cytochrome c into the cytosol [32]. Among them, cytochrome c release is a key step in activation of caspase cascade for initiation of apoptosis. As shown in Fig. 13, the amount of released cytochrome c remarkably increased by the CaP-5MB treatment and laser irradiation, which
might be due to a partial leakage of mitochondrial membrane triggered by the cytotoxicity of CaP-5MB. This result is in good agreement with previous result showing that PDT caused cytochrome c release from the mitochondria into the cytosol prior to caspase activation [32].
4. Conclusions Currently, the engineered nanoparticles have received attention as a possible means of encapsulating and delivering photosensitizers. Such nanostructured materials have enhanced photosensitizers loading and intracellular uptake of photosensitizers due to protecting the photosensitizers from environmental degradation. In the present study, a novel and simple reaction for the preparation of MB-loaded CaP (CaP-MB) nanoparticles was successfully developed by rapid precipitation from water in the presence of polymers which stabilized the nanoparticles. The MB content affected the zeta-potential of CaP-MB nanoparticles, but not the size of nanoparticles. The encapsulation of MB in CaP nanoparticles could effectively protect MB from the enzymatic reduction. Although the MB was encapsulated in CaP nanoparticles, it could be sufficiently excited by laser irradiation to generate singlet oxygen. The resulting CaP-5MB nanoparticles exhibited significant phototoxicity ascribed to apoptotic cell death, while their darktoxicity was negligible in the range of 5–50 lg/mL. In addition, the CaP-5MB treatment and laser irradiation gave rise to the disruption of mitochondrial membrane potential and the increased cytochrome c release. Based on these results, the CaP-MB nanoparticles may contribute to the development of a new generation of photosensitizer carriers for enhanced PDT treatment of cancers.
Acknowledgements
Fig. 13. Effect of CaP-0MB and CaP-5MB on cytochrome c release in MCF-7 cells with or without laser irradiation.
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2013R1A1A2006665).
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