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Vitamin Bc-Bearing Hydrophilic Photosensitizer Conjugate for Photodynamic Cancer Theranostics Jiyoung Kim, Kyoung Sub Kim, Sin-jung Park, Kun Na* The accurate diagnosis and proper therapy for cancer are essential to improve the success rate of cancer treatment. Here, we demonstrated that the vitamin Bc-bearing hydrophilic photosensitizer conjugate folic acid-polyethylene glycol-pheophorbideA (FA-PEG-PheoA) has been synthesized for the intracellular diagnosis and photodynamic therapy of a tumor. The synthesized vitamin Bc-bearing hydrophilic photosensitizer conjugate has been characterized for the folic acid receptor expressing the ability to target tumor cells, which is facilitated by the chemical conjugation with folic acid. The vitamin Bc-bearing hydrophilic photosensitizer conjugate internalization mechanism was identified through a competitive inhibition test with free folic acid. We optimized the laser-sensitive, cytotoxicity changeable, vitamin Bcbearing hydrophilic photosensitizer conjugate concentration, which is non-cytotoxic under normal conditions and specifically cytotoxic toward cancer cells (maximum 69.15%) under laser irradiation conditions used for theranostic agents. The cancer therapeutic and diagnosis effects of synthesized conjugate were confirmed in MDA-MB-231 cells and MDA-MB-231-bearing mice. As a result, the vitamin Bc-bearing hydrophilic photosensitizer conjugate exhibited a highly photodynamic therapeutic effect, which enabled the selective detection of a folic acid receptor expressing cancer using optical imaging.
1. Introduction The early diagnosis and treatment of cancer are important for increasing the survival rate of cancer patients and the success rate of cancer treatment.[1–3] Many researchers have worked intensively to identify the causes and properties of cancer,[4–10] which has led to many improved cancer treatment strategies.[11–17] For instance, chemotherapy,[18] J. Kim, K. S. Kim, S.-j. Park, K. Na Department of Biotechnology, Center for Photomedicine, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon-si Gyeonggi do 420-743, Korea E-mail: [email protected] Macromol. Biosci. 2015, 15, 1081–1090 ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
radiation therapy,[19] targeted therapy,[20] immunotherapy,[21] and hyperthermia[22] have become typical methods for cancer therapy. Recently, various theranostic agents, which involve both diagnostic and therapeutic functions in one combined system, have been developed and improved to specifically visualize and cure cancer cells simultaneously.[23–25] Organic and inorganic materials, including dendrimers,[26] liposomes,[27] polymeric micelles,[28] iron oxide nanoparticles,[29,30] gold nanoparticles,[31] gold nanorods,[32] and carbon nanotubes,[33] have been used as carriers of therapeutic agents. However, to fabricate these theranostic agents, researchers need to conduct complicated processes, due to the various components of theranostic agents, such
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DOI: 10.1002/mabi.201500060
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as fluorescent dyes, chemical drugs, and CT/MR imaging materials. In spite of notable developments in medical technologies, cancer still remains an incurable disease.[34] Photosensitizers (PSs) are photosensitive molecules and important components of photodynamic therapy (PDT). [35] The PSs absorb irradiated photon energy, transfer it to nearby oxygen molecules, and produce reactive oxygen species (ROS). [36,37] Upon irradiation, intrinsic fluorescence and ROS generation of PSs can be used to conduct cancertargeted optical imaging and further clinical treatment.[38,39] As mentioned earlier, PDT is a type of treatment that utilizes PSs and light with the appropriate wavelength.[40,41] PDT is promising for the treatment of various cancers and other diseases because it is a minimally invasive and tissue selective treatment, and it has a high efficiency in producing cell death, vascular shutdown, and immune response activation.[42,43] In this study, pheophorbideA (PheoA), a second-generation PDT agent and a derivative of chlorophyll a,[44,45] was used to photodynamically diagnose and apply therapy to cancer cells at the same time. In contrast to conventional theranostic agents, we synthesized folic acid-polyethylene glycol-pheophorbideA (FA-PEG-PheoA) conjugate through simple conjugation processes without the addition of chemical drugs and fluorescent dyes. In addition, the FAPEG-PheoA conjugate includes vitamin Bc, which can bind to the cancer cell membrane overexpressing receptor. The synthesized FA-PEG-PheoA conjugate was confirmed by a 1H NMR spectrometer. The singlet oxygen generation efficacy of FA-PEG-PheoA under laser irradiation was confirmed by a RF-spectrofluorometer. Then, the cellular internalization behavior and imaging efficacy of FA-PEG-PheoA conjugate were observed with CLSM and intracellular fluorescence intensity. The therapeutic efficacy of FA-PEG-PheoA conjugate was observed with MTT and LIVE/DEAD assays. At last, to evaluate the in vivo feasibility of FA-PEG-PheoA, we performed an in vivo cancer targeting study in MDA-MB-231 tumor-bearing mice model with in vivo imaging station. Thus, the synthesized FA-PEG-PheoA conjugate can be easily applied to early diagnosis and treatment of cancer.
2.2. Synthesis and Characterization of PEG-PheoA The conjugation of PEG/PheoA via DCC and NHS-mediated amide formation was previously reported by our group.[16] Briefly, PEG and PheoA were conjugated in DMSO as described below. The PEG powder (400 mg and 66 mmol) was dissolved in DMSO (20 mL). PheoA (52 mg and 87 mmol corresponding to a molar ratio of PheoA to PEG of 1.3) was dissolved in DMSO (10 mL), followed by the addition of a 1.3 molar equivalent of DCC and NHS, while the PEG was dissolved in DMSO via vigorous stirring. After allowing the PEG and PheoA solutions to completely dissolve, they were mixed slowly, and the reaction mixture was gently stirred for 24 h at room temperature. The reaction mixture was dialyzed (Mw cutoff: 3 500 Da) for 3 d against distilled water to remove unconjugated PheoA and DMSO. The final solution was flash-frozen dry and lyophilized. The 1H NMR spectrum was recorded in deuterated dimethyl sulfoxide (DMSO-d6) at room temperature using a 500 MHz Bruker NMR Spectrometer (Bruker, Germany).
2.3. Purification of PEG-PheoA The purification of PEG-PheoA via open column chromatography was previously reported by our group.[38] For the purification, hydrophobic chromatographic columns were used; the crude products (PEG-PheoA) were chromatographed using an open column filled with Sephadex LH-20 as the stationary phase, and methanol served as the mobile phase. After loading the crude products, the flow rate was set to 0.5 mL min 1 with the mobile phase starting from 50% methanol (5:5 MeOH in water) (0–50 min) and proceeding to 80% methanol (8:2 MeOH in water) (51–95 min). Each of the fractions was collected and obtained as pure products (PEG-PheoA and PheoA-PEG-PheoA) and their absorbances were measured at 665 nm.
2. Experimental Section
2.4. Synthesis and Characterization of FA-PEG-PheoA
2.1. Materials
The PEG-PheoA and FA were conjugated in DMSO, as described below. The FA (30 mg and 67 mmol) was dissolved in DMSO (3 mL). PEG-PheoA (30 mg and 4.5 mmol, corresponding to a molar ratio of FA to PEG-PheoA of 15) was dissolved in DMSO (10 mL), followed by the addition of 2 molar equivalents of DCC and NHS, while the PEGPheoA was dissolved in DMSO via vigorous stirring.[46] After allowing the FA and PEG-PheoA solutions to completely dissolve, they were mixed slowly, and the reaction mixture was gently stirred for 24 h at room temperature. The reaction mixture was dialyzed (Mw cutoff: 3 500 Da) for 3 d against distilled water to remove unconjugated reactants and DMSO. The final solution was
PheophorbideA (PheoA) was purchased from Frontier Scientific, Inc. (Salt Lake City, UT, USA). Polyethylene glycol diamine (PEG) with a molecular weight of 6 000 Da, folic acid (FA), 1, 3-dicyclohexyl carbodiimide (DCC), N-hydroxysuccinimide (NHS), potassium bromide (KBr), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), methanol (MeOH), dimethyl sulfoxide anhydrous (DMSO), Sephadex LH-20, and 9, 10-dimethylanthracene (DMA) were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). The dialysis membrane was obtained from Spectrum
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Laboratories, Inc. (Rancho Dominguez, CA, USA). RPMI 1640 medium without folic acid, fetal bovine serum (FBS), antibiotics (penicillin/streptomycin), and Dulbecco’s phosphate buffered saline (DPBS) were obtained from Gibco BRL (Invitrogen Corp., Carlsbad, CA, USA). A LIVE/DEAD Viability/Cytotoxicity kit was purchased from Molecular Probes, Inc. (OR, USA). MDA-MB-231 (human breast carcinoma cell line) was obtained from the Korean Cell Line Bank (KCLB no. 30026).
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flash-frozen dry and lyophilized. The 1H NMR spectrum was recorded in deuterated dimethyl sulfoxide (DMSO-d6) at room temperature using a 500 MHz Bruker NMR Spectrometer (Bruker, Germany).
2.5. Measurement of Singlet Oxygen Generation (SOG) The generation of singlet oxygen (1O2) was observed chemically by the detection of 9, 10-dimethylanthracene (DMA, singlet oxygen quencher) utilizing fluorescent spectroscopy as an independent method. DMA (20 mM) was mixed with FA-PEG-PheoA in distilled water (DW). The solution was irradiated with a 670 nm laser source at 3 Jcm 2. Singlet oxygen-induced reduction of DMA fluorescence intensity (Ex 360 nm and Em 380-550 nm) was recorded at 0–40, and 45 min using an RF-spectrofluorometer (RF-5301PC, Shimadzu, Japan). The FA-PEG-PheoA in the DW without irradiation was used as the standard.
2.6. Cell Culture and Incubation Conditions MDA-MB-231 (human breast carcinoma) cells obtained from the Korean Cell Line Bank (KCLB no. 30026) were cultured in 10 mL of RPMI 1640 medium without a folic acid supplementation with 10% FBS and 1% penicillin/streptomycin. The cells were cultured at 37 8C in 100% humidity and at 5% CO2. PEG-PheoA and FA-PEG-PheoA were dissolved in serum-free (SF) medium. All of the reported concentrations refer to free PheoA equivalents. The untreated cells were irradiated or kept in the dark and used as a reference standard.
2.7. In Vitro Cytotoxicity of FA-PEG-PheoA MDA-MB-231 (1 104 cells well 1) were seeded onto 96-well plates and allowed to attach for 1 d. Next, FA-PEG-PheoA (diluted to various concentrations (0–1 mg mL 1)) in 100 mL of SF medium was added to each well and the plates were returned to the incubator for 3 h. After incubation, the wells were rinsed twice with DPBS, and fresh culture medium was added to each well. The cells were then incubated for an additional 24 h. The cell viability was assessed using the MTT assay. The resulting formazan crystals were dissolved in DMSO (100 mL), and absorbance intensity was measured at 570 nm using a microplate reader (Bio-Tek, VT, USA).
2.8. In Vitro Cellular Uptake Test To verify the concentration, where one can visualize the cellular uptake of FA-PEG-PheoA, MDA-MB-231 cells (1 105 cells well 1 in a 12-well plate) were treated with FA-PEG-PheoA in various concentrations (0–1 mg mL 1) for 3 h at 37 8C. The cells were then washed twice with DPBS, fixed with 4% paraformaldehyde, and visualized using a confocal laser scanning microscope (CLSM, LSM 710 Meta; Zeiss, Germany).
2.9. Competition Intracellular Uptake Test To identify the FA receptor-mediated internalization and competitive inhibition concentration by free FA, MDA-MB-231 cells
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(1 105 cells well 1 in a 12-well plate) were treated with FA-PEGPheoA in various concentrations of free FA (0.00–0.25 mg mL 1) for 3 h at 37 8C. The cells were then washed, harvested, and dissolved with DMSO. Each 100 mL of FA-PEG-PheoA was then loaded on the plate. The fluorescence spectra were analyzed at room temperature using a plate reader (Tecan Genios, NC, USA) with an excitation wavelength of 600 nm and an emission wavelength of 675 nm. The cells were then washed twice with DPBS, fixed with 4% paraformaldehyde, and visualized using a CLSM. To observe the subcellular localizations of PEG-PheoA and FA-PEGPheoA, MDA-MB-231 cells (1 105 cells well 1 in a 12-well plate) were treated with PEG-PheoA and FA-PEG-PheoA in the presence or absence of free FA for 3 h at 37 8C. The cells were then washed twice with DPBS, fixed with 4% paraformaldehyde, and visualized using CLSM. An optimal pinhole size of 120 mm was selected to exclude fluorescent light emitted from out-of-focus planes above and below the focusing plane. An objective with a magnification of 1 200 was used for image capture. A laser line with a wavelength of 630 nm was used for the excitation of DAPI and a He-Ne laser line was used for excitation of PheoA. A long-pass filter (LP 650 nm) was used at the emission end for detection of PheoA. Fluorescence images were analyzed using LSM Image Browser software (Zeiss). To investigate the cellular uptake of PEG-PheoA and FA-PEGPheoA, MDA-MB-231 cells were exposed with PEG-PheoA and FAPEG-PheoA in the presence or absence of free FA. The cells were incubated for 3 h, washed, harvested, and dissolved with DMSO. Each 100 mL of PEG-PheoA and FA-PEG-PheoA was then loaded on the plate. Fluorescence spectra were analyzed at room temperature using a plate reader (Tecan Genios, NC, USA) with an excitation wavelength of 600 nm and emission wavelength of 675 nm.
2.10. In Vitro Cytotoxicity of PEG-PheoA and FA-PEGPheoA with Laser Irradiation To assess the cytotoxicity of PEG-PheoA and FA-PEG-PheoA, MDAMB-231 cells (1 104 cells well 1) were added to a 96-well plate in 100 mL complement medium and incubated overnight. The samples, in 100 mL SF medium, were added to each well and the plates were returned to the incubator for 3 h. After incubation, the wells were rinsed three times with DPBS to remove any PEGPheoA or FA-PEG-PheoA that had not been internalized into the cells. Then, complement medium (100 mL) was added to the wells, and each well was irradiated using the 670 nm laser source (0–4 Jcm 2). The cells were then incubated for an additional 24 h. After incubation, the cell viability was assessed using the MTT assay. The resulting formazan crystals were dissolved in DMSO (100 mL) and transferred to a new plate. The absorbance intensity was measured at 570 nm using a microplate reader (Bio-Tek, VT, USA). For the live and dead cell assay, the LIVE/DEAD Viability/ Cytotoxicity Assay kit (Molecular Probes, USA) provides a two-color fluorescence cell viability assay. The optimal dyes are 2 mM Calcein AM and 4 mM EthD-1; the former stains live cells green, whereas the latter stains dead cells red. MDA-MB-231 cells were seeded in 35-mm cell culture dishes at a density of 1 106 cells well 1, respectively. The cell culture dishes were incubated overnight at 37 8C in a 5% CO2 incubator. Next, the medium was removed and the cells were incubated in SF medium containing PEG-PheoA or FA-PEG-PheoA for 3 h. The medium was then removed and the cells
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were rinsed twice with DPBS. Laser irradiation (0–4 Jcm 2) was performed with a 670 nm laser source. Then, the cells were incubated in complement medium for 4 h. The cell viability was observed by fluorescence microscopy (Zeiss, Germany).
tissues, the mice were sacrificed at 24 h tail vein injection and each tissue was excised. The fluorescence intensity of PheoA in each tissue was measured using a 12-bit CCD camera.
2.14. Statistical Analysis
2.11. In Vivo Animal Experiments All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Catholic University of Korea (Republic of Korea) in accordance with the ‘‘Principles of Laboratory Animal Care,’’ NIH publication no. 85-23 (revised in 1985). We used 4–6-week-old female Balb/c nude mice (Orient Bio, Seongnam, Korea) for the tumor diagnosis.
The data are expressed as the mean standard deviation (SD). The differences between the values were assessed using Student’s ttest.
3. Results and Discussion 3.1. Synthesis and Characterization of PEG-PheoA
2.12. In Vivo Tumor Diagnosis Using Optical Imaging MDA-MB-231 cells (1 10 cells mice ) were subcutaneously transplanted into Balb/c nude mice (five weeks of age). When the tumors grew to approximately 10 mm3 in volume, PheoA and FAPEG-PheoA (PheoA, 0.2 mg kg 1) were intravenously injected into the MDA-MB-231 bearing mice tail vein. The optical images were obtained using a 12-bit CCD camera (Image Station 4 000 MM; Kodak, New Haven, CT, USA) equipped with a special C-mount lens and a long wave emission filter (600–700 nm; Omega Optical). 6
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2.13. Ex Vivo Biodistribution Study To compare the biodistribution of PheoA and FA-PEG-PheoA in normal tissues (heart, lung, spleen, liver, and kidney) and tumor
To prove our goal, the polyethylene glycol-pheophorbideA (PEG-PheoA) was synthesized via a conventional carbodiimide reaction, as shown in Figure 1, and according to a previous report of our group.[16] The photosensitizer PEGylation is a process that provides a hydrophilic property and enhances photoactivity in water.[42,43] The synthesized PEG-PheoA was confirmed using 1H NMR analysis (Figure 2a). We performed purification of the synthesized PEG-PheoA using a hydrophobic chromatographic Sephadex LH-20 column via a separation process mentioned in our group report.[38] The PEG-PheoA and PheoA-PEG-PheoA were detected with a UV/vis spectrophotometer and eluted in the first and second peak, respectively (Figure 2b). The PheoA-PEG-PheoA has very low solubility in water
Figure 1. Synthetic route of PEG-PheoA and FA-PEG-PheoA.
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Figure 2. In vitro chemical properties of PEG-PheoA and FA-PEG-PheoA. (a) 1H NMR spectrum of PEG-PheoA. The synthesized PEG-PheoA was confirmed when –CH of PheoA appeared at 8.92, 9.48, and 9.81 ppm. (b) Purification of PEG-PheoA by the hydrophobic interaction chromatography of Sephadex LH-20 resin. The absorbance of purified PEG-PheoA and PheoA-PEG-PheoA at 667 nm and photographs of the PEG-PheoA (1) and PheoA-PEG-PheoA (2) solution. (c) 1H NMR spectrum of FA-PEG-PheoA. The synthesized FA-PEG-PheoA was confirmed when the –CH of FA appeared at 7.64 and 8.64 ppm. (d) Singlet oxygen generation (1O2) of FA-PEG-PheoA detected by DMA in DW. All of the irradiations were performed using 670 nm of light at 3 Jcm 2.
compared to PEG-PheoA, which is unsuitable for a clinical application. Different from PheoA-PEG-PheoA, biarmed mPEG-PheoA conjugate was self-assembled, accumulated in tumor site, and used for PDT.[47] In this study, we applied vitamin Bc to the hydrophilic photosensitizer to enable targeting of the FA receptor in overexpressed cancer cells and furthermore photodynamic diagnosis and therapy of cancer.[48,49]
difference in the DMA fluorescence, indicating that it does not produce singlet oxygen under aqueous conditions.[50] In this experiment, mixed DMA with FA-PEG-PheoA dispersed in DW without irradiation was used as the standard. FAPEG-PheoA completely dissolved in DW resulted in a decline in DMA fluorescence intensity, indicating the generation of singlet oxygen upon laser irradiation. This result denotes that FA-PEG-PheoA conjugate can be used to apply therapy under laser irradiation.
3.2. Synthesis and Characterization of FA-PEG-PheoA FA-PEG-PheoA was synthesized with Purified PEG-PheoA and confirmed using 1H NMR (Figure 2c). In Figure 2d, the singlet oxygen generation efficacy of FA-PEG-PheoA was detected using DMA (singlet oxygen quencher) as a detector. DMA reacts irreversibly with 1O2 in many organic solvents and water, which causes a decrease in the intensity of the DMA absorption band at 360 nm. Generally, free PheoA in DW aggregated easily and did not show a
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3.3. In Vitro Cytotoxicity Test of FA-PEG-PheoA To utilize FA-PEG-PheoA as diagnostic agent, we optimized the nontoxic concentration of FA-PEG-PheoA in MDA-MB231 cells.[51] A quantitative in vitro cell cytotoxicity test was performed using the methyl thiazolyl tetrazolium (MTT) colorimetric assay. For various concentrations of FA-PEGPheoA conjugate (0–1 mg mL 1) in MDA-MB-231 cells, the results showed a concentration dependent cytotoxicity
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Figure 3. In vitro cytotoxicity and cellular localization of FA-PEG-PheoA at various concentrations. (a) In vitro cytotoxicity test of FA-PEGPheoA against MDA-MB-231 cells after treatment with FA-PEG-PheoA in various concentrations. (b) CLSM-imaged cellular internalization of various FA-PEG-PheoA concentrations (0–1 mg mL 1) in MDA-MB-231 cells with 3 h of incubation. The scale bar is 20 mm.
(Figure 3a). Overall, the cytotoxicity was indicated when the FA-PEG-PheoA concentration was more than 0.05 mg mL 1.
3.4. In Vitro Cellular Uptake Test To identify the appropriate concentration of FA-PEG-PheoA for cancer cell diagnosis, cellular internalization of the FAPEG-PheoA conjugate was examined using CLSM, based on the fluorescence of PheoA (Figure 3b). The fluorescence of FAPEG-PheoA was observed when the concentration was more than 0.05 mg mL 1. The previous MTT assay result and CLSM images suggest that 0.05 mg mL 1 of FA-PEG-PheoA is nontoxic and suitable for cancer cell diagnosis. In this study, the dose of FA-PEG-PheoA was fixed at 0.05 mg mL 1.
3.5. Cellular Internalization Mechanism of FA-PEGPheoA To prove the FA receptor-mediated cellular internalization mechanism of FA-PEG-PheoA and optimal concentration of
free FA, we monitored cellular internalization through CLSM and intracellular fluorescence intensity in MDAMB-231 cells with various concentrations of free FA (0–0.25 mg mL 1) (Figure 4a). FA is known to have a specific binding affinity to the FA receptor, which is overexpressed in various cancer cells and is used as a targeting moiety in this study.[46,48–50] As the concentration of free FA increases, the uptake of FA-PEG-PheoA in MDAMB-231 cells decreased. In addition, the red fluorescence signal of FA-PEG-PheoA was also decreased. In Figure 4b, the intracellular fluorescence intensity of FA-PEG-PheoA decreased as the free FA concentration increased. These results indicate that the free FA competitively inhibited FA receptor-mediated internalization of FA-PEG-PheoA conjugate in MDA-MB-231 cells. To ensure the competitive inhibition of free FA, we fixed the concentration of free FA at 0.25 mg mL 1. Next, the cellular internalization of PEG-PheoA and FA-PEGPheoA in the presence or absence of free FA was confirmed using CLSM. Regardless of free FA pre-treatment, PEG-PheoA showed a weak red fluorescence signal due to low cellular internalization efficiency (Figure 5a).
Figure 4. Change of intracellular fluorescence intensity by free FA. (a) CLSM image and (b) intracellular fluorescence intensity of FA-PEGPheoA with various concentrations of free FA (0.00–0.25 mg mL 1). The scale bar is 20 mm.
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Figure 5. In vitro cellular internalization and intracellular fluorescence intensity of PEG-PheoA and FA-PEG-PheoA in the presence or absence of free FA (conc; 0.25 mg mL 1). CLSM-imaged cellular internalization of (a) PEG-PheoA and (b) FA-PEG-PheoA in MDA-MB-231 cells with the presence or absence of free FA and 3 h of incubation. The scale bar is 20 mm. (c) Fluorescence intensity of PEG-PheoA and FA-PEG-PheoA in MDA-MB-231 cells.
However, the FA-PEG-PheoA showed a free FA dependent fluorescence increase and decrease (Figure 5b). In free FA pre-treatment, the FA-PEG-PheoA fluorescence signal was decreased by competitive inhibition of free FA. In contrast, the fluorescence signal of free FA non-treated FA-PEG-PheoA was significantly increased. These results indicate that FA performed the role of selective targeting moiety and functioned as an internalization route for the FA receptor in overexpressed cancer cells. In addition, these results were reconfirmed with intracellular fluorescence intensity in MDA-MB-231 cells (Figure 5c). Likewise CLSM images, these results suggested that the fluorescence intensity of free FA non-treated FA-PEGPheoA was approximately fourfold higher than free FAtreated FA-PEG-PheoA.
shows that the laser power dependent cytotoxicity change of FA-PEG-PheoA treated cells. Specifically, when FA-PEG-PhoeA-treated cells were irradiated with 4 Jcm 2, the cytotoxicity increased to 70%. And these results were also confirmed with the LIVE/DEAD assay (Figure 6d). As shown in the LIVE/DEAD assay, without laser irradiation, only live cells were indicated by green fluorescence. However, at a laser power above 1 Jcm 2, the LIVE/DEAD images show dead cells and DNA fragments as a red fluorescence and damaged live cells as a dim green fluorescence. These results demonstrated that the FAPEG-PheoA, which was internalized in MDA-MB-231 cells through the FA receptor, could generate singlet oxygen molecules under laser irradiation, which induced cell membrane peroxidation and cell death.[39]
3.6. In Vitro Cytotoxicity Test with Laser Irradiation
3.7. In Vivo Tumor Diagnosis Efficacy and Body Distribution of FA-PEG-PheoA
To investigate the therapeutic efficacy of FA-PEG-PheoA conjugate compared with PEG-PheoA, we measured the cytotoxicity through various doses of irradiation in MDAMB-231 cells. The viability of irradiated MDA-MB-231 cells was determined by MTT and LIVE/DEAD assays (Figure 6). The MDA-MB-231 cells treated with PEG-PheoA (conc; 0.05 mg mL 1) and FA-PEG-PheoA (conc; 0.05 mg mL 1) were irradiated with 0–4 J/cm2 of various laser powers. As shown in Figure 6a and b, cytotoxicity was not observed after treatment with PEG-PheoA, regardless of the photo irradiation power. The PEG-PheoA has a low internalization efficacy in MDA-MB-231 cells because of the hydrophilic nature of PEGylated PSs; PEG-PheoA hardly passed through the hydrophobic cell membrane and does not have cancer cell specific targeting moiety. In contrast, the FA-PEG-PheoA exhibited significantly enhanced cytotoxicity in MDA-MB-231 cells. Figure 6c
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To confirm the in vivo tumor diagnosis efficacy, we injected PheoA and FA-PEG-PheoA intravenously into MDA-MB-231 tumor bearing Balb/c nude mice. After injection of PheoA and FA-PEG-PheoA (PheoA conc; 0.2 mg kg 1) into the tail vein, the time-dependent biodistribution of PheoA and FA-PEG-PheoA was observed using an in vivo optical imaging station (Figure 7a). In the case of PheoA, a strong fluorescence signal was detected in the liver tissues, but tumor specificity was not observed. Hydrophobic PheoA is not dispersed in the aqueous phase and aggregated in body fluid easily. Therefore, aggregated PheoA was rapidly cleared from the body by the first-pass effect and hardly accumulated in the tumor tissues. In contrast, the fluorescence signal of the tumor in FA-PEGPheoA-treated mice gradually increased up to 6 h postinjection. After 24 h post-injection, the fluorescence signal
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Figure 6. In vitro cytotoxicity of PEG-PheoA and FA-PEG-PheoA with laser irradiation. The in vitro cytotoxicity test (a) and LIVE/DEAD (live; green, dead; red) assay (b) after treatment with PEG-PheoA (conc; 0.05 mg mL 1). The in vitro cytotoxicity test (c) and LIVE/DEAD assay (d) after treatment with FA-PEG-PheoA (PheoA conc; 0.05 mg mL 1). The scale bar is 50 mm.
of FA-PEG-PheoA decreased due to clearance through the hepatic route. Unlike PheoA, water soluble FA-PEG-PheoA is easily dispersed in body fluid and is transported in the blood stream. So, the FA-PEG-PheoA has increased interaction opportunity with FA receptor overexpressing tumor tissues. Moreover, the enhanced tumor accumulation of FA-PEG-PheoA compared to PheoA was observed with CLSM (Figure 7b). The PheoA-injected tumor tissue section showed a minimum red fluorescence signal. However, the strong red fluorescence signal, which indicates high tumor specificity, was observed in the tumor tissue section of FA-PEG-PheoA injected mice. We also assayed the body distribution of PheoA and FA-PEGPheoA in the dissected tumors and other major organs (heart, lung, spleen, liver, and kidney) (Figure 7c). Consistent with in vivo optical imaging, ex vivo fluorescence images at 24 h after FA-PEG-PheoA injection showed a higher fluorescence signal in the tumor region than that of the PheoA-treated group. These results demonstrated that vitamin Bc-bearing hydrophilic photosensitizer, FAPEG-PheoA, showed high water solubility and tumor accumulation efficiency, which are essential characteristics for tumor diagnosis.
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4. Conclusion In this study, we demonstrated that vitamin Bc-bearing hydrophilic photosensitizer conjugate was effective for cancer diagnosis and therapy without additional chemical drugs or the use of fluorescent dyes. The synthesized FAPEG-PheoA conjugate proved FA receptor-mediated internalization and FA receptor-overexpressed cancer cellselective imaging efficacy with non-cytotoxicity. Moreover, under laser irradiation, the FA-PEG-PheoA conjugate generated singlet oxygen molecules that induced cell death by cell membrane peroxidation and exhibited cancer cell therapeutic efficacy. Based on these results, we conclude that the FA-PEG-PheoA conjugate enables both cancer diagnosis and therapy simultaneously and has great potential for biological studies and clinical treatments and therapy of various cancers.
Acknowledgments: This work was supported by the Strategic Research through the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (no. 20110028726), the Strategic core materials technology development
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Figure 7. In vivo tumor diagnosis efficacy of FA-PEG-PheoA. (a) In vivo near-infrared fluorescence imaging of MDA-MB-231 tumor-bearing Balb/c nude mice after tail vein injection of PheoA (conc; 0.2 mg mL 1) and FA-PEG-PheoA (PheoA conc; 0.2 mg mL 1) using an in vivo imaging station. (b) CLSM image of the PheoA and FA-PEG-PheoA in sectioned tumor tissue. The images of the tumor tissue were taken after sacrifice and 24 h post-injection (nucleus; blue, PheoA; red). (c) Ex vivo fluorescence images of tumors and major organs in MDA-MB-231 tumor-bearing Balb/c nude mice after 24 h tail vein injection of PheoA and FA-PEG-PheoA.
(10047756, Development of tetra-pyrrole type for color, lightemitting, detecting device) funded by the Ministry of Trade, Industry & Energy (MI, Korea) and research funds from The Catholic University of Korea (Research Fund 2015).
Received: February 24, 2015; Revised: March 14, 2015; Published online: March 31, 2015; DOI: 10.1002/mabi.201500060 Keywords: cancer; photodynamic diagnosis; photodynamic therapy; photosensitizer; vitamin Bc [1] K. S. Kim, W. Park, J. Hu, Y. H. Bae, K. Na, Biomaterials 2014, 35, 337. [2] R. Weissleder, Science 2006, 312, 1168. [3] S. Ramaswamy, P. Tamayo, R. Rifkin, S. Mukherjee, C. H. Yeang, M. Angelo, C. Ladd, M. Reich, E. Latulippe, J. P. Mesirov, T. Poggio, W. Gerald, M. Loda, E. S. Lander, T. R. Golub, Proc. Natl. Acad. Sci. USA. 2001, 98, 15149. [4] O. Warburg, Science 1956, 123, 309. [5] H. Kitano, Nat. Rev. Cancer 2004, 4, 227. [6] P. Polakis, Genes Dev. 2000, 14, 1837. [7] A. Mantovani, P. Allavena, A. Sica, F. Balkwill, Nature 2008, 454, 436.
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Vitamin Bc-Bearing Hydrophilic Photosensitizer Conjugate for Photodynamic Cancer Theranostics Jiyoung Kim, Kyoung Sub Kim, Sin-jung Park, Kun Na* The accurate diagnosis and proper therapy for cancer are essential to improve the success rate of cancer treatment. Here, we demonstrated that the vitamin Bc-bearing hydrophilic photosensitizer conjugate folic acid-polyethylene glycol-pheophorbideA (FA-PEG-PheoA) has been synthesized for the intracellular diagnosis and photodynamic therapy of a tumor. The synthesized vitamin Bc-bearing hydrophilic photosensitizer conjugate has been characterized for the folic acid receptor expressing the ability to target tumor cells, which is facilitated by the chemical conjugation with folic acid. The vitamin Bc-bearing hydrophilic photosensitizer conjugate internalization mechanism was identified through a competitive inhibition test with free folic acid. We optimized the laser-sensitive, cytotoxicity changeable, vitamin Bcbearing hydrophilic photosensitizer conjugate concentration, which is non-cytotoxic under normal conditions and specifically cytotoxic toward cancer cells (maximum 69.15%) under laser irradiation conditions used for theranostic agents. The cancer therapeutic and diagnosis effects of synthesized conjugate were confirmed in MDA-MB-231 cells and MDA-MB-231-bearing mice. As a result, the vitamin Bc-bearing hydrophilic photosensitizer conjugate exhibited a highly photodynamic therapeutic effect, which enabled the selective detection of a folic acid receptor expressing cancer using optical imaging.
1. Introduction The early diagnosis and treatment of cancer are important for increasing the survival rate of cancer patients and the success rate of cancer treatment.[1–3] Many researchers have worked intensively to identify the causes and properties of cancer,[4–10] which has led to many improved cancer treatment strategies.[11–17] For instance, chemotherapy,[18] J. Kim, K. S. Kim, S.-j. Park, K. Na Department of Biotechnology, Center for Photomedicine, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon-si Gyeonggi do 420-743, Korea E-mail: [email protected] Macromol. Biosci. 2015, 15, 1081–1090 ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
radiation therapy,[19] targeted therapy,[20] immunotherapy,[21] and hyperthermia[22] have become typical methods for cancer therapy. Recently, various theranostic agents, which involve both diagnostic and therapeutic functions in one combined system, have been developed and improved to specifically visualize and cure cancer cells simultaneously.[23–25] Organic and inorganic materials, including dendrimers,[26] liposomes,[27] polymeric micelles,[28] iron oxide nanoparticles,[29,30] gold nanoparticles,[31] gold nanorods,[32] and carbon nanotubes,[33] have been used as carriers of therapeutic agents. However, to fabricate these theranostic agents, researchers need to conduct complicated processes, due to the various components of theranostic agents, such
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as fluorescent dyes, chemical drugs, and CT/MR imaging materials. In spite of notable developments in medical technologies, cancer still remains an incurable disease.[34] Photosensitizers (PSs) are photosensitive molecules and important components of photodynamic therapy (PDT). [35] The PSs absorb irradiated photon energy, transfer it to nearby oxygen molecules, and produce reactive oxygen species (ROS). [36,37] Upon irradiation, intrinsic fluorescence and ROS generation of PSs can be used to conduct cancertargeted optical imaging and further clinical treatment.[38,39] As mentioned earlier, PDT is a type of treatment that utilizes PSs and light with the appropriate wavelength.[40,41] PDT is promising for the treatment of various cancers and other diseases because it is a minimally invasive and tissue selective treatment, and it has a high efficiency in producing cell death, vascular shutdown, and immune response activation.[42,43] In this study, pheophorbideA (PheoA), a second-generation PDT agent and a derivative of chlorophyll a,[44,45] was used to photodynamically diagnose and apply therapy to cancer cells at the same time. In contrast to conventional theranostic agents, we synthesized folic acid-polyethylene glycol-pheophorbideA (FA-PEG-PheoA) conjugate through simple conjugation processes without the addition of chemical drugs and fluorescent dyes. In addition, the FAPEG-PheoA conjugate includes vitamin Bc, which can bind to the cancer cell membrane overexpressing receptor. The synthesized FA-PEG-PheoA conjugate was confirmed by a 1H NMR spectrometer. The singlet oxygen generation efficacy of FA-PEG-PheoA under laser irradiation was confirmed by a RF-spectrofluorometer. Then, the cellular internalization behavior and imaging efficacy of FA-PEG-PheoA conjugate were observed with CLSM and intracellular fluorescence intensity. The therapeutic efficacy of FA-PEG-PheoA conjugate was observed with MTT and LIVE/DEAD assays. At last, to evaluate the in vivo feasibility of FA-PEG-PheoA, we performed an in vivo cancer targeting study in MDA-MB-231 tumor-bearing mice model with in vivo imaging station. Thus, the synthesized FA-PEG-PheoA conjugate can be easily applied to early diagnosis and treatment of cancer.
2.2. Synthesis and Characterization of PEG-PheoA The conjugation of PEG/PheoA via DCC and NHS-mediated amide formation was previously reported by our group.[16] Briefly, PEG and PheoA were conjugated in DMSO as described below. The PEG powder (400 mg and 66 mmol) was dissolved in DMSO (20 mL). PheoA (52 mg and 87 mmol corresponding to a molar ratio of PheoA to PEG of 1.3) was dissolved in DMSO (10 mL), followed by the addition of a 1.3 molar equivalent of DCC and NHS, while the PEG was dissolved in DMSO via vigorous stirring. After allowing the PEG and PheoA solutions to completely dissolve, they were mixed slowly, and the reaction mixture was gently stirred for 24 h at room temperature. The reaction mixture was dialyzed (Mw cutoff: 3 500 Da) for 3 d against distilled water to remove unconjugated PheoA and DMSO. The final solution was flash-frozen dry and lyophilized. The 1H NMR spectrum was recorded in deuterated dimethyl sulfoxide (DMSO-d6) at room temperature using a 500 MHz Bruker NMR Spectrometer (Bruker, Germany).
2.3. Purification of PEG-PheoA The purification of PEG-PheoA via open column chromatography was previously reported by our group.[38] For the purification, hydrophobic chromatographic columns were used; the crude products (PEG-PheoA) were chromatographed using an open column filled with Sephadex LH-20 as the stationary phase, and methanol served as the mobile phase. After loading the crude products, the flow rate was set to 0.5 mL min 1 with the mobile phase starting from 50% methanol (5:5 MeOH in water) (0–50 min) and proceeding to 80% methanol (8:2 MeOH in water) (51–95 min). Each of the fractions was collected and obtained as pure products (PEG-PheoA and PheoA-PEG-PheoA) and their absorbances were measured at 665 nm.
2. Experimental Section
2.4. Synthesis and Characterization of FA-PEG-PheoA
2.1. Materials
The PEG-PheoA and FA were conjugated in DMSO, as described below. The FA (30 mg and 67 mmol) was dissolved in DMSO (3 mL). PEG-PheoA (30 mg and 4.5 mmol, corresponding to a molar ratio of FA to PEG-PheoA of 15) was dissolved in DMSO (10 mL), followed by the addition of 2 molar equivalents of DCC and NHS, while the PEGPheoA was dissolved in DMSO via vigorous stirring.[46] After allowing the FA and PEG-PheoA solutions to completely dissolve, they were mixed slowly, and the reaction mixture was gently stirred for 24 h at room temperature. The reaction mixture was dialyzed (Mw cutoff: 3 500 Da) for 3 d against distilled water to remove unconjugated reactants and DMSO. The final solution was
PheophorbideA (PheoA) was purchased from Frontier Scientific, Inc. (Salt Lake City, UT, USA). Polyethylene glycol diamine (PEG) with a molecular weight of 6 000 Da, folic acid (FA), 1, 3-dicyclohexyl carbodiimide (DCC), N-hydroxysuccinimide (NHS), potassium bromide (KBr), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), methanol (MeOH), dimethyl sulfoxide anhydrous (DMSO), Sephadex LH-20, and 9, 10-dimethylanthracene (DMA) were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). The dialysis membrane was obtained from Spectrum
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Laboratories, Inc. (Rancho Dominguez, CA, USA). RPMI 1640 medium without folic acid, fetal bovine serum (FBS), antibiotics (penicillin/streptomycin), and Dulbecco’s phosphate buffered saline (DPBS) were obtained from Gibco BRL (Invitrogen Corp., Carlsbad, CA, USA). A LIVE/DEAD Viability/Cytotoxicity kit was purchased from Molecular Probes, Inc. (OR, USA). MDA-MB-231 (human breast carcinoma cell line) was obtained from the Korean Cell Line Bank (KCLB no. 30026).
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flash-frozen dry and lyophilized. The 1H NMR spectrum was recorded in deuterated dimethyl sulfoxide (DMSO-d6) at room temperature using a 500 MHz Bruker NMR Spectrometer (Bruker, Germany).
2.5. Measurement of Singlet Oxygen Generation (SOG) The generation of singlet oxygen (1O2) was observed chemically by the detection of 9, 10-dimethylanthracene (DMA, singlet oxygen quencher) utilizing fluorescent spectroscopy as an independent method. DMA (20 mM) was mixed with FA-PEG-PheoA in distilled water (DW). The solution was irradiated with a 670 nm laser source at 3 Jcm 2. Singlet oxygen-induced reduction of DMA fluorescence intensity (Ex 360 nm and Em 380-550 nm) was recorded at 0–40, and 45 min using an RF-spectrofluorometer (RF-5301PC, Shimadzu, Japan). The FA-PEG-PheoA in the DW without irradiation was used as the standard.
2.6. Cell Culture and Incubation Conditions MDA-MB-231 (human breast carcinoma) cells obtained from the Korean Cell Line Bank (KCLB no. 30026) were cultured in 10 mL of RPMI 1640 medium without a folic acid supplementation with 10% FBS and 1% penicillin/streptomycin. The cells were cultured at 37 8C in 100% humidity and at 5% CO2. PEG-PheoA and FA-PEG-PheoA were dissolved in serum-free (SF) medium. All of the reported concentrations refer to free PheoA equivalents. The untreated cells were irradiated or kept in the dark and used as a reference standard.
2.7. In Vitro Cytotoxicity of FA-PEG-PheoA MDA-MB-231 (1 104 cells well 1) were seeded onto 96-well plates and allowed to attach for 1 d. Next, FA-PEG-PheoA (diluted to various concentrations (0–1 mg mL 1)) in 100 mL of SF medium was added to each well and the plates were returned to the incubator for 3 h. After incubation, the wells were rinsed twice with DPBS, and fresh culture medium was added to each well. The cells were then incubated for an additional 24 h. The cell viability was assessed using the MTT assay. The resulting formazan crystals were dissolved in DMSO (100 mL), and absorbance intensity was measured at 570 nm using a microplate reader (Bio-Tek, VT, USA).
2.8. In Vitro Cellular Uptake Test To verify the concentration, where one can visualize the cellular uptake of FA-PEG-PheoA, MDA-MB-231 cells (1 105 cells well 1 in a 12-well plate) were treated with FA-PEG-PheoA in various concentrations (0–1 mg mL 1) for 3 h at 37 8C. The cells were then washed twice with DPBS, fixed with 4% paraformaldehyde, and visualized using a confocal laser scanning microscope (CLSM, LSM 710 Meta; Zeiss, Germany).
2.9. Competition Intracellular Uptake Test To identify the FA receptor-mediated internalization and competitive inhibition concentration by free FA, MDA-MB-231 cells
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(1 105 cells well 1 in a 12-well plate) were treated with FA-PEGPheoA in various concentrations of free FA (0.00–0.25 mg mL 1) for 3 h at 37 8C. The cells were then washed, harvested, and dissolved with DMSO. Each 100 mL of FA-PEG-PheoA was then loaded on the plate. The fluorescence spectra were analyzed at room temperature using a plate reader (Tecan Genios, NC, USA) with an excitation wavelength of 600 nm and an emission wavelength of 675 nm. The cells were then washed twice with DPBS, fixed with 4% paraformaldehyde, and visualized using a CLSM. To observe the subcellular localizations of PEG-PheoA and FA-PEGPheoA, MDA-MB-231 cells (1 105 cells well 1 in a 12-well plate) were treated with PEG-PheoA and FA-PEG-PheoA in the presence or absence of free FA for 3 h at 37 8C. The cells were then washed twice with DPBS, fixed with 4% paraformaldehyde, and visualized using CLSM. An optimal pinhole size of 120 mm was selected to exclude fluorescent light emitted from out-of-focus planes above and below the focusing plane. An objective with a magnification of 1 200 was used for image capture. A laser line with a wavelength of 630 nm was used for the excitation of DAPI and a He-Ne laser line was used for excitation of PheoA. A long-pass filter (LP 650 nm) was used at the emission end for detection of PheoA. Fluorescence images were analyzed using LSM Image Browser software (Zeiss). To investigate the cellular uptake of PEG-PheoA and FA-PEGPheoA, MDA-MB-231 cells were exposed with PEG-PheoA and FAPEG-PheoA in the presence or absence of free FA. The cells were incubated for 3 h, washed, harvested, and dissolved with DMSO. Each 100 mL of PEG-PheoA and FA-PEG-PheoA was then loaded on the plate. Fluorescence spectra were analyzed at room temperature using a plate reader (Tecan Genios, NC, USA) with an excitation wavelength of 600 nm and emission wavelength of 675 nm.
2.10. In Vitro Cytotoxicity of PEG-PheoA and FA-PEGPheoA with Laser Irradiation To assess the cytotoxicity of PEG-PheoA and FA-PEG-PheoA, MDAMB-231 cells (1 104 cells well 1) were added to a 96-well plate in 100 mL complement medium and incubated overnight. The samples, in 100 mL SF medium, were added to each well and the plates were returned to the incubator for 3 h. After incubation, the wells were rinsed three times with DPBS to remove any PEGPheoA or FA-PEG-PheoA that had not been internalized into the cells. Then, complement medium (100 mL) was added to the wells, and each well was irradiated using the 670 nm laser source (0–4 Jcm 2). The cells were then incubated for an additional 24 h. After incubation, the cell viability was assessed using the MTT assay. The resulting formazan crystals were dissolved in DMSO (100 mL) and transferred to a new plate. The absorbance intensity was measured at 570 nm using a microplate reader (Bio-Tek, VT, USA). For the live and dead cell assay, the LIVE/DEAD Viability/ Cytotoxicity Assay kit (Molecular Probes, USA) provides a two-color fluorescence cell viability assay. The optimal dyes are 2 mM Calcein AM and 4 mM EthD-1; the former stains live cells green, whereas the latter stains dead cells red. MDA-MB-231 cells were seeded in 35-mm cell culture dishes at a density of 1 106 cells well 1, respectively. The cell culture dishes were incubated overnight at 37 8C in a 5% CO2 incubator. Next, the medium was removed and the cells were incubated in SF medium containing PEG-PheoA or FA-PEG-PheoA for 3 h. The medium was then removed and the cells
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were rinsed twice with DPBS. Laser irradiation (0–4 Jcm 2) was performed with a 670 nm laser source. Then, the cells were incubated in complement medium for 4 h. The cell viability was observed by fluorescence microscopy (Zeiss, Germany).
tissues, the mice were sacrificed at 24 h tail vein injection and each tissue was excised. The fluorescence intensity of PheoA in each tissue was measured using a 12-bit CCD camera.
2.14. Statistical Analysis
2.11. In Vivo Animal Experiments All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Catholic University of Korea (Republic of Korea) in accordance with the ‘‘Principles of Laboratory Animal Care,’’ NIH publication no. 85-23 (revised in 1985). We used 4–6-week-old female Balb/c nude mice (Orient Bio, Seongnam, Korea) for the tumor diagnosis.
The data are expressed as the mean standard deviation (SD). The differences between the values were assessed using Student’s ttest.
3. Results and Discussion 3.1. Synthesis and Characterization of PEG-PheoA
2.12. In Vivo Tumor Diagnosis Using Optical Imaging MDA-MB-231 cells (1 10 cells mice ) were subcutaneously transplanted into Balb/c nude mice (five weeks of age). When the tumors grew to approximately 10 mm3 in volume, PheoA and FAPEG-PheoA (PheoA, 0.2 mg kg 1) were intravenously injected into the MDA-MB-231 bearing mice tail vein. The optical images were obtained using a 12-bit CCD camera (Image Station 4 000 MM; Kodak, New Haven, CT, USA) equipped with a special C-mount lens and a long wave emission filter (600–700 nm; Omega Optical). 6
1
2.13. Ex Vivo Biodistribution Study To compare the biodistribution of PheoA and FA-PEG-PheoA in normal tissues (heart, lung, spleen, liver, and kidney) and tumor
To prove our goal, the polyethylene glycol-pheophorbideA (PEG-PheoA) was synthesized via a conventional carbodiimide reaction, as shown in Figure 1, and according to a previous report of our group.[16] The photosensitizer PEGylation is a process that provides a hydrophilic property and enhances photoactivity in water.[42,43] The synthesized PEG-PheoA was confirmed using 1H NMR analysis (Figure 2a). We performed purification of the synthesized PEG-PheoA using a hydrophobic chromatographic Sephadex LH-20 column via a separation process mentioned in our group report.[38] The PEG-PheoA and PheoA-PEG-PheoA were detected with a UV/vis spectrophotometer and eluted in the first and second peak, respectively (Figure 2b). The PheoA-PEG-PheoA has very low solubility in water
Figure 1. Synthetic route of PEG-PheoA and FA-PEG-PheoA.
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Figure 2. In vitro chemical properties of PEG-PheoA and FA-PEG-PheoA. (a) 1H NMR spectrum of PEG-PheoA. The synthesized PEG-PheoA was confirmed when –CH of PheoA appeared at 8.92, 9.48, and 9.81 ppm. (b) Purification of PEG-PheoA by the hydrophobic interaction chromatography of Sephadex LH-20 resin. The absorbance of purified PEG-PheoA and PheoA-PEG-PheoA at 667 nm and photographs of the PEG-PheoA (1) and PheoA-PEG-PheoA (2) solution. (c) 1H NMR spectrum of FA-PEG-PheoA. The synthesized FA-PEG-PheoA was confirmed when the –CH of FA appeared at 7.64 and 8.64 ppm. (d) Singlet oxygen generation (1O2) of FA-PEG-PheoA detected by DMA in DW. All of the irradiations were performed using 670 nm of light at 3 Jcm 2.
compared to PEG-PheoA, which is unsuitable for a clinical application. Different from PheoA-PEG-PheoA, biarmed mPEG-PheoA conjugate was self-assembled, accumulated in tumor site, and used for PDT.[47] In this study, we applied vitamin Bc to the hydrophilic photosensitizer to enable targeting of the FA receptor in overexpressed cancer cells and furthermore photodynamic diagnosis and therapy of cancer.[48,49]
difference in the DMA fluorescence, indicating that it does not produce singlet oxygen under aqueous conditions.[50] In this experiment, mixed DMA with FA-PEG-PheoA dispersed in DW without irradiation was used as the standard. FAPEG-PheoA completely dissolved in DW resulted in a decline in DMA fluorescence intensity, indicating the generation of singlet oxygen upon laser irradiation. This result denotes that FA-PEG-PheoA conjugate can be used to apply therapy under laser irradiation.
3.2. Synthesis and Characterization of FA-PEG-PheoA FA-PEG-PheoA was synthesized with Purified PEG-PheoA and confirmed using 1H NMR (Figure 2c). In Figure 2d, the singlet oxygen generation efficacy of FA-PEG-PheoA was detected using DMA (singlet oxygen quencher) as a detector. DMA reacts irreversibly with 1O2 in many organic solvents and water, which causes a decrease in the intensity of the DMA absorption band at 360 nm. Generally, free PheoA in DW aggregated easily and did not show a
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3.3. In Vitro Cytotoxicity Test of FA-PEG-PheoA To utilize FA-PEG-PheoA as diagnostic agent, we optimized the nontoxic concentration of FA-PEG-PheoA in MDA-MB231 cells.[51] A quantitative in vitro cell cytotoxicity test was performed using the methyl thiazolyl tetrazolium (MTT) colorimetric assay. For various concentrations of FA-PEGPheoA conjugate (0–1 mg mL 1) in MDA-MB-231 cells, the results showed a concentration dependent cytotoxicity
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Figure 3. In vitro cytotoxicity and cellular localization of FA-PEG-PheoA at various concentrations. (a) In vitro cytotoxicity test of FA-PEGPheoA against MDA-MB-231 cells after treatment with FA-PEG-PheoA in various concentrations. (b) CLSM-imaged cellular internalization of various FA-PEG-PheoA concentrations (0–1 mg mL 1) in MDA-MB-231 cells with 3 h of incubation. The scale bar is 20 mm.
(Figure 3a). Overall, the cytotoxicity was indicated when the FA-PEG-PheoA concentration was more than 0.05 mg mL 1.
3.4. In Vitro Cellular Uptake Test To identify the appropriate concentration of FA-PEG-PheoA for cancer cell diagnosis, cellular internalization of the FAPEG-PheoA conjugate was examined using CLSM, based on the fluorescence of PheoA (Figure 3b). The fluorescence of FAPEG-PheoA was observed when the concentration was more than 0.05 mg mL 1. The previous MTT assay result and CLSM images suggest that 0.05 mg mL 1 of FA-PEG-PheoA is nontoxic and suitable for cancer cell diagnosis. In this study, the dose of FA-PEG-PheoA was fixed at 0.05 mg mL 1.
3.5. Cellular Internalization Mechanism of FA-PEGPheoA To prove the FA receptor-mediated cellular internalization mechanism of FA-PEG-PheoA and optimal concentration of
free FA, we monitored cellular internalization through CLSM and intracellular fluorescence intensity in MDAMB-231 cells with various concentrations of free FA (0–0.25 mg mL 1) (Figure 4a). FA is known to have a specific binding affinity to the FA receptor, which is overexpressed in various cancer cells and is used as a targeting moiety in this study.[46,48–50] As the concentration of free FA increases, the uptake of FA-PEG-PheoA in MDAMB-231 cells decreased. In addition, the red fluorescence signal of FA-PEG-PheoA was also decreased. In Figure 4b, the intracellular fluorescence intensity of FA-PEG-PheoA decreased as the free FA concentration increased. These results indicate that the free FA competitively inhibited FA receptor-mediated internalization of FA-PEG-PheoA conjugate in MDA-MB-231 cells. To ensure the competitive inhibition of free FA, we fixed the concentration of free FA at 0.25 mg mL 1. Next, the cellular internalization of PEG-PheoA and FA-PEGPheoA in the presence or absence of free FA was confirmed using CLSM. Regardless of free FA pre-treatment, PEG-PheoA showed a weak red fluorescence signal due to low cellular internalization efficiency (Figure 5a).
Figure 4. Change of intracellular fluorescence intensity by free FA. (a) CLSM image and (b) intracellular fluorescence intensity of FA-PEGPheoA with various concentrations of free FA (0.00–0.25 mg mL 1). The scale bar is 20 mm.
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Figure 5. In vitro cellular internalization and intracellular fluorescence intensity of PEG-PheoA and FA-PEG-PheoA in the presence or absence of free FA (conc; 0.25 mg mL 1). CLSM-imaged cellular internalization of (a) PEG-PheoA and (b) FA-PEG-PheoA in MDA-MB-231 cells with the presence or absence of free FA and 3 h of incubation. The scale bar is 20 mm. (c) Fluorescence intensity of PEG-PheoA and FA-PEG-PheoA in MDA-MB-231 cells.
However, the FA-PEG-PheoA showed a free FA dependent fluorescence increase and decrease (Figure 5b). In free FA pre-treatment, the FA-PEG-PheoA fluorescence signal was decreased by competitive inhibition of free FA. In contrast, the fluorescence signal of free FA non-treated FA-PEG-PheoA was significantly increased. These results indicate that FA performed the role of selective targeting moiety and functioned as an internalization route for the FA receptor in overexpressed cancer cells. In addition, these results were reconfirmed with intracellular fluorescence intensity in MDA-MB-231 cells (Figure 5c). Likewise CLSM images, these results suggested that the fluorescence intensity of free FA non-treated FA-PEGPheoA was approximately fourfold higher than free FAtreated FA-PEG-PheoA.
shows that the laser power dependent cytotoxicity change of FA-PEG-PheoA treated cells. Specifically, when FA-PEG-PhoeA-treated cells were irradiated with 4 Jcm 2, the cytotoxicity increased to 70%. And these results were also confirmed with the LIVE/DEAD assay (Figure 6d). As shown in the LIVE/DEAD assay, without laser irradiation, only live cells were indicated by green fluorescence. However, at a laser power above 1 Jcm 2, the LIVE/DEAD images show dead cells and DNA fragments as a red fluorescence and damaged live cells as a dim green fluorescence. These results demonstrated that the FAPEG-PheoA, which was internalized in MDA-MB-231 cells through the FA receptor, could generate singlet oxygen molecules under laser irradiation, which induced cell membrane peroxidation and cell death.[39]
3.6. In Vitro Cytotoxicity Test with Laser Irradiation
3.7. In Vivo Tumor Diagnosis Efficacy and Body Distribution of FA-PEG-PheoA
To investigate the therapeutic efficacy of FA-PEG-PheoA conjugate compared with PEG-PheoA, we measured the cytotoxicity through various doses of irradiation in MDAMB-231 cells. The viability of irradiated MDA-MB-231 cells was determined by MTT and LIVE/DEAD assays (Figure 6). The MDA-MB-231 cells treated with PEG-PheoA (conc; 0.05 mg mL 1) and FA-PEG-PheoA (conc; 0.05 mg mL 1) were irradiated with 0–4 J/cm2 of various laser powers. As shown in Figure 6a and b, cytotoxicity was not observed after treatment with PEG-PheoA, regardless of the photo irradiation power. The PEG-PheoA has a low internalization efficacy in MDA-MB-231 cells because of the hydrophilic nature of PEGylated PSs; PEG-PheoA hardly passed through the hydrophobic cell membrane and does not have cancer cell specific targeting moiety. In contrast, the FA-PEG-PheoA exhibited significantly enhanced cytotoxicity in MDA-MB-231 cells. Figure 6c
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To confirm the in vivo tumor diagnosis efficacy, we injected PheoA and FA-PEG-PheoA intravenously into MDA-MB-231 tumor bearing Balb/c nude mice. After injection of PheoA and FA-PEG-PheoA (PheoA conc; 0.2 mg kg 1) into the tail vein, the time-dependent biodistribution of PheoA and FA-PEG-PheoA was observed using an in vivo optical imaging station (Figure 7a). In the case of PheoA, a strong fluorescence signal was detected in the liver tissues, but tumor specificity was not observed. Hydrophobic PheoA is not dispersed in the aqueous phase and aggregated in body fluid easily. Therefore, aggregated PheoA was rapidly cleared from the body by the first-pass effect and hardly accumulated in the tumor tissues. In contrast, the fluorescence signal of the tumor in FA-PEGPheoA-treated mice gradually increased up to 6 h postinjection. After 24 h post-injection, the fluorescence signal
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Figure 6. In vitro cytotoxicity of PEG-PheoA and FA-PEG-PheoA with laser irradiation. The in vitro cytotoxicity test (a) and LIVE/DEAD (live; green, dead; red) assay (b) after treatment with PEG-PheoA (conc; 0.05 mg mL 1). The in vitro cytotoxicity test (c) and LIVE/DEAD assay (d) after treatment with FA-PEG-PheoA (PheoA conc; 0.05 mg mL 1). The scale bar is 50 mm.
of FA-PEG-PheoA decreased due to clearance through the hepatic route. Unlike PheoA, water soluble FA-PEG-PheoA is easily dispersed in body fluid and is transported in the blood stream. So, the FA-PEG-PheoA has increased interaction opportunity with FA receptor overexpressing tumor tissues. Moreover, the enhanced tumor accumulation of FA-PEG-PheoA compared to PheoA was observed with CLSM (Figure 7b). The PheoA-injected tumor tissue section showed a minimum red fluorescence signal. However, the strong red fluorescence signal, which indicates high tumor specificity, was observed in the tumor tissue section of FA-PEG-PheoA injected mice. We also assayed the body distribution of PheoA and FA-PEGPheoA in the dissected tumors and other major organs (heart, lung, spleen, liver, and kidney) (Figure 7c). Consistent with in vivo optical imaging, ex vivo fluorescence images at 24 h after FA-PEG-PheoA injection showed a higher fluorescence signal in the tumor region than that of the PheoA-treated group. These results demonstrated that vitamin Bc-bearing hydrophilic photosensitizer, FAPEG-PheoA, showed high water solubility and tumor accumulation efficiency, which are essential characteristics for tumor diagnosis.
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4. Conclusion In this study, we demonstrated that vitamin Bc-bearing hydrophilic photosensitizer conjugate was effective for cancer diagnosis and therapy without additional chemical drugs or the use of fluorescent dyes. The synthesized FAPEG-PheoA conjugate proved FA receptor-mediated internalization and FA receptor-overexpressed cancer cellselective imaging efficacy with non-cytotoxicity. Moreover, under laser irradiation, the FA-PEG-PheoA conjugate generated singlet oxygen molecules that induced cell death by cell membrane peroxidation and exhibited cancer cell therapeutic efficacy. Based on these results, we conclude that the FA-PEG-PheoA conjugate enables both cancer diagnosis and therapy simultaneously and has great potential for biological studies and clinical treatments and therapy of various cancers.
Acknowledgments: This work was supported by the Strategic Research through the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (no. 20110028726), the Strategic core materials technology development
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Figure 7. In vivo tumor diagnosis efficacy of FA-PEG-PheoA. (a) In vivo near-infrared fluorescence imaging of MDA-MB-231 tumor-bearing Balb/c nude mice after tail vein injection of PheoA (conc; 0.2 mg mL 1) and FA-PEG-PheoA (PheoA conc; 0.2 mg mL 1) using an in vivo imaging station. (b) CLSM image of the PheoA and FA-PEG-PheoA in sectioned tumor tissue. The images of the tumor tissue were taken after sacrifice and 24 h post-injection (nucleus; blue, PheoA; red). (c) Ex vivo fluorescence images of tumors and major organs in MDA-MB-231 tumor-bearing Balb/c nude mice after 24 h tail vein injection of PheoA and FA-PEG-PheoA.
(10047756, Development of tetra-pyrrole type for color, lightemitting, detecting device) funded by the Ministry of Trade, Industry & Energy (MI, Korea) and research funds from The Catholic University of Korea (Research Fund 2015).
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