pH-Responsive Nanophotosensitizer for an Enhanced Photodynamic

Article Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX

pH-Responsive Nanophotosensitizer for an Enhanced Photodynamic Therapy of Colorectal Cancer Overexpressing EGFR Wen-Yu Chu,†,‡ Ming-Hsien Tsai,†,‡ Cheng-Liang Peng,# Ying-Hsia Shih,# Tsai-Yueh Luo,# Shu-Jyuan Yang,*,†,§,∥ and Ming-Jium Shieh*,†,⊥ †

Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, No. 1, Section 1, Jen-Ai Road, Taipei 100, Taiwan # Isotope Application Division, Institute of Nuclear Energy Research, P.O. Box 3-27, Longtan, Taoyuan 325, Taiwan § Gene’e Tech Co. Ltd. 2F., No. 661, Bannan Road, Zhonghe Dist., New Taipei City 235, Taiwan ∥ Apius Bio Inc. 1F., No. 92, Daxin Street, Yonghe Dist., New Taipei City 234, Taiwan ⊥ Department of Oncology, National Taiwan University Hospital and College of Medicine, No. 7, Chung-Shan South Road, Taipei 100, Taiwan S Supporting Information *

ABSTRACT: Photodynamic therapy (PDT) has been shown to kill cancer cells and improve survival and quality of life in cancer patients, and numerous new approaches have been considered for maximizing the efficacy of PDT. In this study, a new multifunctional nanophotosensitizer Ce6/GE11-(pH)micelle was developed to target epidermal growth factor receptor (EGFR) overexpressing colorectal cancer (CRC) cells. This nanophotosensitizer was synthesized using a micelle comprising pHresponsive copolymers (PEGMA−PDPA), biodegradable copolymers (mPEG−PCL), and maleimide-modified biodegradable copolymers (Mal−PEG−PCL) to entrap the potential hydrophobic photosensitizer chlorin e6 (Ce6) and to present EGFRtargeting peptides (GE11) on its surface. In the presence of Ce6/GE11-(pH)micelles, Ce6 uptake by EGFR-overexpressing CRC cells significantly increased due to GE11 specificity. Moreover, Ce6 was released from Ce6/GE11-(pH)micelles in tumor environments, leading to improved elimination of cancer cells in PDT. These results indicate enhanced efficacy of PDT using Ce6/GE11-(pH)micelle, which is a powerful nanophotosensitizer with high potential for application to future PDT for CRC. KEYWORDS: photodynamic therapy, epidermal growth factor receptor, Ce6, micelle

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PDT using specific NIR light.5−7 However, Ce6 (original form) is not currently used in clinical practice due to its hydrophobicity and low tumor-selectivity.8−10 Considering the outstanding ability of Ce6 to generate ROS and eliminate cancer cells during PDT, we designed and synthesized a new multifunctional nanophotosensitizer using a pH-responsive micelle that entraps Ce6 and bears EGFRtargeting peptides (GE11) on its surface. The resulting

INTRODUCTION Photodynamic therapy (PDT) can produce targeted tissue damage via photochemical reactions of photosensitizers, and its effects against cancer cells have resulted in improved survival times and quality of life for cancer patients.1,2 During PDT, the photosensitizer is exposed to light at specific wavelengths whereupon it reacts with oxygen to generate reactive oxygen species (ROS), such as singlet oxygen, which efficiently induce cancer cell death.3 Chlorin e6 (Ce6) having long-lived photoexcited triplet state is a potential hydrophobic photosensitizer for PDT.4 After light excitation, Ce6 efficiently generates ROS, and its high light absorption in the nearinfrared (NIR) red spectral region allows improved depths of © XXXX American Chemical Society

Received: November 5, 2017 Revised: February 20, 2018 Accepted: February 26, 2018

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DOI: 10.1021/acs.molpharmaceut.7b00925 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics

Scheme 1. Schematic Diagram of Accumulation Mechanisms of Ce6/GE11-(pH)Micelle According to the EPR Effect and EGFRTargeting and Successful Ce6 Release in Slightly Acidic Tumor Microenvironments or Lysosomes after EGFR-Mediated Endocytosis

Figure 1. Schematic diagram of PEGMA−PDPA, mPEG−PCL, and Mal−PEG−PCL and their fabrication into Ce6/GE11-(pH)micelles with Ce6 and GE11.

nanophotosensitizer Ce6/GE11-(pH)micelle will likely improve the efficacy of PDT against epidermal growth factor receptor (EGFR)-overexpressing colorectal cancer (CRC). Specifically, the Ce6/GE11-(pH)micelle is expected to increase Ce6 release and accumulation in tumor microenvironments and lysosomes after EGFR-mediated endocytosis (Scheme 1). Nanocarrier micelles have been shown to entrap various hydrophobic drugs and deliver them to tumors, providing enhanced permeation and retention (EPR) effect-mediated passive targeting and increased accumulation in tumors.11,12 To

further increase Ce6 accumulation in EGFR-overexpressing tumor cells, we conjugated Ce6-loaded pH-responsive micelles with EGFR target peptides. EGFR is overexpressed on various human epithelial cancer cells, including breast, lung, and ovarian cancer cells,13−16 and recognized as an important player in CRC initiation and progression.17 EGFR has been exploited in the design of drug delivery strategies for antitumor therapy.18,19 Subsequent studies have shown specifically enhanced drug uptake into EGFR-overexpressing cancer cells and efficient inhibition of tumor growth with minimal drug B


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Molecular Pharmaceutics concentrations in other tissues.20−23 In addition to passive and active targeting, the present micelle preparation was pHresponsive and similar to other pH-responsive copolymer micelles with pKa values of 6.2−6.9,24,25 leading to disassembly and release of encapsulated drug in tumor microenvironments (pH 5.8−6.5)26 and lysosomes (pH 4.0−5.5).27 This approach increased Ce6 distribution areas in tumors and enhanced antiproliferative activity. The present micelle preparation was synthesized using the pH-responsive copolymer poly(ethylene glycol) methacrylateco-2-(diisopropylamino)ethyl methacrylate (PEGMA−PDPA) and the biodegradable copolymers methoxypoly(ethylene glycol)/poly(ε-caprolactone) (mPEG−PCL) and maleimidemodified PEG−PCL (Mal−PEG−PCL). In initial experiments, we analyzed chemical structures, molecular weights (MW), and polydispersity (PD) of PEGMA−PDPA, mPEG−PCL, and Mal−PEG−PCL. In subsequent experiments, we compared PEGMA−PDPAs (1/2 and 1/4) with different PDPA molecular weights (MW) and confirmed that the designed PEGMA−PDPA had a pKa around 6.2−6.9. Subsequently, PEGMA−PDPA micelles, mPEG−PCL micelles, unloaded pHresponsive micelles ((pH)micelles), unloaded pH-responsive micelles with maleimide modification (Mal-(pH)micelle), Ce6loaded pH-responsive micelles (Ce6/(pH)micelles), and Ce6loaded pH-responsive micelles with maleimide modification (Ce6/Mal- (pH) micelles) were generated to validate the anticancer efficacy of Ce6/GE11-(pH)micelle. Basic characteristics, including size, drug encapsulation efficiency (EE), drug content (DC), critical micelle concentration (CMC), absorption and fluorescence (FL) spectra, pH-responsiveness, and singlet oxygen-generating abilities were determined. Subsequently, cell uptake, cytotoxicity, and photocytotoxicity were evaluated in vitro in HCT-116 cells with high EGFR expression and SW620 cells with low EGFR expression.28,29 Finally, in vivo and ex vivo distributions in mice bearing HCT-116 and SW620 xenograft tumors and in vivo PDT efficacy in mice bearing HCT-116 tumors were evaluated.

Bruker AVIII-400 instrument (Bruker, Billerica, MA, USA) and gel permeation chromatography (GPC). MW and chemical structures were determined using GPC and 1H NMR data. To evaluate pH-responsiveness, potentiometric titration curves of PEGMA−PDPAs were determined by monitoring pH from 3.0 to 11.0 in increments of 0.1 N NaOH using a pH meter (Mettler Toledo, Columbus, OH, USA). Preparation of (pH)Micelle, Ce6/(pH)Micelle, PEGMA− PDPA Micelle, and mPEG−PCL Micelle. Unloaded pHresponsive micelles ((pH)micelles) and Ce6-loaded pHresponsive micelles (Ce6/(pH)micelles) were prepared using a solvent evaporation method.31 Briefly, PEGMA−PDPA (5 mg), mPEG−PCL (5 mg), and Ce6 (0, 2, 1, 0.5 mg) were dissolved in THF (0.25 mL). THF containing polymers and Ce6 was then dropped into PBS (2.5 mL), and then the solutions were stirred overnight to evaporate THF. PEGMA−PDPA, mPEG− PCL, and Ce6-loaded mPEG−PCL micelles were prepared using the solvent evaporation method and were compared with (pH) micelle and Ce6/(pH)micelle. All micelles were filtered through 0.22 μm filters before use in experiments. Preparation of Mal-(pH)Micelle, Ce6/Mal-(pH)Micelle, and Ce6/GE11-(pH)Micelle. Unloaded pH-responsive micelles with maleimide modification (Mal-(pH)micelle) and Ce6-loaded Ce6/Mal-(pH)micelles were prepared using Mal−PEG−PCL (1 mg), PEGMA−PDPA (5 mg), and mPEG−PCL (5 mg) at a weight ratio of 1/5/5. EGFR-targeting Ce6/GE11-(pH)micelles were prepared by reacting Ce6/Mal-(pH)micelles with the EGFR specific peptides GE11 (YHWYGYTPQNVI-GGGGC) or GE11-FITC (FITC-YHWYGYTPQNVI-GGGGC) via thiolmaleimide conjugation.32 GE11 or GE11-FITC were conjugated to Ce6/Mal-(pH)micelles by mixing with Ce6/ Mal-(pH)micelles at a GE11/maleimide molar ratio of 5/1 at 4 °C for 24 h. Subsequently, unconjugated GE11 or GE11−FITC peptides were removed and collected using PD-10 desalting columns (GE Healthcare, Uppsala, Sweden). GE11 or GE11− FITC contents of Ce6/Mal-(pH)micelles were then analyzed using Pierce MicroBCA Protein Assay kits (Thermal, USA), and average numbers of GE11 peptides per micelle were calculated by dividing the number of GE11 peptides in each fraction by the calculated average number of micelles.33 Mal-(pH)micelle, Ce6/Mal-(pH)micelle, Ce6/GE11-(pH)micelle, and FITC-labeled Ce6/GE11-(pH)micelles were filtered through 0.22 μm filters before use in experiments. Characteristics of Micelles. Sizes and polydispersity indexes (PdI) were determined using dynamic light scattering (DLS) with a Zetasizer Nano ZS90 apparatus (Malvern Instruments, Worcestershire, UK). Morphological features were observed using transmission electron microscopy (TEM; Hitachi H-7650, Tokyo, Japan). Drug encapsulation efficiencies (EE) and drug contents (DC) were calculated according to the concentration of Ce6, which was determined according to fluorescence (FL) at excitation and emission wavelengths of 403 and 670 nm, respectively, using a FL spectrophotometer (Varian, Palo Alto, CA, USA). Critical micelle concentrations (CMC) were determined using pyrene as a fluorescent probe.34 In addition, thermal properties of (pH)micelles and equivalent mixtures of PEGMA−PDPA (5 mg) and mPEG−PCL micelles (5 mg) were evaluated using differential scanning calorimetry (DSC, LT-Modulate DSC 2920, TA Instrument, USA) at −20−100 °C at a heating rate of 5 °C/min. pH-Responsiveness of (pH)Micelle and Ce6/(pH)Micelle. To evaluate pH-responsiveness of (pH)micelle, sizes and PdI values in PBS were determined at pH 9.5, 7.4, 6, and 4.5, and

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EXPERIMENTAL SECTION Materials. 2-(Diisopropylamino)ethyl methacrylate) (DPA) was purchased from Scientific Polymer Products (Ontario, NY, USA). Azobis(isobutyronitrile) (AIBN), poly(ethylene glycol) methacrylate (PEGMA; MW, 360), ε-caprolactone, and other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA). Chlorin e6 was kindly provided by BiolitecAG (Jena, Germany). GE11 (YHWYGYTPQNVI-GGGGC) and GE11FITC (FITC-YHWYGYTPQNVI-GGGGC) peptides were purchased from OmicsBio, Ltd. (Taiwan) Syntheses of PEGMA−PDPA, mPEG−PCL, and Mal− PEG−PCL. Methods for synthesis of the pH-responsive copolymer PEGMA−PDPA, the biodegradable copolymer mPEG−PCL, and the maleimide-terminated poly(ethylene glycol)-poly(ε-caprolactone) Mal−PEG−PCL are illustrated in Figure 1. PEGMA−PDPA was synthesized via free radical polymerization.30 PEGMA−PDPA copolymers were synthesized at PEGMA/DPA ratios (w/w) of 1/2 and 1/4. In these preparations, PEGMA (0.25 g) was reacted with DPA (0.5 g; 1 g) at 70 °C for 24 h in the presence of an AIBN initiator (9.25 mg) and was then dialyzed against ddwater for 3 days to remove monomers and purify PEGMA−PDPA. Mal−PEG− PCL and mPEG−PCL copolymers were prepared using ringopening polymerization.23 PEGMA−PDPA, mPEG−PCL, and Mal−PEG−PCL were then characterized using 1H NMR with a C


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imaging system, an IVIS imaging system (Xenogen, Alameda, CA, USA), and FL microscopy. In these experiments, female BALB/c athymic (nut/nut) mice of 5−6 weeks of age were purchased from the National Laboratory Animal Center (Taipei, Taiwan). The right flanks and left flanks of mice were inoculated subcutaneously with 5 × 106 HCT-116 and SW620 cells, respectively. After xenograft tumor volumes reached 500−600 mm3, mice received intravenous injections of Ce6/(pH)micelle or Ce6/GE11-(pH)micelle at a Ce6 concentration of 5 mg/kg, and at 3 or 24 h postintravenous injection, mice were monitored using an IVIS imaging system to evaluate in vivo biodistributions of Ce6/ (pH) micelle and Ce6/ GE11-(pH)micelle. The IVIS image system was used with an exposure time of 1 min at excitation and emission wavelengths of 640 and 680 nm, respectively. Heart, liver, spleen, lung, kidney, HCT-116-tumor, and SW620-tumor tissues were excised at 24 h postintravenous injection and were monitored using an IVIS image system. Tissue sections of organs and tumors were observed under FL microscopy to evaluate ex vivo distributions of Ce6/(pH)micelle and Ce6/GE11-(pH)micelle. Vasculature in tumor sections was stained using a Blood Vessel Staining Kit (Chemicon) before FL microscopy observations. Quantitative analyses of FL intensities in FL images were performed using Living Image software (Caliper Life Sciences Inc., Hopkinton, MA, USA). In Vivo PDT Anticancer Efficacy. In vivo PDT antitumor efficacy was evaluated in mice bearing HCT-116 xenograft tumors by measuring changes in tumor volumes. Mice were implanted subcutaneously with 5 × 106 HCT-116 cells. When tumor volumes reached 150−200 mm3 (day 0), mice were intravenously injected with PBS (control), Ce6/(pH)micelle, or Ce6/GE11-(pH)micelle at a Ce6 concentration of 5 mg/kg. At 24 h postinjection, tumors were treated with a 670 nm-diode laser light (634 mW/cm2) for 10 min. Body weights and tumor volumes of mice were then measured from day 0 to 22. Tumor volumes were calculated as follows: Vtumor = 1/2 × length × width2. In vivo PDT efficacy was evaluated using histological and immunohistochemical analyses. In these experiments, HCT116-tumor-bearing mice were injected with Ce6/(pH)micelle or Ce6/GE11-(pH)micelle at a Ce6 concentration of 5 mg/kg and were then sacrificed at 24 h post-light irradiation (48 h postinjection) for harvest of tumor tissues. For histological analysis, tissues were fixed in 3% formaldehyde, were paraffinembedded, and were stained with hematoxylin and eosin (H&E).35 For immunohistochemical analyses, tissues were embedded in optimal cutting temperature compound (Tissue Tek, Sakura, Torrance, CA), were frozen immediately, and were then sectioned and stained with DAPI and NADPHdiaphorase, or proliferating cell nuclear antigen (PCNA).36 Sections were observed using FL microscopy. Statistical Analysis. Differences were identified using Student’s t tests and were considered significant when P < 0.05.

CMC values were determined at pH 7.4 and 6.6. In addition, pH-responsiveness of Ce6/(pH)micelle was evaluated according to absorbance and FL spectra in Tris buffer solutions at pH 5, 6, 7, and 8 at a concentration of 2 μg/mL, and was compared with the pH-responsiveness of Ce6-loaded mPEG−PCL micelle and free Ce6. In Vitro Singlet Oxygen-Generating Capacities of Ce6/ GE11-(pH)Micelle. Singlet oxygen generation was evaluated using Singlet Oxygen Sensor Green reagent (Molecular Probes. Eugene, OR, USA). 30 In these experiments, Ce6/ GE11-(pH)micelles (2 μg/mL) and singlet oxygen sensor green (1 mM) were dispersed in Tris buffer solution at pH 7.4 and 6.6. Solutions were then exposed to 14.6 mW/cm2 light at 660 nm for 0, 60, 120, 150, 180, 210, and 240 s. After light exposure, FL intensities of sensor green in solution were measured using a FL spectrophotometer (excitation, 488 nm; emission, 524). Cell Culture. HCT-116 and SW620 CRC cells were cultured in McCoy’s 5A modified medium (Sigma-Aldrich) and 4-mM L-glutamine-supplemented Dulbecco’s modified Eagle’s medium (Gibco BRL, Grand Island, NJ, USA), respectively. Both cell types were supplemented with 10% (v/ v) heat-activated fetal bovine serum (Gibco BRL) and 1% (v/v) penicillin−streptomycin−amphotericin B antibiotic−antimytotic solutions (Sigma-Aldrich). Determinations of Cellular Uptake and Intracellular Localization. Cellular uptake and intracellular localization in HCT-116 and SW620 cells were evaluated using FL spectrophotometry and FL microscopy. For cellular uptake assays, HCT-116 and SW620 cells (105 cells/3.5 cm dish) were incubated with free Ce6, Ce6/ ( p H ) micelle, Ce6/ GE11-(pH)micelle, or Ce6/GE11-(pH)micelle with four-fold excesses of free GE11 peptide (four times more than GE11 on Ce6/GE11-(pH)micelles) at a Ce6 concentration of 2 μg/mL for 0.17, 0.5, 1, and 5 h. Cells were then lysed in 0.1 mL aliquots of lysis buffer, and Ce6 uptake was determined using a FL spectrophotometer at excitation and emission wavelengths of 403 and 670 nm, respectively. Data were normalized to protein contents, which were determined using bicinchoninic acid (BCA) protein assay kits. To determine intracellular localization, cells were incubated with Ce6/(pH)micelle or Ce6/ GE11-(pH)micelle at a Ce6 concentration of 2 μg/mL for 5 h and were then washed with PBS. Nuclei were then stained with 4′,6-diamidino-2-phenylindole (DAPI), and cells were observed using FL microscopy. In Vitro Cytotoxicity and Photocytotoxicity. Cytotoxicities and photocytotoxicities of free Ce6, Ce6/(pH)micelle, and Ce6/GE11-(pH)micelle in HCT-116 cells were evaluated using a laser light at a wavelength of 660 nm (14.6 mW/cm2). Briefly, HCT-116 cells were incubated in 96-well plates at a density of 104 cell/well for 24 h. Cells were then incubated with free Ce6, Ce6/(pH)micelles, or Ce6/GE11-(pH)micelles at Ce6 concentrations of 0, 0.5, 1, 2, 4, 8, and 16 μg/mL for 24 h, and were then washed with PBS three times prior to determining cell viability using MTT assays.24 To determine photocytotoxicity, cells were incubated with Ce6/(pH) micelles and Ce6/ GE11-(pH)micelles at Ce6 concentrations of 0, 0.5, 1, or 2 μg/mL for 1, 5, and 24 h. Cells were then washed with PBS and exposed to laser light for 5, 15, and 25 s, and cell viability was finally determined using MTT assays. In Vivo and ex Vivo Distribution of Micelles. In vivo and ex vivo biodistribution experiments were performed in mice bearing HCT-116 and SW620 xenograft tumors using a FL

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RESULTS AND DISCUSSION Synthesis of PEGMA−PDPA, mPEG−PCL, and Mal− PEG−PCL. Chemical structures of PEGMA−PDPA (1/2), mPEG−PCL, and Mal−PEG−PCL were examined using 1H NMR. As shown in Figure S1, 1H NMR spectra of PEGMA− PDPA (1/2) revealed resonances of PEGMA (δHa = 3.629 ppm) and PDPA (δHe = 0.989 ppm, δHc = 2.611 ppm, δHd = 2.971 ppm, and δHb = 3.811 ppm). In addition, 1H NMR spectra of mPEG−PCL (Figure S2) showed resonances of PCL D


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Molecular Pharmaceutics (δHc = 2.286 ppm, δHd = 1.65 ppm, δHe = 1.368 ppm, δHf = 4.033 ppm) and PEG (δHa = 3.375 ppm, δHb = 3.637 ppm).37 1 H NMR spectra of Mal−PEG−PCL (Figure S3) showed resonances of PCL and PEG with those of the maleimide group at 6.67 ppm. Furthermore, GPC and 1H NMR analyses showed that the MW of PEGMA−PDPA (1/2), mPEG−PCL, and Mal−PEG−PCL were 10900, 15400, and 14300 g/mol, respectively, and PD were 1.58, 1.31, and 1.42, respectively (Table S1). These data indicate that PEGMA−PDPA (1/2), mPEG−PCL, and Mal−PEG−PCL were successfully synthesized. Moreover, potentiometric acid−base titration curves of PEGMA−PDPA (1/2 and 1/4) showed that PEGMA−PDPA (1/2) and PEGMA−PDPA (1/4) have sharp protonation transitions at pH 6.89 and 6.81(Figure S4), indicating respective pKa values of the designed pH-responsive polymers. These data suggested that the designed PEGM−PDPAs and the micelles self-assembled from PEGMA−PDPAs are pHresponsive at pH lower than 6.8. PEGMA−PDPA (1/2) was used to self-assemble the designed pH-responsive micelle, (pH) micelle, in the following experiments. Characteristics of Designed Micelles. The designed micelles, including (pH) micelle, Ce6/ (pH) micelle, Ce6/ GE11-(pH)micelle, Mal-(pH)micelle, and Ce6/Mal-(pH)micelle, were characterized using TEM and DLS. DLS data showed that the sizes of all designed micelles ranged from 91.1 to 105.7 nm (Table 1), indicating that all designed micelles were successfully

morphology indicated that these micelles are almost uniform and spherical (Figure 2), as shown in DLS analyzes. In further experiments, EE of the designed Ce6-loaded micelles were examined. As shown in Table 1, EE of Ce6/(pH)micelles at D/P of 1/5, 1/10, and 1/20 were at least 74%, indicating successful entrapment of Ce6. The Ce6/(pH)micelle had the highest EE at a D/P of 1/10 and was therefore considered optimal. Hence, Ce6/(pH)micelle at a D/P of 1/10 was further added to Mal−PEG−PCL to prepare Ce6/Mal-(pH)micelle. Moreover, Ce6/Mal-(pH)micelle had a high EE (85.9%), indicating good encapsulation ability (Table 1). However, after conjugation of Ce6/Mal-(pH)micelle with the EGFR-targeting peptide GE11 (Ce6/GE11-(pH)micelle), EE decreased slightly to 78.8%, reflecting slight drug loss with the removal of unconjugated GE11. Moreover, average numbers of GE11 conjugated per 105 nm diameter micelles were between 4.81 and 7.08 × 105 on Ce6/GE11-(pH)micelles, indicating successful conjugation of GE11 on Ce6/GE11-(pH)micelles. Subsequently, absorbance spectra of free Ce6, Ce6/(pH)micelle, and Ce6/GE11-(pH)micelle were measured and strong absorbance was observed at a wavelength of 660 nm in both designed Ce6-loaded micelles (Figure 3), indicating

Table 1. Characteristics of Designed Micelles micelle

D/Pa

EE (%)b

DC (%)c

1/5 1/10 1/20

75.52 86.25 74.35

12.09 7.82 3.54

1/10 1/10

85.9 78.8

7.81 7.16

(pH)

micelle Ce6/(pH)micelle Ce6/(pH)micelle Ce6/(pH)micelle Mal-(pH)micelle Ce6/Mal-(pH)micelle Ce6/GE11-(pH)micelle

size/nm (PdI)d 91.1 96.6 96.7 103.1 105.7 110.0 105.1

(0.239) (0.109) (0.125) (0.154) (0.222) (0.184) (0.293)

a

D/P ratio = weight of Ce6/weight of polymer. bCe6 encapsulation efficiency (%) = (weight of Ce6 loaded in the micelles/weight of added Ce6) × 100%. cCe6 drug content (%) = (weight of Ce6 loaded in micelles)/(weight of Ce6-loaded micelles) × 100%. dSizes and polydispersity indexes (PdI) were determined using dynamic light scattering (DLS).

Figure 3. Absorption spectra of free Ce6, Ce6/(pH)micelle, and Ce6/ GE11-(pH)micelle.

that after being loaded into the present micelles, Ce6 efficiently absorbs NIR-light for PDT. These data are in strict agreement with previously published studies associated with designing

prepared in the nanoscale, presumably allowing the micelles to accumulate in tumors as a result of the EPR effect. TEM images of (pH)micelle, Ce6/(pH)micelle, and Ce6/GE11-(pH)micelle

Figure 2. Transmission electron micrograph (TEM) images of (a)

(pH)

micelle, (b) Ce6/(pH)micelle, (c) Ce6/GE11-(pH)micelle; scale bar, 0.2 μm. E


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Figure 4. (A) CMC of (pH)micelle at pH 6.5 and 7.4 was determined using pyrene. (B) DLS sizes and PdI values of (pH)micelle at pH 3, 4.5, 6, 7.4, and 9.5. (C) FL spectra of Ce6/(pH)micelle and free Ce6 (equivalent to 2 μg/mL of Ce6) at pH 5, 6, 7, and 8 were measured at an excitation wavelength of 403 nm. (D) Singlet oxygen generation of Ce6/GE11-(pH)micelle at pH 6.4 and 7.4.

hydrophobic photosensitizer-encapsulated nanocarriers.30,38 However, free Ce6 rarely absorbed in the NIR region, reflecting low-solubility and aggregation in water. CMC were measured using pyrene, and PEGMA−PDPA micelle, mPEG−PCL micelle, (pH)micelle, and Mal-(pH)micelle had CMC values of 0.94, 0.28, 0.3, and 0.47 mg/L, respectively (Figure S5). Lower CMC indicates that micelles remain stable at low copolymer concentrations and resist dissociation in blood.39 Hence, the stability of (pH)micelle in blood is considered better than that of PEGMA−PDPA micelles. In addition, miscibility of (pH)micelle was examined using DSC. These analyses show that (pH)micelle of mPEG−PCL and PEGMA−PDPA had melting points of only 54.55 °C, whereas mixed mPEG−PCL and PEGMA−PDPA micelles had melting points at 50.92 and 53.76 °C (Figure S6). These data demonstrate that (pH)micelle is a single-component micelle with good miscibility.40 pH-Responsiveness and Singlet Oxygen-Generating Abilities of (pH)Micelle and Ce6/(pH)Micelle. CMC and DLS analyses indicated good pH-responsiveness of (pH)micelles. As shown in Figure 4A, CMC of (pH)micelles increased from 0.3 to

3.2 mg/L with pH decreasing from 7.4 to 6.5. Moreover, higher CMC values indicated worse micelle stability. DLS data showed significant positive trends between sizes and PdI values with pH decreases from 7.4 to 3 (Figure 4B), suggesting unstable aggregation of (pH)micelle at pH lower than 7.4. These observations likely reflect partial protonation of amine groups in PEGMA−PDPA of (pH)micelles, leading to greater internal hydrophilicity of (pH)micelles.41 FL spectra of Ce6/(pH)micelles were compared with those of Ce6-loaded mPEG−PCL micelles. As shown in Figure 4C, the FL intensity of Ce6/(pH)micelle decreased with decreasing pH values, reflecting poor dispersion in unstable Ce6/(pH)micelles. In contrast, FL intensities of Ce6-loaded mPEG−PCL micelles did not change significantly with decreases in pH (Figure S7), indicating relative stability of Ce6-loaded mPEG−PCL micelles and little pH responsiveness. Free Ce6 had relatively weak change in FL intensity at pH 5−8 due to low-solubility in aqueous buffers. In contrast, Ce6/(pH)micelles had strong FL and were pH-responsive. In ROS assays, Ce6/(pH)micelle exhibited high singlet oxygen-generating capacity. As shown in Figure 4D, the FL F


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Figure 5. Cellular uptake; Ce6 uptake by (A) HCT-116 and (B) SW620 cells incubated with free Ce6, Ce6/(pH)micelle, and Ce6/GE11-(pH)micelle for 0.16, 0.5, 1, and 5 h; EGFR competition was examined in HCT-116 cells incubated with Ce6/GE11-(pH)micelle in the presence of free GE11 peptides. FL images of (C) HCT-116 and (D) SW620 cells incubated with Ce6/(pH)micelle and Ce6/GE11-(pH)micelle for 1 and 5 h; scale bar, 10 μm; *P < 0.05. Intracellular localization (E); FL microscopy images of HCT-116 cells after 5 h treatments with FITC-labeled Ce6/GE11-(pH)micelle or FITC-labeled Ce6/(pH)micelle; nuclei were stained with DAPI. Scale bar, 20 μm. G


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Figure 6. Photocytotoxicity; (A) cell viability of HCT-116 cells treated with Ce6/GE11-(pH)micelle (equivalent to 0−2 μg/mL of Ce6) for 24 h after 5, 15, and 25 s exposures to NIR-light irradiation at 14.6 mW/cm2. Cell viability of HCT-116 cells treated with Ce6/(pH)micelle or Ce6/ GE11-(pH)micelle for 1 (B), 5 (C), or 24 h (D) with 25 s exposures to NIR-light irradiation at 0.365 J/cm2; *P < 0.05.

intensity of Ce6/(pH)micelle increased with light irradiation times at all tested pH values, 7.4 and 6.4. These data indicate that although Ce6/(pH)micelles become unstable in tumor microenvironments, they might efficiently generate singlet oxygen and limit proliferation of tumor cells during PDT. Cellular Uptake and Localization. Cellular uptake rates of free Ce6, Ce6/(pH)micelle, Ce6/GE11-(pH)micelle, and Ce6/ GE11-(pH)micelle in combination with free GE11 peptides were determined according to Ce6 FL in HCT-116 CRC cells with high levels of EGFR expression, and in SW620 CRC cells with low levels of EGFR expression. As shown in Figures 5A,B, Ce6 uptake increased with time in HCT-116 and SW620 cells. However, after 1- and 5-h incubation, Ce6 uptake from Ce6/ GE11-(pH)micelle was 1.7 times higher than that from Ce6/(pH)micelle in HCT-116 cells (Figure 5C). In addition, Ce6/GE11-(pH)micelle produced significantly higher FL intensities than Ce6/(pH)micelle, suggesting that with active EGFR-targeting, Ce6/GE11-(pH)micelles are rapidly internalized via receptor-mediated endocytosis due to targeted ligand coating of nanomedicines for the cancer biomarkers EGFR. In

contrast, uptake of Ce6/(pH)micelle may occur via membrane based processes such as fusion and pinocytosis, which are known to be comparatively time-dependent and delayed processes.42,43 No significant differences in Ce6 uptake were observed between Ce6/(pH)micelle and Ce6/GE11-(pH)micelle in SW620 cells at all incubation times (Figure 5B), as indicated by the FL images shown in Figure 5D. These observations indicate that GE11 peptides do not target CRC cells that do not overexpress EGFR, and uptake of Ce6/GE11-(pH)micelle is specific to CRC cells overexpressing EGFR. Furthermore, specific uptake was verified in EGFR competition experiments in HCT-116 cells treated with Ce6/GE11-(pH)micelles in the presence of free GE11 peptides. Specifically, the presence of free GE11 peptides significantly decreased Ce6 uptake from Ce6/ GE11-(pH)micelles after 5 h incubation (Figure 5A), reflecting competitive binding of GE11 peptides to EGFR. These results confirm that Ce6/GE11-(pH)micelles specifically bind EGFR on CRC cells, resulting in enhanced Ce6 uptake. H


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Figure 7. In vivo and ex vivo FL imaging of HCT-116 (right) and SW620 (left) tumor-bearing mice following administration of Ce6/ GE11-(pH)micelle and Ce6/(pH)micelle; (A) in vivo FL images of tumor-bearing mice treated with Ce6/GE11-(pH)micelle and Ce6/(pH)micelle. (B) Ex vivo FL images of organs and tumors at 24 h postinjection of Ce6/GE11-(pH)micelle; total fluorescent photon counts of Ce6 in HCT-116-tumor (C) and SW620-tumor (D) were quantified using FL images (Figure 8A); *P < 0.05. FL microscopy images (E) of HCT-116 and SW620 tumors at 3 and 24 h postinjection of Ce6/GE11-(pH)micelle; scale bar, 50 μm. I


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Figure 8. In vivo PDT efficacy of PBS (control), Ce6/(pH)micelle, and Ce6/GE11-(pH)micelle in HCT-116 tumor-bearing mice; (A) tumor volumes of mice treated with PDT using PBS (control), Ce6/(pH)micelle, or Ce6/GE11-(pH)micelle. (B) Representative photos of HCT-116 tumor-bearing mice treated with PDT using PBS, Ce6/GE11-(pH)micelle, and Ce6/(pH)micelle (5 mg/kg of Ce6). Laser irradiation energy in PDT was 400 J/cm2. The arrows indicate laser irradiation sites.

In Vivo and ex Vivo Biodistribution. In vivo biodistribution studies of Ce6/GE11-(pH)micelle and Ce6/(pH)micelle were performed in mice with HCT-116 xenograft tumors (high EGFR expression) on the right flanks and SW620 xenograft tumors (low EGFR expression) on the left flanks. In these experiments, Ce6 was monitored using noninvasive and realtime FL imaging.45 The in vivo FL images shown in Figure 7A show that Ce6/GE11-(pH)micelle accumulates more effectively in HCT-116-tumors than in SW620-tumors. However, Ce6/ GE11-(pH)micelle also accumulated more efficiently than Ce6/(pH)micelle in HCT-116-tumors. Moreover, FL image analyses indicated that, at 3 and 24 h postinjection, FL intensities of Ce6/GE11-(pH)micelle in HCT-116-tumors were significantly higher than those of Ce6/(pH)micelle (Figure 7B). In contrast, FL intensities of these micelles did not differ in SW620-tumors (Figure 7C), indicating high EGFR-targeting specificity of Ce6/GE11-(pH)micelle. In further investigations, ex vivo biodistribution of Ce6/ GE11-(pH)micelle was examined using FL imaging and FL microscopy after harvesting tumors and organs from mice at 3 and 24 h postinjection. These ex vivo FL images are in clear agreement with in vivo FL analyses, with preferential accumulation of Ce6/GE11-(pH)micelle in HCT-116-tumors relative to that in other organs and SW620-tumors at 24 h postinjection (Figure 7D). Furthermore, significantly higher uniform FL signals of Ce6 from Ce6/GE11-(pH)micelle were observed in sections of HCT-116-tumor at 3 and 24 h postinjection (Figure 7E). These findings indicate EGFR selectivity of Ce6/GE11-(pH)micelle and reflect differing microenvironments of these two CRC xenograft tumors.46 Moreover, in HCT-116-tumor sections, Ce6/GE11-(pH)micelle resulted in a higher FL intensity than Ce6/(pH)micelle at 3 and 24 h postinjection (Figure S10). In conclusion, Ce6/ GE11-(pH)micelle delivers high volumes of Ce6 to HCT-116tumors and might maximize the effectiveness of PDT. In Vivo PDT Efficacy. In vivo PDT efficacies of PBS (control), Ce6/GE11-(pH)micelle, and Ce6/(pH)micelle were examined in mice bearing HCT-116 xenograft tumors by monitoring tumor volumes and body weights over 22 days. PDT was conducted with a NIR laser light (670 nm, 634 mW/

In further studies, intracellular localization of Ce6/ GE11-(pH)micelle and Ce6/(pH)micelle was determined in HCT-116 cells using FITC-labeled Ce6/GE11-(pH)micelle and FITC-labeled Ce6/(pH)micelle. In these experiments, FL intensities of both FITC (green) and Ce6 (red) from FITClabeled Ce6/GE11-(pH)micelle were higher than those from FITC-labeled Ce6/(pH)micelle (Figure 5E), indicating that FITC-labeled Ce6/GE11-(pH)micelles are more rapidly internalized by HCT-116 cells. Furthermore, merged FL images showed that the localization of FITC-labeled GE11-(pH)micelle in the cytoplasm of HCT-116 cells differed from that of Ce6, indicating that Ce6 was successfully released from Ce6/ GE11-(pH)micelles and confirming the pH sensitivity of (pH)micelles. Cytotoxicity and Photocytotoxicity. Cytotoxicity and photocytotoxicity of free Ce6, Ce6/GE11-(pH)micelle, and Ce6/(pH)micelle were examined using MTT assays. No significant toxicity of (pH)micelle was observed in HCT-116 or SW620 cells in the presence or absence of 0.365 J/cm2 dose of light (Figure S8), indicating good biocompatibility of the (pH) micelle. In addition, free Ce6, Ce6/(pH)micelle, and Ce6/ GE11-(pH)micelle showed negligible cytotoxicity in HCT-116 and SW620 cells without light dosing (Figure S9). In contrast, Ce6/GE11-(pH)micelle was highly antiproliferative against HCT-116 cells under NIR-light irradiation, and significant decreases in cell viability were observed with increased light doses and Ce6 concentrations (Figure 6A). These results show that these nanophotosensitizers are only cytotoxic in the presence of light at the appropriate wavelength for the generation of singlet oxygen.44 In photocytotoxicity experiments, PDT responses to Ce6/(pH)micelle and Ce6/GE11-(pH)micelle differed significantly after 1 and 5 h treatments (Figure 6B,C), likely reflecting increased receptor-mediated endocytosis of Ce6/ GE11-(pH)micelle by HCT-116 cells with high EGFR expression levels. However, no significant differences in cell viability were observed between Ce6/(pH)micelle and Ce6/GE11-(pH)micelle after incubation for 24 h due to time-dependent passive uptake (Figure 6D). J


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Figure 9. Histological and immunohistochemical analyses; tumor sections were analyzed using hematoxylin and eosin (H&E), NADPH-diaphorase (A), and PCNA (B) staining after treatments of mice with PDT using Ce6/GE11-(pH)micelle, or Ce6/(pH)micelle. (C) Numbers of PCNA-positive cells per field at 200× magnification were quantified as in panel (B). Data are presented as means ± standard deviations (SD) in 10 distinct regions from three tumors per group; *P < 0.05.

cm2) for 10 min at 24 h postinjection. These experiments (Figure 8A) indicated that Ce6/GE11-(pH)micelle had the best PDT efficacy as no significant changes in tumor volumes between day 0 and day 22 were observed following treatments with Ce6/GE11-(pH)micelle. However, significant increases in tumor volumes were not observed in mice treated with Ce6/(pH)micelle until day 13, but tumor rapidly grew thereafter, to about 850 mm3 on day 22. Tumor volumes differed significantly between PBS and Ce6/GE11-(pH)micelle treated mice on day 22. Furthermore, the images presented in Figure 8B show clear hemorrhagic injury at tumor sites on Ce6/ GE11-(pH)micelle-treated mice on day 5, suggesting outstanding therapeutic PDT efficacy of Ce6/GE11-(pH)micelle. Although tumor injury was observed in mice treated with Ce6/(pH)micelle on day 5, healing of necrotic scar tissue was observed, and tumor-surrounding scar tissues had recurred on day 13, leading to subsequent rapid regrowth of tumors. Body weights did not change or differ between mice treated with PBS (control), Ce6/(pH)micelle, or Ce6/GE11-(pH)micelle (Figure S11),

indicating acceptable in vivo toxicity of the designed nanophotosensitizers. Finally, histological and immunohistochemical analyses of subcutaneous tumors included H&E, NADPH-diaphorase, and PCNA staining. As shown in Figure 9A, H&E stained sections showed varying levels of damage between tumors treated with Ce6/(pH)micelle and Ce6/GE11-(pH)micelle, as indicated by coagulation, vacuolation, and loss of nuclear staining. Similarly, NADPH-diaphorase staining showed loss of NADPH-diaphorase activities in the interior of tumors treated with Ce6/ GE11-(pH)micelle, indicating prominent necrosis. However, only partial necrosis and vacuolation were observed in tumors treated with Ce6/(pH)micelle. Subsequently, cell proliferation in non-necrotic regions of tumors was analyzed using PCNA staining. In these experiments, high cell proliferation (PCNApositive cells, 88% ± 6%) was observed in control groups, whereas relatively low proliferation was observed in Ce6/ GE11-(pH)micelle (PCNA-positive cells, 22% ± 4%) and Ce6/(pH)micelle (PCNA-positive cells, 40 ± 5%) treated tumors (Figure 9B,C). These results indicate that Ce6/ K


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Molecular Pharmaceutics GE11-(pH)micelle produces significant antitumor effects during PDT and has excellent PDT efficacy. Ce6/GE11-(pH)micelle coated with EGFR-targeting probes could be a powerful nanophotosensitizer and may lead to excellent PDT efficacy in CRC treatment.

acid; H&E, hematoxylin and eosin; PCNA, proliferating cell nuclear antigen

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(1) Chen, K.-C.; Hsieh, Y.-S.; Tseng, Y.-F.; Shieh, M.-J.; Chen, J.-S.; Lai, H.-S.; Lee, J.-M. Pleural Photodynamic Therapy and Surgery in Lung Cancer and Thymoma Patients with Pleural Spread. PLoS One 2015, 10 (7), e0133230. (2) Schmidt-Erfurth, U.; Hasan, T. Mechanisms of action of photodynamic therapy with verteporfin for the treatment of agerelated macular degeneration. Surv. Ophthalmol. 2000, 45 (3), 195− 214. (3) Li, S.; Chang, K.; Sun, K.; Tang, Y.; Cui, N.; Wang, Y.; Qin, W.; Xu, H.; Wu, C. Amplified Singlet Oxygen Generation in Semiconductor Polymer Dots for Photodynamic Cancer Therapy. ACS Appl. Mater. Interfaces 2016, 8 (6), 3624−3634. (4) Chin, W.-W.-L.; Heng, P.-W.-S.; Thong, P.-S.-P.; Bhuvaneswari, R.; Hirt, W.; Kuenzel, S.; Soo, K.-C.; Olivo, M. Improved formulation of photosensitizer chlorin e6 polyvinylpyrrolidone for fluorescence diagnostic imaging and photodynamic therapy of human cancer. Eur. J. Pharm. Biopharm. 2008, 69 (3), 1083−1093. (5) Wang, X.; Liu, K.; Yang, G.; Cheng, L.; He, L.; Liu, Y.; Li, Y.; Guo, L.; Liu, Z. Near-infrared light triggered photodynamic therapy in combination with gene therapy using upconversion nanoparticles for effective cancer cell killing. Nanoscale 2014, 6 (15), 9198−205. (6) Luk, B.-T.; Zhang, L. Current Advances in Polymer-Based Nanotheranostics for Cancer Treatment and Diagnosis. ACS Appl. Mater. Interfaces 2014, 6 (24), 21859−21873. (7) Wang, X.; Liu, K.; Yang, G.; Cheng, L.; He, L.; Liu, Y.; Li, Y.; Guo, L.; Liu, Z. Near-infrared light triggered photodynamic therapy in combination with gene therapy using upconversion nanoparticles for effective cancer cell killing. Nanoscale 2014, 6 (15), 9198−205. (8) Kostenich, G.-A.; Zhuravkin, I.-N.; Zhavrid, E.-A. Experimental grounds for using chlorin e6 in the photodynamic therapy of malignant tumors. J. Photochem. Photobiol., B 1994, 22 (3), 211−217. (9) Huang, L.; St. Denis, T.-G.; Xuan, Y.; Huang, Y.-Y.; Tanaka, M.; Zadlo, A.; Sarna, T.; Hamblin, M.-R. Paradoxical potentiation of methylene blue-mediated antimicrobial photodynamic inactivation by sodium azide: Role of ambient oxygen and azide radicals. Free Radical Biol. Med. 2012, 53 (11), 2062−2071. (10) Liu, Y.; Ma, K.; Jiao, T.; Xing, R.; Shen, G.; Yan, X. WaterInsoluble Photosensitizer Nanocolloids Stabilized by Supramolecular Interfacial Assembly towards Photodynamic Therapy. Sci. Rep. 2017, 7, 42978. (11) Greish, K. Enhanced Permeability and Retention (EPR) Effect for Anticancer Nanomedicine Drug Targeting. Methods Mol. Biol. 2010, 624, 25−37. (12) Sabra, S.; Abdelmoneem, M.; Abdelwakil, M.; Taha Mabrouk, M.; Anwar, D.; Mohamed, R.; Khattab, S.; Bekhit, A.; Elkhodairy, K.; Freag, M.; Elzoghby, A. Self-assembled nanocarriers based on amphiphilic natural polymers for anti-cancer drug delivery applications. Curr. Pharm. Des. 2017, 23, 5213. (13) Arteaga, C.-L. Epidermal Growth Factor Receptor Dependence in Human Tumors: More Than Just Expression? Oncologist 2002, 7 (Suppl 4), 31−39. (14) Scaltriti, M.; Baselga, J. The epidermal growth factor receptor pathway: a model for targeted therapy. Clin. Cancer Res. 2006, 12 (18), 5268−5272. (15) Sheng, Q.; Liu, J. The therapeutic potential of targeting the EGFR family in epithelial ovarian cancer. Br. J. Cancer 2011, 104 (8), 1241−1245. (16) Wang, Z.; Qiao, R.; Tang, N.; Lu, Z.; Wang, H.; Zhang, Z.; Xue, X.; Huang, Z.; Zhang, S.; Zhang, G.; Li, Y. Active targeting theranostic iron oxide nanoparticles for MRI and magnetic resonance-guided focused ultrasound ablation of lung cancer. Biomaterials 2017, 127, 25−35. (17) Levi, O.; Shapira, A.; Tal, B.; Benhar, I.; Eliaz, N. Isolating epidermal growth factor receptor overexpressing carcinoma cells from

CONCLUSIONS We synthesized and characterized the multifunctional nanophotosensitizer Ce6/GE11-(pH)micelle for PDT in EGFRoverexpressing CRC. The resulting nanophotosensitizer comprised a pH-responsive micelle of PEGMA−PDPA, mPEG−PCL, and Mal−PEG−PCL, which entrapped the photosensitizer Ce6 and was coated with the EGFR-targeting peptide GE11. Subsequent in vitro and in vivo experiments confirmed that Ce6/GE11-(pH)micelle specifically targets and accumulates in EGFR-overexpressing cancer cells/tumors, reflecting passive and active targeting functions. Furthermore, Ce6/GE11-(pH)micelle allowed detection of EGFR-overexpressing tumors using FL imaging, and the ensuing analyses showed significant suppression of tumor growth. These results warrant further consideration of Ce6/GE11-(pH)micelle as a nanophotosensitizer for FL imaging and PDT treatment of EGFRoverexpressing CRC in the future.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.7b00925. 1 H NMR spectra, acid−base titration curves, CMC at different pH values, DSC thermograms, FL spectra, cytotoxicity, FL microscopy images of HCT16-tumor sections, and mice body weights (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] *E-mail: [email protected]. Tel: 886-2-23123456, ext. 67142. ORCID

Shu-Jyuan Yang: 0000-0002-4881-0040 Ming-Jium Shieh: 0000-0003-2921-4443 Author Contributions ‡

The first two authors (M.-H.T. and W.-Y.C.) contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by the National Taiwan University Hospital, TAIWAN (106-S3583). ABBREVIATIONS PDT, photodynamic therapy; EGFR, epidermal growth factor receptor; CRC, colorectal cancer; Ce6, chlorin e6; ROS, reactive oxygen species; NIR, near-infrared; EPR, enhanced permeation and retention; PD, polydispersity; MW, molecular weights; EE, encapsulation efficiency; DC, drug content; CMC, critical micelle concentration; FL, fluorescence; GPC, gel permeation chromatography; TEM, transmission electron microscopy; PdI, polydispersity indexes; BCA, bicinchoninic L


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pH-Responsive Nanophotosensitizer for an Enhanced Photodynamic

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