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A Cancer-Recognizing Polymeric Photosensitizer Based on the Tumor Extracellular pH Response of Conjugated Polymers for Targeted Cancer Photodynamic Therapya Songhee Jeong,y Wooram Park,y Chung-Sung Lee, Kun Na*
Herein, a cancer-recognizing polymeric photosensitizer (CRPP) was demonstrated not only for high water solubility but also for pH-responsive targeted photodynamic cancer therapy. The synthesized CRPP exhibited high water solubility and the pH-dependent charge-switching property. From an in vitro cellular internalization study with HCT-116 human colon cancer cells, significantly enhanced cellular uptake as detected for CRPP at pH 6.5 compared to the cellular uptake of CRPP at pH 7.4, which led to enhanced cytotoxicity in the cancer cells. Finally, the CRPP was found to exhibit high tumor-targeting efficacy in an in vivo tumor model and was finally excreted through the renal route.
1. Introduction Photodynamic therapy (PDT) is known as a prospective treatment method and has been used for the treatment of a variety of oncological, dermatological, cardiovascular, and ophthalmic diseases.[1–3] PDT is based on combinations of photosensitizers (PS), light, and molecular oxygen. Under light irradiation at an appropriate wavelength, the PS forms reactive oxygen species (ROSs), such as singlet oxygen (1O2) or free radicals, resulting in irreversible damage of tumor cells.[4] However, the water-insolubility S. Jeong, W. Park, C.-S. Lee, 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] Supporting Information is available from the Wiley Online Library or from the author. y These authors contributed equally to this work. a
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and limited tumor selectivity of PS have been major drawbacks for clinical application.[5–7] In this regard, various types of PSs based on the polymer conjugation technique have been developed to overcome the limitation of conventional photosensitizing agents.[8–13] Here, we propose a cancer-recognizing polymeric photosensitizer (CRPP) that is pH-responsive to targeted cancer PDT (Figure 1a,b). CRPP was synthesized by a three-step synthetic process (Figure 2a). First, methoxy-polyethylene glycol-block-poly(benzyl-L-aspartic acid) (mPEG-poly(Bz-LAsp)) was synthesized using a ring opening polymerization (ROP). Second, the second-generation PS chlorin e6 (Ce6) was conjugated with mPEG-poly(Bz-L-Asp). Finally, pHresponsive imidazole groups were introduced by an aminolysis reaction.[11,14,15] We hypothesized that the higher water solubility of the CRPP would have significantly higher fluorescence and singlet-oxygen generation (SOG) than conventional PS under aqueous conditions. In addition, CRPP can also have a pH-dependent charge-switching property due to the ionizable groups (i.e., the imidazole
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DOI: 10.1002/mabi.201400361
Cancer-Recognizing Polymeric Photosensitizer www.mbs-journal.de
addition, an in vitro cellular-uptake study was performed with the HCT-116 human colon cancer cell line to evaluate the pH-dependent cellular uptake behavior of CRPP using flow cytometry and confocal laser scanning microscopy (CLSM). The in vitro therapeutic potential of CRPP was also confirmed at various pH values (e.g., pH 7.4 and 6.5) using an MTT cytotoxicity assay. Finally, to evaluate the in vivo feasibility of CRPP, we performed an in vivo cancer-specific targeting study in a CT26 tumor-bearing mice model using an in vivo image station (IVIS).
2. Experimental Section 2.1. Materials b-Benzyl-L-aspartic acid (BLA), triphosgene, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dichloromethane (DCM), 1,3Figure 1. Schematic representation of the cancer-recognizing polymeric photosensitizer dicyclohexylcarbodiimide (DCC), and tetrahy(CRPP). a) Chemical structure of CRPP and b) schematic representation of the pH- drofuran (THF), 3-(4,5-dimethyl-2-thiazolyl)2,5-diphenyl-2H-tetrazolium bromide (MTT), dependent charge switching behavior of CRPP and chemical structural representation of the protonation of the imidazole groups in CRPP at an acidic pH. Schematic triethylamine (TEA), and 1-(3-aminopropyl) representations of (c) the tumor-accumulation behavior of CRPP and (d) its imidazole (API) was purchased from Sigma– enhanced cellular internalization via electrostatic interactions between the positively Aldrich (Sigma–Aldrich Korea, Seoul, Korea). charged CRPP and negatively charged cellular membrane; then, the internalized Methoxy polyethylene glycol amine (mPEGCRPP can generate singlet oxygen under laser irradiation, which leads to the killing amine, M ; 5 000) was purchased from Sunbio, w of tumor cells. Inc. (Anyang, Korea). Chlorin e6 (Ce6) was purchased from Frontier Scientific, Inc. (UT, USA). A human colon cancer HCT-116 cell line was obtained from the Korean Cell Line Bank (KCLB No.10247). groups) in the polymer backbone (Figure 1b). Consequently, RPMI 1640 medium, fetal bovine serum (FBS), antibiotics (penicillin CRPP can be stable in the bloodstream because the charge of and streptomycin), and Dulbecco’s phosphate-buffered saline CRPP is negative under normal, physiological pH (pH 7.4) (DPBS) were obtained from Gibco BRL (Invitrogen Corp., Carlsbad, (Figure 1c). However, after accumulation in a tumor site via CA, USA). SOSG was purchased from Molecular Probes, Inc. the enhanced permeability and retention (EPR) effect, the (Eugene, OR, USA). b-Benzyl-L-aspartic acid N-carboxyanhydride CRPP can become positively charged at the tumoral acidic (BLA-NCA) was synthesized by the Fuchs–Farthing method using extracellular pH (pHe, pH 6.5) through protonation of triphosgene.[18]
the imidazole groups. Because the cellular membrane generally exhibits a net negative charge due to the phosphate group of phosphatidylserine,[16,17] the cellular uptake of CRPP should be increased as a result of the strengthened interaction between the positively charged CRPP and the tumor cells via electrostatic interactions (Figure 1d). Therefore, a significantly enhanced PDT effect can be achieved against tumor cells. In this paper, a pH-responsive polymeric PS was designed and synthesized and then characterized using 1H NMR, fluorescence spectroscopy, and dynamic light scattering (DLS). We also measured the SOG behavior of the synthesized polymeric PS in an aqueous solution using singlet oxygen sensor green (SOSG) as a singlet oxygen probe. In
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2.2. Synthesis of CRPP (mPEG-Poly(Bz-L-Asp)-Ce6) mPEG-poly(Bz-L-Asp)-Ce6 was synthesized using a modification of the method reported previously.[9,11,15] To synthesis mPEGpoly(Bz-L-Asp), BLA-NCA (1.5 g, 6 mmol) was polymerized in a mixture of DMF (10 mL) and CH2Cl2 (50 mL) at room temperature by initiation from the terminal primary amine group of mPEGamine (M n ¼ 5 kDa, 1 g, 200 mmol). mPEG-poly(Bz-L-Asp) was purified by precipitation in ether (1 L) three times and then dried in vacuo. To synthesize mPEG-poly(Bz-L-Asp)-Ce6, Ce6 was conjugated to the terminal amine group of mPEG-poly(Bz-L-Asp) by the conventional carbodiimide reaction. mPEG-poly(Bz-L-Asp) (0.5 g, 43 mmol) and a mixture of Ce6 (31 mg, 52 mmol),
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2.3. Measurement of the ZetaPotential The zeta-potential was determined using DLS (Zetasizer Nano ZS, Malvern Instruments Ltd., UK). DLS was performed at 25 8C in an aqueous solution (sample concentration, 1 mg mL1) with the sampling time and analysis set to automatic.
2.4. Optical Photography and Fluorescence Analysis
Figure 2. Chemical synthesis of CRPP (mPEG-poly(API-L-Asp)-Ce6, n ¼ 114, m ¼ 30). a) Synthetic route of CRPP and b) 1H NMR analysis of CRPP in DMSO-d6. dicyclohexylcarbodiimide (16 mg, 78 mmol), and N-hydroxysuccinimide (9 mg, 78 mmol) were dissolved separately in DMF (10 mL), and the solutions were stirred thoroughly for 3 h prior to the condensation reaction. The two reactant solutions were then mixed and stirred at room temperature. After 24 h, the reaction mixture was purified by precipitation in ether (0.2 L) three times and then dried in vacuo. Finally, mPEG-poly(API-LAsp)-Ce6 was synthesized via aminolysis of mPEG-poly(Bz-LAsp)-Ce6 with API. mPEG-poly(Bz-L-Asp)-Ce6 (0.2 g, 16 mmol) was dissolved in DMSO (5 mL), followed by reaction with API (40 mg, 320 mmol) and TEA (32 mg, 320 mmol) under nitrogen atmosphere at room temperature for 12 h. The reaction mixture was added dropwise into a cooled aqueous solution of 0.1 N HCl (20 mL) and was dialyzed against a 0.01 N HCl solution three times (Spectra/Por; MWCO: 1 kDa). The final solution was lyophilized, and the formation of mPEG-poly(API-L-Asp)-Ce6 was verified by 1H NMR. The 1H NMR spectra were recorded in DMSO-d6 at room temperature using a Bruker NMR Spectrometer (Bruker, Germany) at 500 MHz. 1 H NMR (500 MHz, DMSO-d6) for mPEG-poly(API-L-Asp)-Ce6 (Figure 2b): d ¼ 7.6 ppm (1H, s, —NCH5 5N— of imidazole ring), 7.1 ppm (1H, s, —NCH5 5CH— of imidazole ring), 6.8 ppm (1H, s, —CHCH5 5N— of imidazole ring), 4.5 ppm (1H, m, —NHCHC5 5O—), 3.9 ppm (2H, m, —NCH2CH2), 3.5 ppm (182H, s, —CH2CH2O— of mPEG chain), 3.2 ppm (3H, CH3O— of mPEG chain and CH3C— of Ce6), 3.0 ppm (2H, m, —NHCH2CH2—), d 2.8–2.5 ppm (2H, m, —CHCH2C5 5O—), and 1.8 ppm (2H, m, —CH2CH2CH2—).
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Free Ce6 and CRPP (Ce6, 1.6 104 M) were dissolved in distilled water and DMSO, respectively. Optical photographs were recorded using a digital camera (nx1000, Samsung Electronics Co., Korea) at room temperature. The fluorescence intensity was measured in 96-well plate using a plate reader (Tecan Genios, Durham, NC, USA) with an excitation wavelength of 650 nm and an emission wavelength of 675 nm at room temperature. The fluorescence images were captured using a 12-bit CCD camera (Image Station 4000 MM; Kodak, New Haven, CT, USA) with a special Cmount lens and a long-wave emission filter (600–700 nm; Omega Optical, Brattleboro, VT, USA).
2.5. Measurement of Singlet Oxygen Generation (SOG) The SOG was detected chemically using SOSG (S-36002, Invitrogen/ Molecular Probes) as a probe. The SOSG probe works via intramolecular electron transfer, which quenches the fluorescence from the light-emitting chromophore prior to reaction with singlet oxygen. Reaction with singlet oxygen results in the formation of the endoperoxide, prohibiting electron transfer and thus leading to the recovery of fluorescence.[19,20] Free Ce6 or CRPP was dissolved in distilled water (Ce6, 1.0 105 M) and mixed with a SOSG solution (2 mM). The mixture was then irradiated with 10 mW cm2 of a 670 nm laser source (fiber-coupled laser system, LaserLab1, Korea) over time. The fluorescence intensity of SOSG (lex ¼ 494 nm, lem ¼ 534 nm) was recorded using fluorescence spectroscopy (RF5301; Shimadzu, Japan).
2.6. In Vitro Cell Culture and Incubation Conditions HCT-116 (human colon cancer) and CT-26 (murine colon cancer) cells were obtained from the Korean Cell Line Bank (HCT-116; KCLB No. 10247, CT-26; KCLB No. 80009) and were cultured in RPMI-1640 (HCT-116) and DMEM (CT-26) supplemented with 10% heatinactivated FBS and penicillin/streptomycin (100 U mL1 and 100 mg mL1, respectively), which is called the complement medium (CM medium) in this study. The cells were cultured at
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37 8C in a humidified atmosphere containing 5% CO2 and were subcultured in a new medium every 2–3 d. CRPP or free Ce6 was dissolved in a serum-free (SF) medium.
2.7. In Vitro Cellular-Uptake Test To verify the cellular uptake of CRPP at two different pH values (pH 7.4 and 6.5), HCT-116 cells were seeded in a six-well cell culture plate at a density of 1.0 106 cells per well and were incubated for 12 h at 37 8C in 5% CO2. The medium was then removed, and the cells were incubated in serum-free (SF) medium containing CRPP of free Ce6 (Ce6, 3.2 106 M) for 10 min at pH 7.4 or 6.5. The cells were rinsed, harvested, and resuspended with DPBS. The cellular uptake was quantitatively analyzed using flow cytometery (Beckman, San Jose, CA, USA). For each sample, 10 000 cells (gated events) were counted, and the Ce6 fluorescence was detected with logarithmic settings (FL4; Em ¼ 670 nm). Each experiment was analyzed statistically using the CXP analysis program (Beckman).
2.8. Confocal Laser Scanning Microscopy (CLSM) Analysis To observe the cellular localization of CRPP at two different pH values (pH 7.4 and 6.5), HCT-116cells (1.0 105 cells per well in a 35mm dish) were treated with CRPP or free Ce6 (Ce6, 3.2 106 M) for 10 min at pH 7.4 or 6.5. The cells were then washed twice with DPBS, fixed with 4% paraformaldehyde and stained with DAPI. The cells were mounted in a mounting medium (Dako, Glostrup, Denmark) and visualized using a confocal laser scanning microscope (LSM 710 Meta; Carl Zeiss, Germany). A laser line with a wavelength of 633 nm was used for excitation. A long-pass filter (LP 650 nm) was used at the emission end for detection. The fluorescence images were analyzed using the LSM Image Browser software (Carl Zeiss, Germany).
2.9. In Vitro Cytotoxicity Study HCT-116 cells were seeded in black 96-well culture plates at a density of 2.0 104 cells per well and were incubated for 12 h. CRPP or free Ce6 (Ce6, 1.6 105 M) was added to each well in SF medium (100 mL), and the plates were returned to the incubator for 10 min. After incubation, the wells were rinsed twice with DPBS to remove materials that had not been internalized by the cells. One hundred microliters of the complement medium were added to the wells, and each well was irradiated using a 670 nm laser source (3.6 J cm2, fiber-coupled laser system, LaserLab1, Korea). The cells were incubated for a further 24 h. The cell viability was assessed using the MTT assay according to our previous reports.[6,21,22]
cells red. HCT-116 cells were seeded in 35-mm cell culture dishes at a density of 1 105 per well. The cells were incubated for 12 h at 37 8C in 5% CO2. After incubation, the medium was removed, and the cells were incubated in SF medium containing CRPP or free Ce6 (Ce6, 1.6 105 M) at pH 7.4 and 6.5 for 10 min. The medium was removed, and the cells were rinsed twice with DPBS. Then, irradiation (3.6 J cm2) was performed with a 670 nm laser source (fiber-coupled laser system, LaserLab1, Korea). The cells were then incubated in the complement medium for 1 h. The cell viability was observed using fluorescence microscopy (Carl Zeiss, Germany). The fluorescence images were analyzed using LSM Image Browser software (Carl Zeiss).
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 four- to six-week-old female BALB/c nude mice (Oriental Bio, Seongnam, Korea) for fluorescence optical imaging.
2.12. In Vivo Optical Imaging CT26 cells (1.0 105 cells) were subcutaneously transplanted into male Balb/c mice. When the tumors grew to approximately 10 mm3 in volume, CRPP or free Ce6 (Ce6, 0.2 mg kg1) was intravenously injected into the CT26-bearing mice. The images were obtained using a 12-bit CCD camera (Image Station 4000 MM; Kodak, New Haven, CT) with a special C-mount lens and a long-wave emission filter (600–700 nm; Omega Optical).
2.13. Ex Vivo Biodistribution Study To compare the biodistribution of CRPP and free Ce6 in normal tissues (heart, lung, spleen, liver, kidney, and muscle) and tumor tissue, the mice were sacrificed at 24 h post-injection, and each tissue was excised. The fluorescence intensity of Ce6 in each tissue was measured using a 12-bit CCD camera (Image Station 4000 MM; Kodak, New Haven, CT) with a special C-mount lens and a longwave emission filter (600–700 nm; Omega Optical).
2.14. Statistical Analysis The data are expressed as the mean standard deviation (SD). The differences between the values were assessed using Student’s t-test.
3. Results and Discussion 2.10. Live/Dead Assay The live/dead viability/cytotoxicity assay kit (Molecular Probes, USA) was used as a two-color fluorescence cell-viability assay.[11] The optimal dyes used were calcein AM (2 mM) and EthD-1 (4 mM); the former stains live cells green, whereas the latter stains dead
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3.1. Synthesis and Physicochemical Characterization of CRPP CRPP was synthesized by combining the ring-opening polymerization (ROP) of b-benzyl-L-aspartate N-carboxyanhydride
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(Bz-L-Asp-NCA) and an aminolysis reaction (Figure 2a). First, poly(Bz-L-Asp) was polymerized from the primary amine group of mPEG-amine to generate mPEG-poly(Bz-LAsp). Then, PS (Chlolin e6, Ce6) was conjugated with mPEG-poly(Bz-L-Asp) using conversional carbodiimide coupling chemistry. The successful synthesis of mPEGpoly(Bz-L-Asp)-Ce6 conjugate was confirmed by GPC analysis (Figure S1, Supporting Information): an increased molecular weight of polymer was observed after PSconjugation. Finally, the pH-responsive groups (i.e., API) were incorporated into mPEG-poly(Bz-L-Asp) though an aminolysis reaction between the primary amine group of API (nucleophilic) and the benzyl ester group of mPEGpoly(Bz-L-Asp). The chemical structure of CRPP was analyzed using 1H NMR analysis (Figure 2b). The degree of polymerization (DP) of the Bz-L-Asp units was calculated to be 30. In addition, we confirmed the successful incorporation of zwitterionic groups (i.e., imidazole and carboxyl groups) on CRPP (Table 1). To characterize the physicochemical property of CRPP, we first confirmed the solubility of CRPP in water and an organic solvent. Figure 3a,b shows the solubility of free Ce6 and CRPP (Ce6, 1.6 104 M) in water and the organic solvent (i.e., DMSO), respectively. Free Ce6 was precipitated under aqueous conditions due to the strong hydrophobicity (Figure 3a(1)); this low water solubility is a major limitation to the use of conventional PS as a pharmaceutical agent. However, CRPP exhibited high water solubility (Figure 3a(2)). As shown in Figure 3b, both compounds could be dissolved well in the organic solvent. To investigate the photoactivity of CRPP (Ce6, 1.0 105 M) in an aqueous solution, the fluorescence emission and SOG property were examined using fluorescence spectroscopy. Whereas the fluorescence emission (lex ¼ 670, lem ¼ 675) of CRPP was much higher than that of free Ce6 in water (Figure 3c,d). The SOG of CRPP was also evaluated chemically using SOSG as a probe with laser irradiation (670 nm). As shown in Figure 3e, CRPP showed significantly increased SOG compared to free
Ce6. These results indicate that the introduction of hydrophilic PEG and ionizable groups (e.g., imidazole and carboxyl groups) effectively enhanced the photoactivity of PS under aqueous conditions. Next, to confirm the pH-dependent charge-switching property of CRPP, the zeta-potential was measured as a function of the pH value (Figure 3f). The zeta-potential of CRPP was changed from negative (approximately 16 mV) to positive (approximately þ12 mV) as the pH of the solution decreased from pH 7.4 to 6.5. We suppose that the negative value at pH 7.4 was attributed to the unmodified carboxyl groups and PEG of CRPP, whereas that was offset by the protonation of the imidazole groups at pH 6.5. 3.2. In Vitro Cellular-Internalization Behavior of CRPP at Various pH Values To investigate the cellular internalization behavior of CRPP at pH 7.4 and 6.5, we used flow cytometry and CLSM with human colon cancer (HCT-116) cells (the dose of Ce6 was equally treated with 2 mg mL1) (Figure 4a,b). As a control test, the cellular-uptake test of free Ce6 was performed at pH 7.5 and 6.5. As shown in Figure 4a, a high level of cellular uptake was observed for the free Ce6 regardless of the pH due to its nonspecific cellular-uptake behavior though diffusion.[6,23] However, significantly enhanced cellular uptake was detected for CRPP at pH 6.5 compared to the cellular uptake of CRPP at pH 7.4. This observation was further confirmed by CLSM under the same conditions (Figure 4b). Consistent with data from flow cytometry, the strong red fluorescence of free Ce6 was observed at both pH values. In contrast, HCT-116 cells treated with CRPP showed higher red fluorescence at pH 6.5 than that at pH 7.4. These results can be attributed to the charge-switching property of CRPP, which becomes positively charged at pH 6.5. As a result, the interaction between the positively charged CRPP and the negatively charged cellular membrane could be strengthened, which led to enhanced cellular internalization.
Table 1. Characterization of CRPP.
Actual content [mole, wt%] Code
mPEGa)
Ce6b)
Deprot-L-Aspc)
API-g-L-Aspd)
M n e)
PZC (pH)f)
ZP at pH 7.4g)
ZP at pH 6.5h)
CRPP
1 (43)
1 (5)
10 (11)
20 (41)
11 700
7.0
16 1
þ12 1
a)
Actual content of mPEG in the synthesized compound, determined by 1H NMR; b)Actual content of Ce6 in the synthesized compound, determined by 1H NMR and UV–Vis spectrophotometer (Figure S2, Supporting Information); c)Actual content of deprotected-L-aspartic acid (Deprot-L-Asp) in the synthesized compound, determined by 1H NMR; d)Actual content of 1-(3-aminopropyl) imidazole-grafted-L-aspartic acid (API-g-L-ASP) in the synthesized compound, determined by 1H NMR; e)Number average molecular weight (M n ), determined by 1H NMR; f) Point of zero charge (PZC, pH value) of the synthesized compound, determined by DLS; g)Zeta potential value (ZP) of the synthesized compound at pH 7.4, determined by DLS; h)Zeta potential value (ZP) of the synthesized compound at pH 6.5, determined by DLS.
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Figure 3. Physicochemical characterization of CRPP. a and b) Photographs of (1) free Ce6 and (2) CRPP in (a) water or (b) DMSO (the yellow arrow indicates aggregates of free Ce6). c) Fluorescence emission intensity of free Ce6 and CRPP in water. d) Fluorescence image from wells containing free Ce6 and CRPP in water. e) Singlet oxygen generation (SOG) of free Ce6 and CRPP in water with laser irradiation. f) Variation in the zeta-potential measurements of CRPP as a function of pH (n ¼ 3).
3.3. In Vitro Cytotoxicity of CRPP at Various pH Values To verify the feasibility of using CRPP for PDT, an in vivo cytotoxicity test was performed using the MTT assay at pH 7.4 and 6.5 (dose of Ce6 was equally treated with 10 mg mL1, Figure 5a). Under laser irradiation (3.6 J cm2), CRPP showed significantly enhanced cytotoxicity at pH 6.5 compared to that at pH 7.4 ( p < 0.001), whereas the free Ce6 exhibited significant toxicity regardless of the pH value due to its nonspecific cellular-uptake behavior. The results of the live/dead assay were consistent with those of the MTT assay (Figure 5b). Significantly increased cell death (red fluorescence) was observed in the cancer cells treated with CRPP at pH 6.5 under laser irradiation, in contrast to the cells
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after the same treatment but at an incubation pH of 7.4. However, the free Ce6-treated groups showed strong red fluorescence under both pH conditions. These results indicate that the charge-switchable property of the CRPP enhanced its cellular internalization into cancer cells, which led to remarkably enhanced efficiency in the eradication of cancer cells.
3.4. In Vivo Tumor-Targeting Ability and Body Distribution of CRPP in the CT26 Tumor Model Finally, to evaluate the feasibility of using CRPP for in vivo application, we injected CRPP intravenously into Balb/c nude mice bearing CT26 tumors and examined its
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Figure 4. In vitro cellular internalization of CRPP at various pH values. a) Flow cytometry quantification of the cellular internalization of free Ce6 and CRPP at pH 7.4 and 6.5. b) CLSM image of an HCT-116 cells treated with free Ce6 or CRPP at pH 7.4 and 6.5 (scale bar ¼ 100 mm).
distribution in the body using an IVIS (Figure 6). The fluorescence signals from the tumor in CRPP-treated mice gradually increased up to 6 h post-injection, whereas in the case of free Ce6, a relatively weak fluorescence signal was detected at all of the time points (Figure 6a). We also assayed the distribution in the body of CRPP in the dissected tumors and other major organs (heart, lung, spleen, liver, kidney, and muscle) (Figure 6b). Consistent with real-time in vivo optical imaging, ex vivo fluorescence images at 24 h after CRPP injection showed a higher fluorescence signal in the tumor region than that of the free Ce6-treated group. In addition, CRPP exhibited a much stronger fluorescence signal in the kidney region than that of free Ce6. According to previous reports,[24–26] the majority of clearance for small molecules and nanoparticles injected into the bloodstream is through either the renal (urine) or hepatic (bile to feces) route. Because neutral, geometrically balanced, and polyionic (referred to as zwitterionic) charged imaging proves that they tend to be rapidly cleared through the renal route,[25] zwitterionic charged group (i.e., imidazole and carboxyl groups)-containing CRPP could be eliminated from
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Figure 5. In vitro cytotoxicity of CRPP at various pH values. a) Cellviability test (MTT assay) and b) live/dead assay of HCT-116 cells treated with free Ce6 or CRPP under laser irradiation at pH 7.4 and 6.5 (dose of Ce6 ¼ 10 mg mL1, dose of laser power ¼ 3.6 J cm2, n ¼ 4, p < 0.001).
the body exclusively though renal filtration. Altogether, these results support that, following accumulation in the tumor tissue, the CRPP responded to the acidic tumor environment and became positively charged, which enhanced its accumulation in the tumor cells, and it was finally secreted by the renal route.
4. Conclusion We have demonstrated a cancer-recognizing polymeric photosensitizer (CRPP) that is pH-responsive to targeted cancer therapy. The CRPP was synthesized by a facile twostep processes combining ROP and aminolysis reactions. The newly synthesized CRPP exhibited high water solubility. In particular, CRPP is capable of reversing its charge from negative to positive at the tumor extracellular pH (pHe, pH 6.5) to facilitate cellular internalization, which led to enhanced cytotoxicity in cancer cells. As a result, CRPP was found to exhibit highly effective tumor-targeting efficacy in an in vivo tumor model and was finally excreted through the renal route. These properties indicate that the creation of CRPP can be applied to the design of an advanced photosensitizer for PDT. Given the promising results of
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Received: August 7, 2014; Revised: September 3, 2014; Published online: September 24, 2014; DOI: 10.1002/mabi.201400361 Keywords: cancer; drug delivery; photodynamic therapy; photosensitizer; pH-responsive polymer
Figure 6. In vivo tumour targeting and biodistribution of CRPP. a) In vivo optical fluorescence imaging of CT26 tumor-bearing Balb/c nude mice after intravenous injection of free Ce6 and CRPP using IVIS (equivalent to 0.2 mg kg1, Ce6). b) Ex vivo fluorescence images of tumors and organs in CT26 tumorbearing Balb/c nude mice after 24 h post-injection of free Ce6 and CRPP.
these initial in vitro and in vivo studies, additional investigations are now warranted to rigorously examine the potential therapeutic efficacy of CRPP with in vivo preclinical animal studies.
Acknowledgements: 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) and the Strategic core materials technology development (10047756, Development of tetra-pyrrole type for color, light-emitting, detecting device) funded By the Ministry of Trade, industry & Energy (MI, Korea).
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[1] R. R. Allison, C. H. Sibata, Photodiagn. Photodyn. Ther. 2010, 7, 61. [2] P. Agostinis, K. Berg, K. A. Cengel, T. H. Foster, A. W. Girotti, S. O. Gollnick, S. M. Hahn, M. R. Hamblin, A. Juzeniene, D. Kessel, CA. Cancer J. Clin. 2011, 61, 250. [3] F. Li, S.-J. Park, D. Ling, W. Park, J. Y. Han, K. Na, K. Char, J. Mater. Chem. B 2013, 1, 1678. [4] I. Roy, T. Y. Ohulchanskyy, H. E. Pudavar, E. J. Bergey, A. R. Oseroff, J. Morgan, T. J. Dougherty, P. N. Prasad, J. Am. Chem. Soc. 2003, 125, 7860. [5] H. Kim, S. Mun, Y. Choi, J. Mater. Chem. B 2013, 1, 429. [6] W. Park, S.-J. Park, K. Na, Biomaterials 2011, 32, 8261. [7] M. Hurtgen, A. Debuigne, M. Hoebeke, C. Passirani, N. Lautram, ^me, C. Detrembleur, A. Mouithys-Mickalad, P. H. Guelluy, C. Jero Macromol. Biosci. 2013, 13, 106. [8] B.-C. Bae, K. Na, Biomaterials 2010, 31, 6325. [9] C.-S. Lee, W. Park, S.-J. Park, K. Na, Biomaterials 2013, 34, 9227. [10] D. Ling, B.-C. Bae, W. Park, K. Na, Biomaterials 2012, 33, 5478. [11] C.-S. Lee, W. Park, Y. U. Jo, K. Na, Chem. Commun. 2014, 50, 4354. [12] J. Lu, W. Zhang, L. Yuan, W. Ma, X. Li, W. Lu, Y. Zhao, G. Chen, Macromol. Biosci. 2014, 14, 340. [13] H. Cho, L. Li, Y. H. Bae, K. M. Huh, H. C. Kang, Macromol. Biosci. 2014, DOI: 10.1002/mabi.201400145. [14] M. Nakanishi, J.-S. Park, W.-D. Jang, M. Oba, K. Kataoka, React. Funct. Polym. 2007, 67, 1361. [15] D. Ling, W. Park, S.-J. Park, Y. Lu, K. S. Kim, M. J. Hackett, B. H. Kim, H. Yim, Y. S. Jeon, K. Na, J. Am. Chem. Soc. 2014, 136, 5647. [16] T. Yeung, G. E. Gilbert, J. Shi, J. Silvius, A. Kapus, S. Grinstein, Science 2008, 319, 210. [17] A. Verma, F. Stellacci, Small 2010, 6, 12. [18] W. H. Daly, D. Poche, Tetrahedron Lett. 1988, 29, 5859. [19] Z. Zhu, Z. Tang, J. A. Phillips, R. Yang, H. Wang, W. Tan, J. Am. Chem. Soc. 2008, 130, 10856. [20] P. Huang, J. Lin, X. Wang, Z. Wang, C. Zhang, M. He, K. Wang, F. Chen, Z. Li, G. Shen, D. Cui, X. Chen, Adv. Mater. 2012, 24, 5104. [21] H. Park, W. Park, K. Na, Biomaterials 2014, 35, 7963. [22] K. S. Kim, W. Park, J. Hu, Y. H. Bae, K. Na, Biomaterials 2014, 35, 337. [23] H. Park, K. Na, Biomaterials 2013, 34, 6992. [24] H. S. Choi, J. V. Frangioni, Mol. Imaging 2010, 9, 291. [25] H. S. Choi, S. L. Gibbs, J. H. Lee, S. H. Kim, Y. Ashitate, F. Liu, H. Hyun, G. Park, Y. Xie, S. Bae, Nat. Biotechnol. 2013, 31, 148. [26] H. S. Choi, Y. Ashitate, J. H. Lee, S. H. Kim, A. Matsui, N. Insin, M. G. Bawendi, M. Semmler-Behnke, J. V. Frangioni, A. Tsuda, Nat. Biotechnol. 2010, 28, 1300.
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A Cancer-Recognizing Polymeric Photosensitizer Based on the Tumor Extracellular pH Response of Conjugated Polymers for Targeted Cancer Photodynamic Therapya Songhee Jeong,y Wooram Park,y Chung-Sung Lee, Kun Na*
Herein, a cancer-recognizing polymeric photosensitizer (CRPP) was demonstrated not only for high water solubility but also for pH-responsive targeted photodynamic cancer therapy. The synthesized CRPP exhibited high water solubility and the pH-dependent charge-switching property. From an in vitro cellular internalization study with HCT-116 human colon cancer cells, significantly enhanced cellular uptake as detected for CRPP at pH 6.5 compared to the cellular uptake of CRPP at pH 7.4, which led to enhanced cytotoxicity in the cancer cells. Finally, the CRPP was found to exhibit high tumor-targeting efficacy in an in vivo tumor model and was finally excreted through the renal route.
1. Introduction Photodynamic therapy (PDT) is known as a prospective treatment method and has been used for the treatment of a variety of oncological, dermatological, cardiovascular, and ophthalmic diseases.[1–3] PDT is based on combinations of photosensitizers (PS), light, and molecular oxygen. Under light irradiation at an appropriate wavelength, the PS forms reactive oxygen species (ROSs), such as singlet oxygen (1O2) or free radicals, resulting in irreversible damage of tumor cells.[4] However, the water-insolubility S. Jeong, W. Park, C.-S. Lee, 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] Supporting Information is available from the Wiley Online Library or from the author. y These authors contributed equally to this work. a
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and limited tumor selectivity of PS have been major drawbacks for clinical application.[5–7] In this regard, various types of PSs based on the polymer conjugation technique have been developed to overcome the limitation of conventional photosensitizing agents.[8–13] Here, we propose a cancer-recognizing polymeric photosensitizer (CRPP) that is pH-responsive to targeted cancer PDT (Figure 1a,b). CRPP was synthesized by a three-step synthetic process (Figure 2a). First, methoxy-polyethylene glycol-block-poly(benzyl-L-aspartic acid) (mPEG-poly(Bz-LAsp)) was synthesized using a ring opening polymerization (ROP). Second, the second-generation PS chlorin e6 (Ce6) was conjugated with mPEG-poly(Bz-L-Asp). Finally, pHresponsive imidazole groups were introduced by an aminolysis reaction.[11,14,15] We hypothesized that the higher water solubility of the CRPP would have significantly higher fluorescence and singlet-oxygen generation (SOG) than conventional PS under aqueous conditions. In addition, CRPP can also have a pH-dependent charge-switching property due to the ionizable groups (i.e., the imidazole
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DOI: 10.1002/mabi.201400361
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addition, an in vitro cellular-uptake study was performed with the HCT-116 human colon cancer cell line to evaluate the pH-dependent cellular uptake behavior of CRPP using flow cytometry and confocal laser scanning microscopy (CLSM). The in vitro therapeutic potential of CRPP was also confirmed at various pH values (e.g., pH 7.4 and 6.5) using an MTT cytotoxicity assay. Finally, to evaluate the in vivo feasibility of CRPP, we performed an in vivo cancer-specific targeting study in a CT26 tumor-bearing mice model using an in vivo image station (IVIS).
2. Experimental Section 2.1. Materials b-Benzyl-L-aspartic acid (BLA), triphosgene, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dichloromethane (DCM), 1,3Figure 1. Schematic representation of the cancer-recognizing polymeric photosensitizer dicyclohexylcarbodiimide (DCC), and tetrahy(CRPP). a) Chemical structure of CRPP and b) schematic representation of the pH- drofuran (THF), 3-(4,5-dimethyl-2-thiazolyl)2,5-diphenyl-2H-tetrazolium bromide (MTT), dependent charge switching behavior of CRPP and chemical structural representation of the protonation of the imidazole groups in CRPP at an acidic pH. Schematic triethylamine (TEA), and 1-(3-aminopropyl) representations of (c) the tumor-accumulation behavior of CRPP and (d) its imidazole (API) was purchased from Sigma– enhanced cellular internalization via electrostatic interactions between the positively Aldrich (Sigma–Aldrich Korea, Seoul, Korea). charged CRPP and negatively charged cellular membrane; then, the internalized Methoxy polyethylene glycol amine (mPEGCRPP can generate singlet oxygen under laser irradiation, which leads to the killing amine, M ; 5 000) was purchased from Sunbio, w of tumor cells. Inc. (Anyang, Korea). Chlorin e6 (Ce6) was purchased from Frontier Scientific, Inc. (UT, USA). A human colon cancer HCT-116 cell line was obtained from the Korean Cell Line Bank (KCLB No.10247). groups) in the polymer backbone (Figure 1b). Consequently, RPMI 1640 medium, fetal bovine serum (FBS), antibiotics (penicillin CRPP can be stable in the bloodstream because the charge of and streptomycin), and Dulbecco’s phosphate-buffered saline CRPP is negative under normal, physiological pH (pH 7.4) (DPBS) were obtained from Gibco BRL (Invitrogen Corp., Carlsbad, (Figure 1c). However, after accumulation in a tumor site via CA, USA). SOSG was purchased from Molecular Probes, Inc. the enhanced permeability and retention (EPR) effect, the (Eugene, OR, USA). b-Benzyl-L-aspartic acid N-carboxyanhydride CRPP can become positively charged at the tumoral acidic (BLA-NCA) was synthesized by the Fuchs–Farthing method using extracellular pH (pHe, pH 6.5) through protonation of triphosgene.[18]
the imidazole groups. Because the cellular membrane generally exhibits a net negative charge due to the phosphate group of phosphatidylserine,[16,17] the cellular uptake of CRPP should be increased as a result of the strengthened interaction between the positively charged CRPP and the tumor cells via electrostatic interactions (Figure 1d). Therefore, a significantly enhanced PDT effect can be achieved against tumor cells. In this paper, a pH-responsive polymeric PS was designed and synthesized and then characterized using 1H NMR, fluorescence spectroscopy, and dynamic light scattering (DLS). We also measured the SOG behavior of the synthesized polymeric PS in an aqueous solution using singlet oxygen sensor green (SOSG) as a singlet oxygen probe. In
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2.2. Synthesis of CRPP (mPEG-Poly(Bz-L-Asp)-Ce6) mPEG-poly(Bz-L-Asp)-Ce6 was synthesized using a modification of the method reported previously.[9,11,15] To synthesis mPEGpoly(Bz-L-Asp), BLA-NCA (1.5 g, 6 mmol) was polymerized in a mixture of DMF (10 mL) and CH2Cl2 (50 mL) at room temperature by initiation from the terminal primary amine group of mPEGamine (M n ¼ 5 kDa, 1 g, 200 mmol). mPEG-poly(Bz-L-Asp) was purified by precipitation in ether (1 L) three times and then dried in vacuo. To synthesize mPEG-poly(Bz-L-Asp)-Ce6, Ce6 was conjugated to the terminal amine group of mPEG-poly(Bz-L-Asp) by the conventional carbodiimide reaction. mPEG-poly(Bz-L-Asp) (0.5 g, 43 mmol) and a mixture of Ce6 (31 mg, 52 mmol),
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2.3. Measurement of the ZetaPotential The zeta-potential was determined using DLS (Zetasizer Nano ZS, Malvern Instruments Ltd., UK). DLS was performed at 25 8C in an aqueous solution (sample concentration, 1 mg mL1) with the sampling time and analysis set to automatic.
2.4. Optical Photography and Fluorescence Analysis
Figure 2. Chemical synthesis of CRPP (mPEG-poly(API-L-Asp)-Ce6, n ¼ 114, m ¼ 30). a) Synthetic route of CRPP and b) 1H NMR analysis of CRPP in DMSO-d6. dicyclohexylcarbodiimide (16 mg, 78 mmol), and N-hydroxysuccinimide (9 mg, 78 mmol) were dissolved separately in DMF (10 mL), and the solutions were stirred thoroughly for 3 h prior to the condensation reaction. The two reactant solutions were then mixed and stirred at room temperature. After 24 h, the reaction mixture was purified by precipitation in ether (0.2 L) three times and then dried in vacuo. Finally, mPEG-poly(API-LAsp)-Ce6 was synthesized via aminolysis of mPEG-poly(Bz-LAsp)-Ce6 with API. mPEG-poly(Bz-L-Asp)-Ce6 (0.2 g, 16 mmol) was dissolved in DMSO (5 mL), followed by reaction with API (40 mg, 320 mmol) and TEA (32 mg, 320 mmol) under nitrogen atmosphere at room temperature for 12 h. The reaction mixture was added dropwise into a cooled aqueous solution of 0.1 N HCl (20 mL) and was dialyzed against a 0.01 N HCl solution three times (Spectra/Por; MWCO: 1 kDa). The final solution was lyophilized, and the formation of mPEG-poly(API-L-Asp)-Ce6 was verified by 1H NMR. The 1H NMR spectra were recorded in DMSO-d6 at room temperature using a Bruker NMR Spectrometer (Bruker, Germany) at 500 MHz. 1 H NMR (500 MHz, DMSO-d6) for mPEG-poly(API-L-Asp)-Ce6 (Figure 2b): d ¼ 7.6 ppm (1H, s, —NCH5 5N— of imidazole ring), 7.1 ppm (1H, s, —NCH5 5CH— of imidazole ring), 6.8 ppm (1H, s, —CHCH5 5N— of imidazole ring), 4.5 ppm (1H, m, —NHCHC5 5O—), 3.9 ppm (2H, m, —NCH2CH2), 3.5 ppm (182H, s, —CH2CH2O— of mPEG chain), 3.2 ppm (3H, CH3O— of mPEG chain and CH3C— of Ce6), 3.0 ppm (2H, m, —NHCH2CH2—), d 2.8–2.5 ppm (2H, m, —CHCH2C5 5O—), and 1.8 ppm (2H, m, —CH2CH2CH2—).
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Free Ce6 and CRPP (Ce6, 1.6 104 M) were dissolved in distilled water and DMSO, respectively. Optical photographs were recorded using a digital camera (nx1000, Samsung Electronics Co., Korea) at room temperature. The fluorescence intensity was measured in 96-well plate using a plate reader (Tecan Genios, Durham, NC, USA) with an excitation wavelength of 650 nm and an emission wavelength of 675 nm at room temperature. The fluorescence images were captured using a 12-bit CCD camera (Image Station 4000 MM; Kodak, New Haven, CT, USA) with a special Cmount lens and a long-wave emission filter (600–700 nm; Omega Optical, Brattleboro, VT, USA).
2.5. Measurement of Singlet Oxygen Generation (SOG) The SOG was detected chemically using SOSG (S-36002, Invitrogen/ Molecular Probes) as a probe. The SOSG probe works via intramolecular electron transfer, which quenches the fluorescence from the light-emitting chromophore prior to reaction with singlet oxygen. Reaction with singlet oxygen results in the formation of the endoperoxide, prohibiting electron transfer and thus leading to the recovery of fluorescence.[19,20] Free Ce6 or CRPP was dissolved in distilled water (Ce6, 1.0 105 M) and mixed with a SOSG solution (2 mM). The mixture was then irradiated with 10 mW cm2 of a 670 nm laser source (fiber-coupled laser system, LaserLab1, Korea) over time. The fluorescence intensity of SOSG (lex ¼ 494 nm, lem ¼ 534 nm) was recorded using fluorescence spectroscopy (RF5301; Shimadzu, Japan).
2.6. In Vitro Cell Culture and Incubation Conditions HCT-116 (human colon cancer) and CT-26 (murine colon cancer) cells were obtained from the Korean Cell Line Bank (HCT-116; KCLB No. 10247, CT-26; KCLB No. 80009) and were cultured in RPMI-1640 (HCT-116) and DMEM (CT-26) supplemented with 10% heatinactivated FBS and penicillin/streptomycin (100 U mL1 and 100 mg mL1, respectively), which is called the complement medium (CM medium) in this study. The cells were cultured at
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37 8C in a humidified atmosphere containing 5% CO2 and were subcultured in a new medium every 2–3 d. CRPP or free Ce6 was dissolved in a serum-free (SF) medium.
2.7. In Vitro Cellular-Uptake Test To verify the cellular uptake of CRPP at two different pH values (pH 7.4 and 6.5), HCT-116 cells were seeded in a six-well cell culture plate at a density of 1.0 106 cells per well and were incubated for 12 h at 37 8C in 5% CO2. The medium was then removed, and the cells were incubated in serum-free (SF) medium containing CRPP of free Ce6 (Ce6, 3.2 106 M) for 10 min at pH 7.4 or 6.5. The cells were rinsed, harvested, and resuspended with DPBS. The cellular uptake was quantitatively analyzed using flow cytometery (Beckman, San Jose, CA, USA). For each sample, 10 000 cells (gated events) were counted, and the Ce6 fluorescence was detected with logarithmic settings (FL4; Em ¼ 670 nm). Each experiment was analyzed statistically using the CXP analysis program (Beckman).
2.8. Confocal Laser Scanning Microscopy (CLSM) Analysis To observe the cellular localization of CRPP at two different pH values (pH 7.4 and 6.5), HCT-116cells (1.0 105 cells per well in a 35mm dish) were treated with CRPP or free Ce6 (Ce6, 3.2 106 M) for 10 min at pH 7.4 or 6.5. The cells were then washed twice with DPBS, fixed with 4% paraformaldehyde and stained with DAPI. The cells were mounted in a mounting medium (Dako, Glostrup, Denmark) and visualized using a confocal laser scanning microscope (LSM 710 Meta; Carl Zeiss, Germany). A laser line with a wavelength of 633 nm was used for excitation. A long-pass filter (LP 650 nm) was used at the emission end for detection. The fluorescence images were analyzed using the LSM Image Browser software (Carl Zeiss, Germany).
2.9. In Vitro Cytotoxicity Study HCT-116 cells were seeded in black 96-well culture plates at a density of 2.0 104 cells per well and were incubated for 12 h. CRPP or free Ce6 (Ce6, 1.6 105 M) was added to each well in SF medium (100 mL), and the plates were returned to the incubator for 10 min. After incubation, the wells were rinsed twice with DPBS to remove materials that had not been internalized by the cells. One hundred microliters of the complement medium were added to the wells, and each well was irradiated using a 670 nm laser source (3.6 J cm2, fiber-coupled laser system, LaserLab1, Korea). The cells were incubated for a further 24 h. The cell viability was assessed using the MTT assay according to our previous reports.[6,21,22]
cells red. HCT-116 cells were seeded in 35-mm cell culture dishes at a density of 1 105 per well. The cells were incubated for 12 h at 37 8C in 5% CO2. After incubation, the medium was removed, and the cells were incubated in SF medium containing CRPP or free Ce6 (Ce6, 1.6 105 M) at pH 7.4 and 6.5 for 10 min. The medium was removed, and the cells were rinsed twice with DPBS. Then, irradiation (3.6 J cm2) was performed with a 670 nm laser source (fiber-coupled laser system, LaserLab1, Korea). The cells were then incubated in the complement medium for 1 h. The cell viability was observed using fluorescence microscopy (Carl Zeiss, Germany). The fluorescence images were analyzed using LSM Image Browser software (Carl Zeiss).
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 four- to six-week-old female BALB/c nude mice (Oriental Bio, Seongnam, Korea) for fluorescence optical imaging.
2.12. In Vivo Optical Imaging CT26 cells (1.0 105 cells) were subcutaneously transplanted into male Balb/c mice. When the tumors grew to approximately 10 mm3 in volume, CRPP or free Ce6 (Ce6, 0.2 mg kg1) was intravenously injected into the CT26-bearing mice. The images were obtained using a 12-bit CCD camera (Image Station 4000 MM; Kodak, New Haven, CT) with a special C-mount lens and a long-wave emission filter (600–700 nm; Omega Optical).
2.13. Ex Vivo Biodistribution Study To compare the biodistribution of CRPP and free Ce6 in normal tissues (heart, lung, spleen, liver, kidney, and muscle) and tumor tissue, the mice were sacrificed at 24 h post-injection, and each tissue was excised. The fluorescence intensity of Ce6 in each tissue was measured using a 12-bit CCD camera (Image Station 4000 MM; Kodak, New Haven, CT) with a special C-mount lens and a longwave emission filter (600–700 nm; Omega Optical).
2.14. Statistical Analysis The data are expressed as the mean standard deviation (SD). The differences between the values were assessed using Student’s t-test.
3. Results and Discussion 2.10. Live/Dead Assay The live/dead viability/cytotoxicity assay kit (Molecular Probes, USA) was used as a two-color fluorescence cell-viability assay.[11] The optimal dyes used were calcein AM (2 mM) and EthD-1 (4 mM); the former stains live cells green, whereas the latter stains dead
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3.1. Synthesis and Physicochemical Characterization of CRPP CRPP was synthesized by combining the ring-opening polymerization (ROP) of b-benzyl-L-aspartate N-carboxyanhydride
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(Bz-L-Asp-NCA) and an aminolysis reaction (Figure 2a). First, poly(Bz-L-Asp) was polymerized from the primary amine group of mPEG-amine to generate mPEG-poly(Bz-LAsp). Then, PS (Chlolin e6, Ce6) was conjugated with mPEG-poly(Bz-L-Asp) using conversional carbodiimide coupling chemistry. The successful synthesis of mPEGpoly(Bz-L-Asp)-Ce6 conjugate was confirmed by GPC analysis (Figure S1, Supporting Information): an increased molecular weight of polymer was observed after PSconjugation. Finally, the pH-responsive groups (i.e., API) were incorporated into mPEG-poly(Bz-L-Asp) though an aminolysis reaction between the primary amine group of API (nucleophilic) and the benzyl ester group of mPEGpoly(Bz-L-Asp). The chemical structure of CRPP was analyzed using 1H NMR analysis (Figure 2b). The degree of polymerization (DP) of the Bz-L-Asp units was calculated to be 30. In addition, we confirmed the successful incorporation of zwitterionic groups (i.e., imidazole and carboxyl groups) on CRPP (Table 1). To characterize the physicochemical property of CRPP, we first confirmed the solubility of CRPP in water and an organic solvent. Figure 3a,b shows the solubility of free Ce6 and CRPP (Ce6, 1.6 104 M) in water and the organic solvent (i.e., DMSO), respectively. Free Ce6 was precipitated under aqueous conditions due to the strong hydrophobicity (Figure 3a(1)); this low water solubility is a major limitation to the use of conventional PS as a pharmaceutical agent. However, CRPP exhibited high water solubility (Figure 3a(2)). As shown in Figure 3b, both compounds could be dissolved well in the organic solvent. To investigate the photoactivity of CRPP (Ce6, 1.0 105 M) in an aqueous solution, the fluorescence emission and SOG property were examined using fluorescence spectroscopy. Whereas the fluorescence emission (lex ¼ 670, lem ¼ 675) of CRPP was much higher than that of free Ce6 in water (Figure 3c,d). The SOG of CRPP was also evaluated chemically using SOSG as a probe with laser irradiation (670 nm). As shown in Figure 3e, CRPP showed significantly increased SOG compared to free
Ce6. These results indicate that the introduction of hydrophilic PEG and ionizable groups (e.g., imidazole and carboxyl groups) effectively enhanced the photoactivity of PS under aqueous conditions. Next, to confirm the pH-dependent charge-switching property of CRPP, the zeta-potential was measured as a function of the pH value (Figure 3f). The zeta-potential of CRPP was changed from negative (approximately 16 mV) to positive (approximately þ12 mV) as the pH of the solution decreased from pH 7.4 to 6.5. We suppose that the negative value at pH 7.4 was attributed to the unmodified carboxyl groups and PEG of CRPP, whereas that was offset by the protonation of the imidazole groups at pH 6.5. 3.2. In Vitro Cellular-Internalization Behavior of CRPP at Various pH Values To investigate the cellular internalization behavior of CRPP at pH 7.4 and 6.5, we used flow cytometry and CLSM with human colon cancer (HCT-116) cells (the dose of Ce6 was equally treated with 2 mg mL1) (Figure 4a,b). As a control test, the cellular-uptake test of free Ce6 was performed at pH 7.5 and 6.5. As shown in Figure 4a, a high level of cellular uptake was observed for the free Ce6 regardless of the pH due to its nonspecific cellular-uptake behavior though diffusion.[6,23] However, significantly enhanced cellular uptake was detected for CRPP at pH 6.5 compared to the cellular uptake of CRPP at pH 7.4. This observation was further confirmed by CLSM under the same conditions (Figure 4b). Consistent with data from flow cytometry, the strong red fluorescence of free Ce6 was observed at both pH values. In contrast, HCT-116 cells treated with CRPP showed higher red fluorescence at pH 6.5 than that at pH 7.4. These results can be attributed to the charge-switching property of CRPP, which becomes positively charged at pH 6.5. As a result, the interaction between the positively charged CRPP and the negatively charged cellular membrane could be strengthened, which led to enhanced cellular internalization.
Table 1. Characterization of CRPP.
Actual content [mole, wt%] Code
mPEGa)
Ce6b)
Deprot-L-Aspc)
API-g-L-Aspd)
M n e)
PZC (pH)f)
ZP at pH 7.4g)
ZP at pH 6.5h)
CRPP
1 (43)
1 (5)
10 (11)
20 (41)
11 700
7.0
16 1
þ12 1
a)
Actual content of mPEG in the synthesized compound, determined by 1H NMR; b)Actual content of Ce6 in the synthesized compound, determined by 1H NMR and UV–Vis spectrophotometer (Figure S2, Supporting Information); c)Actual content of deprotected-L-aspartic acid (Deprot-L-Asp) in the synthesized compound, determined by 1H NMR; d)Actual content of 1-(3-aminopropyl) imidazole-grafted-L-aspartic acid (API-g-L-ASP) in the synthesized compound, determined by 1H NMR; e)Number average molecular weight (M n ), determined by 1H NMR; f) Point of zero charge (PZC, pH value) of the synthesized compound, determined by DLS; g)Zeta potential value (ZP) of the synthesized compound at pH 7.4, determined by DLS; h)Zeta potential value (ZP) of the synthesized compound at pH 6.5, determined by DLS.
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Figure 3. Physicochemical characterization of CRPP. a and b) Photographs of (1) free Ce6 and (2) CRPP in (a) water or (b) DMSO (the yellow arrow indicates aggregates of free Ce6). c) Fluorescence emission intensity of free Ce6 and CRPP in water. d) Fluorescence image from wells containing free Ce6 and CRPP in water. e) Singlet oxygen generation (SOG) of free Ce6 and CRPP in water with laser irradiation. f) Variation in the zeta-potential measurements of CRPP as a function of pH (n ¼ 3).
3.3. In Vitro Cytotoxicity of CRPP at Various pH Values To verify the feasibility of using CRPP for PDT, an in vivo cytotoxicity test was performed using the MTT assay at pH 7.4 and 6.5 (dose of Ce6 was equally treated with 10 mg mL1, Figure 5a). Under laser irradiation (3.6 J cm2), CRPP showed significantly enhanced cytotoxicity at pH 6.5 compared to that at pH 7.4 ( p < 0.001), whereas the free Ce6 exhibited significant toxicity regardless of the pH value due to its nonspecific cellular-uptake behavior. The results of the live/dead assay were consistent with those of the MTT assay (Figure 5b). Significantly increased cell death (red fluorescence) was observed in the cancer cells treated with CRPP at pH 6.5 under laser irradiation, in contrast to the cells
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after the same treatment but at an incubation pH of 7.4. However, the free Ce6-treated groups showed strong red fluorescence under both pH conditions. These results indicate that the charge-switchable property of the CRPP enhanced its cellular internalization into cancer cells, which led to remarkably enhanced efficiency in the eradication of cancer cells.
3.4. In Vivo Tumor-Targeting Ability and Body Distribution of CRPP in the CT26 Tumor Model Finally, to evaluate the feasibility of using CRPP for in vivo application, we injected CRPP intravenously into Balb/c nude mice bearing CT26 tumors and examined its
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Figure 4. In vitro cellular internalization of CRPP at various pH values. a) Flow cytometry quantification of the cellular internalization of free Ce6 and CRPP at pH 7.4 and 6.5. b) CLSM image of an HCT-116 cells treated with free Ce6 or CRPP at pH 7.4 and 6.5 (scale bar ¼ 100 mm).
distribution in the body using an IVIS (Figure 6). The fluorescence signals from the tumor in CRPP-treated mice gradually increased up to 6 h post-injection, whereas in the case of free Ce6, a relatively weak fluorescence signal was detected at all of the time points (Figure 6a). We also assayed the distribution in the body of CRPP in the dissected tumors and other major organs (heart, lung, spleen, liver, kidney, and muscle) (Figure 6b). Consistent with real-time in vivo optical imaging, ex vivo fluorescence images at 24 h after CRPP injection showed a higher fluorescence signal in the tumor region than that of the free Ce6-treated group. In addition, CRPP exhibited a much stronger fluorescence signal in the kidney region than that of free Ce6. According to previous reports,[24–26] the majority of clearance for small molecules and nanoparticles injected into the bloodstream is through either the renal (urine) or hepatic (bile to feces) route. Because neutral, geometrically balanced, and polyionic (referred to as zwitterionic) charged imaging proves that they tend to be rapidly cleared through the renal route,[25] zwitterionic charged group (i.e., imidazole and carboxyl groups)-containing CRPP could be eliminated from
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Figure 5. In vitro cytotoxicity of CRPP at various pH values. a) Cellviability test (MTT assay) and b) live/dead assay of HCT-116 cells treated with free Ce6 or CRPP under laser irradiation at pH 7.4 and 6.5 (dose of Ce6 ¼ 10 mg mL1, dose of laser power ¼ 3.6 J cm2, n ¼ 4, p < 0.001).
the body exclusively though renal filtration. Altogether, these results support that, following accumulation in the tumor tissue, the CRPP responded to the acidic tumor environment and became positively charged, which enhanced its accumulation in the tumor cells, and it was finally secreted by the renal route.
4. Conclusion We have demonstrated a cancer-recognizing polymeric photosensitizer (CRPP) that is pH-responsive to targeted cancer therapy. The CRPP was synthesized by a facile twostep processes combining ROP and aminolysis reactions. The newly synthesized CRPP exhibited high water solubility. In particular, CRPP is capable of reversing its charge from negative to positive at the tumor extracellular pH (pHe, pH 6.5) to facilitate cellular internalization, which led to enhanced cytotoxicity in cancer cells. As a result, CRPP was found to exhibit highly effective tumor-targeting efficacy in an in vivo tumor model and was finally excreted through the renal route. These properties indicate that the creation of CRPP can be applied to the design of an advanced photosensitizer for PDT. Given the promising results of
Macromol. Biosci. 2014, 14, 1688–1695 ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Cancer-Recognizing Polymeric Photosensitizer www.mbs-journal.de
Received: August 7, 2014; Revised: September 3, 2014; Published online: September 24, 2014; DOI: 10.1002/mabi.201400361 Keywords: cancer; drug delivery; photodynamic therapy; photosensitizer; pH-responsive polymer
Figure 6. In vivo tumour targeting and biodistribution of CRPP. a) In vivo optical fluorescence imaging of CT26 tumor-bearing Balb/c nude mice after intravenous injection of free Ce6 and CRPP using IVIS (equivalent to 0.2 mg kg1, Ce6). b) Ex vivo fluorescence images of tumors and organs in CT26 tumorbearing Balb/c nude mice after 24 h post-injection of free Ce6 and CRPP.
these initial in vitro and in vivo studies, additional investigations are now warranted to rigorously examine the potential therapeutic efficacy of CRPP with in vivo preclinical animal studies.
Acknowledgements: 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) and the Strategic core materials technology development (10047756, Development of tetra-pyrrole type for color, light-emitting, detecting device) funded By the Ministry of Trade, industry & Energy (MI, Korea).
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