Biomaterials 35 (2014) 7963e7969
Contents lists available at ScienceDirect
Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Doxorubicin loaded singlet-oxygen producible polymeric micelle based on chlorine e6 conjugated pluronic F127 for overcoming drug resistance in cancer Hyung Park 1, Wooram Park 1, Kun Na* Center for Photomedicine, Department of Biotechnology, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon-si, Gyeonggi-do 420-743, Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 May 2014 Accepted 22 May 2014 Available online 13 June 2014
Drug resistance remains one of the primary obstacles to the success of cancer chemotherapy. In this work, we demonstrate a singlet-oxygen producible polymeric (SOPP) micelle based on photosensitizer (PS, chlorin e6 (Ce6)) conjugated amphiphilic copolymer (pluronic F127®, PF127) for overcoming drug resistance in cancer by applying photochemical internalization (PCI). The doxorubicin (DOX)-loaded SOPP micelles were self-assembled from Ce6-PF127 conjugates, which have a spherical shape with a uniform size of ~30 nm. Compared with free Ce6, enhanced singlet-oxygen generation efficiency in the DOX-loaded SOPP micelles have been demonstrated in aqueous environments due to their increased water-dispersibility. Under low dose of laser power and anti-cancer drug (DOX) conditions, in vitro and in vivo studies on drug-resistant cancer cells demonstrated that singlet-oxygen-mediated cellular membrane damage (caused by lipid peroxidation) significantly increased the cellular uptake of drug (DOX), which led to overcoming the drug resistance in cancer cells without undesirable side effects. We believe this approach could represent a promising platform for drug-resistant cancer treatment. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Photochemical internalization (PCI) Drug resistance Photosensitizer Doxorubicin Cancer
1. Introduction Drug resistance remains one of the primary obstacles to effective cancer treatment and contributes to treatment failure in more than 90% of patients with metastatic cancer [1e3]. Several mechanisms responsible for drug resistance have been suggested (e.g., enhanced efflux of drugs by transporter, increased detoxification of compounds via the glutathione system, and protein alterations in drug targets such as DNA topoisomerase II) [4,5]. In particular, the overexpression of the ATP binding cassette (ABC) transporters, particularly P-glycoprotein (P-gp), is a key contributing factor to the development of drug resistance [6]. To overcome drug resistance, many strategies have been investigated, including the use of excipients that reduce the function of ABC transporters, or chemical P-gp inhibitors [7,8]. However, because P-gp has an important physiological function in protecting many types of normal tissues (e.g., brain, mammary gland, testis, and papillary dermis), systemic inhibition of P-gp may lead to severe adverse effects [9,10]. More recently, photochemical * Corresponding author. Tel.: þ82 2 2164 4832; fax: þ82 2 2164 4865. E-mail address: [email protected] (K. Na). 1 These authors contributed equally to the work. http://dx.doi.org/10.1016/j.biomaterials.2014.05.063 0142-9612/© 2014 Elsevier Ltd. All rights reserved.
internalization (PCI), an innovative approach based on photodynamic therapy (PDT), has been utilized for new strategies to improve the treatment of drug-resistant cancers [11e14]. The mechanism of PCI is based on the breakdown of the cellular membrane of laser exposed cells by reactive oxygen-induced lipid peroxidation [15e19]. Therefore, PCI systems are expected to improve therapeutic effects dramatically in drug-resistant tumors and effectively reduce systematic toxicity. Furthermore, since the laser dose required for PCI is much lower than that required for PDT, a deeper treatment effect without undesirable side effects can be achieved with PCI than with PDT for the same delivered laser dose [12,20,21]. In this study, we demonstrate a singlet-oxygen producible polymeric (SOPP) micelles based on PS conjugated amphiphilic copolymer for circumventing drug resistance in cancer by applying PCI. The PS conjugated amphiphilic copolymer was composed of chlorin e6 (Ce6) and pluronic F127® (PF127) (Scheme 1a). In our previous report [22], Ce6-PF127 conjugate showed enhanced cellular internalization and cancer-targeting efficacy. As depicted in Scheme 1b, upon irradiation of laser in a particular section of the cancer, the PS moiety would cause singlet-oxygen-mediated cellular membrane damage. We therefore hypothesized that SOPP micelles loaded with anti-cancer drug (doxorubicin, DOX) would
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Scheme 1. Schematic illustration of singlet-oxygen producible polymeric (SOPP) micelles. (a) Synthetic route to PS conjugated amphiphilic copolymer (Ce6-PF127 conjugate), (b) Schematic illustration of formation of the singlet-oxygen producible polymeric (SOPP) micelle and their singlet-oxygen generation upon laser irradiation, and (c) Schematic illustration of strategy for overcoming drug-resistant in cancer cells via singlet-oxygen-mediated cellular membrane damage; see text for details.
exhibit significant enhancement of chemotherapeutic efficiency in drug-resistant cancer cells by the enhancement of cell membrane permeability and rapid accumulation of the anti-cancer drug via the singlet-oxygen-mediated cellular membrane damage. 2. Materials and methods 2.1. Materials Pluronic F 127® (PF127), Doxorubicin (DOX), 1,3-dicyclohexyl carbodiimide (DCC), 4-dimethylaminopyridine (DMAP), Triethylamine (TEA), 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and anhydrous dimethyl sulfoxide (DMSO) were purchased from SigmaeAldrich Co., (St. Louis, MO, USA). Chlorin e6 (Ce6) was purchased from Frontier Scientific, Inc., (Salt Lake City, UT, USA). The dialysis tube was obtained from Spectrum Laboratories Inc., (Rancho Dominguez, CA, USA). RPMI 1640 medium and Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), antibiotics (penicillin/streptomycin), and Dulbecco's phosphate buffer saline (DPBS) were obtained from Gibco BRL (Invitrogen Corp., Carlsbad, CA, USA).
2.4. Characterization of DOX-loaded SOPP micelles To determine the drug-loading content, dried samples were suspended in DMSO and stirred vigorously for 2 h. The UV/Vis absorbance of the supernatant containing DOX extracted from the DOX-loaded SOPP micelles was measured at 470 nm using a UV/Visible spectrophotometer (UV-2450, Shimadzu, Japan). The particle size of DOX-loaded SOPP micelles were determined by dynamic light scattering (DLS, Zetasizer Nano ZS, Malvern Instruments Ltd., UK). The morphology of DOX-loaded SOPP micelles were observed by FE-SEM (S-4700, Hitachi, Japan). A drop of the DOX-loaded SOPP micelles (1 mg/mL) in water was placed on a graphite surface and coated with platinum by sputtering for 4 min at 20 mA. 2.5. Time-resolved singlet-oxygen production measurements Time-resolved singlet-oxygen measurements were performed by direct detection of the near-IR luminescence emission of oxygen at 1270 nm corresponding to a singletetriplet transition state [23]. The samples were excited with 670 nm mJ pulses (5 ms duration) generated by a fiber coupled diode laser operating with 10 kHz repetition rate. The singlet-oxygen luminescence was detected by a PMT detector (model H10330-45, Hamamatsu, Shizuoka, Japan) with high sensitivity in the nearIR region. Three bandpass filters (1220, 1270, and 1320 nm) were placed sequentially in front of the photodetector to sample the luminescence spectrum.
2.2. Synthesis of Ce6-PF127 conjugates Ce6-PF127 conjugate was synthesized by following our previously reported procedures [22]. In brief, PF127 (100 mg) and a mixture of Ce6 (30 mg), 1,3dicyclohexyl carbodiimide (1.5 Ce6 in moles), and 4-dimethylaminopyridine (1.5 Ce6 in moles) were dissolved separately in DMSO (5 mL) and the solutions were stirred thoroughly for 3 h prior to the condensation reaction. After 24 h, the reaction solution was filtered to remove insoluble by-products and the filtrate dialyzed using a dialysis tube (molecular weight cut-off (MWCO), 1 kDa) against deionized water for 2 days. The final solution was lyophilized.
2.6. In vitro drug release test Free DOX and DOX-loaded SOPP micelles (DOX concentration, 1 mg/mL) were suspended in 5 mL of PBS buffer (50 mM, pH 7.4) and then transferred into a dialysis tube (MWCO, 1 kDa). The dialysis tube was placed into the same buffered solution (10 mL), and then the release study was performed at 37 C in an incubator shaker (50 rpm). At certain time intervals, solution outside the dialysis tube was removed for quantitative analysis and replaced with fresh buffer. 2.7. Cell culture and incubation conditions
2.3. Preparation of DOX-loaded SOPP micelles DOX (10 mg) was dissolved in anhydrous DMSO (10 mL) containing TEA (10 mg). After the addition of Ce6-PF127 conjugates (100 mg) into the solution, the mixture was stirred overnight in a cold dark room. The solution was transferred to a wet dialysis tube (MWCO, 1 kDa) and dialyzed against excess borate buffer (pH 9.6) at room temperature for 24 h. The final product was filtered with syringe filter (0.45 mm, Millipore) and then used for in vitro and in vivo studies.
HCT-116 (drug-sensitive cancer), HCT-8 (drug-resistant cancer), and SNU-484 (drug-resistant cancer) cells were obtained from the Korean Cell Line Bank (KCLB No. 10247) and cultured in 10 mL of RPMI 1640 medium that was supplemented with 10% FBS and 1% penicillin/streptomycin. The cells were cultured at 37 C with 100% humidity and 5% CO2, and the medium was replaced every 2e3 days. The micelles were suspended in serum-free (SF) medium. Water-insoluble free DOX was dissolved in DMSO and then diluted in SF medium until the DMSO concentration
H. Park et al. / Biomaterials 35 (2014) 7963e7969 reached
Contents lists available at ScienceDirect
Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Doxorubicin loaded singlet-oxygen producible polymeric micelle based on chlorine e6 conjugated pluronic F127 for overcoming drug resistance in cancer Hyung Park 1, Wooram Park 1, Kun Na* Center for Photomedicine, Department of Biotechnology, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon-si, Gyeonggi-do 420-743, Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 May 2014 Accepted 22 May 2014 Available online 13 June 2014
Drug resistance remains one of the primary obstacles to the success of cancer chemotherapy. In this work, we demonstrate a singlet-oxygen producible polymeric (SOPP) micelle based on photosensitizer (PS, chlorin e6 (Ce6)) conjugated amphiphilic copolymer (pluronic F127®, PF127) for overcoming drug resistance in cancer by applying photochemical internalization (PCI). The doxorubicin (DOX)-loaded SOPP micelles were self-assembled from Ce6-PF127 conjugates, which have a spherical shape with a uniform size of ~30 nm. Compared with free Ce6, enhanced singlet-oxygen generation efficiency in the DOX-loaded SOPP micelles have been demonstrated in aqueous environments due to their increased water-dispersibility. Under low dose of laser power and anti-cancer drug (DOX) conditions, in vitro and in vivo studies on drug-resistant cancer cells demonstrated that singlet-oxygen-mediated cellular membrane damage (caused by lipid peroxidation) significantly increased the cellular uptake of drug (DOX), which led to overcoming the drug resistance in cancer cells without undesirable side effects. We believe this approach could represent a promising platform for drug-resistant cancer treatment. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Photochemical internalization (PCI) Drug resistance Photosensitizer Doxorubicin Cancer
1. Introduction Drug resistance remains one of the primary obstacles to effective cancer treatment and contributes to treatment failure in more than 90% of patients with metastatic cancer [1e3]. Several mechanisms responsible for drug resistance have been suggested (e.g., enhanced efflux of drugs by transporter, increased detoxification of compounds via the glutathione system, and protein alterations in drug targets such as DNA topoisomerase II) [4,5]. In particular, the overexpression of the ATP binding cassette (ABC) transporters, particularly P-glycoprotein (P-gp), is a key contributing factor to the development of drug resistance [6]. To overcome drug resistance, many strategies have been investigated, including the use of excipients that reduce the function of ABC transporters, or chemical P-gp inhibitors [7,8]. However, because P-gp has an important physiological function in protecting many types of normal tissues (e.g., brain, mammary gland, testis, and papillary dermis), systemic inhibition of P-gp may lead to severe adverse effects [9,10]. More recently, photochemical * Corresponding author. Tel.: þ82 2 2164 4832; fax: þ82 2 2164 4865. E-mail address: [email protected] (K. Na). 1 These authors contributed equally to the work. http://dx.doi.org/10.1016/j.biomaterials.2014.05.063 0142-9612/© 2014 Elsevier Ltd. All rights reserved.
internalization (PCI), an innovative approach based on photodynamic therapy (PDT), has been utilized for new strategies to improve the treatment of drug-resistant cancers [11e14]. The mechanism of PCI is based on the breakdown of the cellular membrane of laser exposed cells by reactive oxygen-induced lipid peroxidation [15e19]. Therefore, PCI systems are expected to improve therapeutic effects dramatically in drug-resistant tumors and effectively reduce systematic toxicity. Furthermore, since the laser dose required for PCI is much lower than that required for PDT, a deeper treatment effect without undesirable side effects can be achieved with PCI than with PDT for the same delivered laser dose [12,20,21]. In this study, we demonstrate a singlet-oxygen producible polymeric (SOPP) micelles based on PS conjugated amphiphilic copolymer for circumventing drug resistance in cancer by applying PCI. The PS conjugated amphiphilic copolymer was composed of chlorin e6 (Ce6) and pluronic F127® (PF127) (Scheme 1a). In our previous report [22], Ce6-PF127 conjugate showed enhanced cellular internalization and cancer-targeting efficacy. As depicted in Scheme 1b, upon irradiation of laser in a particular section of the cancer, the PS moiety would cause singlet-oxygen-mediated cellular membrane damage. We therefore hypothesized that SOPP micelles loaded with anti-cancer drug (doxorubicin, DOX) would
7964
H. Park et al. / Biomaterials 35 (2014) 7963e7969
Scheme 1. Schematic illustration of singlet-oxygen producible polymeric (SOPP) micelles. (a) Synthetic route to PS conjugated amphiphilic copolymer (Ce6-PF127 conjugate), (b) Schematic illustration of formation of the singlet-oxygen producible polymeric (SOPP) micelle and their singlet-oxygen generation upon laser irradiation, and (c) Schematic illustration of strategy for overcoming drug-resistant in cancer cells via singlet-oxygen-mediated cellular membrane damage; see text for details.
exhibit significant enhancement of chemotherapeutic efficiency in drug-resistant cancer cells by the enhancement of cell membrane permeability and rapid accumulation of the anti-cancer drug via the singlet-oxygen-mediated cellular membrane damage. 2. Materials and methods 2.1. Materials Pluronic F 127® (PF127), Doxorubicin (DOX), 1,3-dicyclohexyl carbodiimide (DCC), 4-dimethylaminopyridine (DMAP), Triethylamine (TEA), 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and anhydrous dimethyl sulfoxide (DMSO) were purchased from SigmaeAldrich Co., (St. Louis, MO, USA). Chlorin e6 (Ce6) was purchased from Frontier Scientific, Inc., (Salt Lake City, UT, USA). The dialysis tube was obtained from Spectrum Laboratories Inc., (Rancho Dominguez, CA, USA). RPMI 1640 medium and Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), antibiotics (penicillin/streptomycin), and Dulbecco's phosphate buffer saline (DPBS) were obtained from Gibco BRL (Invitrogen Corp., Carlsbad, CA, USA).
2.4. Characterization of DOX-loaded SOPP micelles To determine the drug-loading content, dried samples were suspended in DMSO and stirred vigorously for 2 h. The UV/Vis absorbance of the supernatant containing DOX extracted from the DOX-loaded SOPP micelles was measured at 470 nm using a UV/Visible spectrophotometer (UV-2450, Shimadzu, Japan). The particle size of DOX-loaded SOPP micelles were determined by dynamic light scattering (DLS, Zetasizer Nano ZS, Malvern Instruments Ltd., UK). The morphology of DOX-loaded SOPP micelles were observed by FE-SEM (S-4700, Hitachi, Japan). A drop of the DOX-loaded SOPP micelles (1 mg/mL) in water was placed on a graphite surface and coated with platinum by sputtering for 4 min at 20 mA. 2.5. Time-resolved singlet-oxygen production measurements Time-resolved singlet-oxygen measurements were performed by direct detection of the near-IR luminescence emission of oxygen at 1270 nm corresponding to a singletetriplet transition state [23]. The samples were excited with 670 nm mJ pulses (5 ms duration) generated by a fiber coupled diode laser operating with 10 kHz repetition rate. The singlet-oxygen luminescence was detected by a PMT detector (model H10330-45, Hamamatsu, Shizuoka, Japan) with high sensitivity in the nearIR region. Three bandpass filters (1220, 1270, and 1320 nm) were placed sequentially in front of the photodetector to sample the luminescence spectrum.
2.2. Synthesis of Ce6-PF127 conjugates Ce6-PF127 conjugate was synthesized by following our previously reported procedures [22]. In brief, PF127 (100 mg) and a mixture of Ce6 (30 mg), 1,3dicyclohexyl carbodiimide (1.5 Ce6 in moles), and 4-dimethylaminopyridine (1.5 Ce6 in moles) were dissolved separately in DMSO (5 mL) and the solutions were stirred thoroughly for 3 h prior to the condensation reaction. After 24 h, the reaction solution was filtered to remove insoluble by-products and the filtrate dialyzed using a dialysis tube (molecular weight cut-off (MWCO), 1 kDa) against deionized water for 2 days. The final solution was lyophilized.
2.6. In vitro drug release test Free DOX and DOX-loaded SOPP micelles (DOX concentration, 1 mg/mL) were suspended in 5 mL of PBS buffer (50 mM, pH 7.4) and then transferred into a dialysis tube (MWCO, 1 kDa). The dialysis tube was placed into the same buffered solution (10 mL), and then the release study was performed at 37 C in an incubator shaker (50 rpm). At certain time intervals, solution outside the dialysis tube was removed for quantitative analysis and replaced with fresh buffer. 2.7. Cell culture and incubation conditions
2.3. Preparation of DOX-loaded SOPP micelles DOX (10 mg) was dissolved in anhydrous DMSO (10 mL) containing TEA (10 mg). After the addition of Ce6-PF127 conjugates (100 mg) into the solution, the mixture was stirred overnight in a cold dark room. The solution was transferred to a wet dialysis tube (MWCO, 1 kDa) and dialyzed against excess borate buffer (pH 9.6) at room temperature for 24 h. The final product was filtered with syringe filter (0.45 mm, Millipore) and then used for in vitro and in vivo studies.
HCT-116 (drug-sensitive cancer), HCT-8 (drug-resistant cancer), and SNU-484 (drug-resistant cancer) cells were obtained from the Korean Cell Line Bank (KCLB No. 10247) and cultured in 10 mL of RPMI 1640 medium that was supplemented with 10% FBS and 1% penicillin/streptomycin. The cells were cultured at 37 C with 100% humidity and 5% CO2, and the medium was replaced every 2e3 days. The micelles were suspended in serum-free (SF) medium. Water-insoluble free DOX was dissolved in DMSO and then diluted in SF medium until the DMSO concentration
H. Park et al. / Biomaterials 35 (2014) 7963e7969 reached
