Article pubs.acs.org/Biomac

Photochemically Triggered Cytosolic Drug Delivery Using pHResponsive Hyaluronic Acid Nanoparticles for Light-Induced Cancer Therapy Chung-Sung Lee and Kun Na* Department of Biotechnology, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon-si, Gyeonggi-do 420-743, Korea S Supporting Information *

ABSTRACT: A photochemically triggered cytosolic drug delivery system based on combining tumor-targeting pHresponsive hyaluronic acid (HA) nanoparticles (PHANs) with anticancer therapeutics (doxorubicin; DOX) was successfully developed for light-induced cancer therapy. PHANs were prepared through the self-assembly of a photosensitizer (PS), chlorin e6, and a pH-responsive moiety, poly(diisopropylaminoethyl) aspartamide (PDIPASP),conjugated to HA. DOX encapsulating PHANs (DOX@PHANs) have a uniform spherical shape,a sub-100 nm size distribution and a negative surface charge. The pH-responsiveness of PHANs leads to their disassembly due to the protonation of PDIPASP, which triggers DOX release. Competitive cellular uptake and confocal microscopy studies revealed CD44 receptor-mediated endocytosis, endosomal escape capability and efficient drug targeting. Compared to treatment with free DOX or PHANs, the combined treatment with DOX@PHANs and spatiotemporally defined irradiation remarkably improved the anticancer efficacy both in vitro and in vivo studies. Therefore, this strategy shows promise for the photochemically triggered cytosolic drug delivery of therapeutic agents for light-induced cancer therapy.

1. INTRODUCTION Over recent decades, nanoparticles (NPs) have increasingly been developed as carriers for bio- and chemotherapeutic agents.1,2 Many reports have demonstrated that NPs can increase the solubility and bioavailability of drugs, thus, allowing them to reach their target sites.3−5 However, conventional NP-based systems have some limitations, such as reduced intracellular delivery and endolysosomal capture; therapeutics in NP-based delivery systems must overcome these barriers to effectively reach their intracellular targets.6−10 Thus, the improvement of delivery systems with better cellular internalization strategies of therapeutics is facing important challenges. Hence, efforts to improve intracellular drug delivery and endosomal escape using stimuli-responsive polymers that respond to certain physiological conditions in vivo, such as acidity,11−14 temperature,15,16 enzymes,17,18 and redox substances19 have been extensively investigated in recent years. The most frequently exploited physiological stimulus is pH; the lysosome, with a pH of approximately 5.0, has been used to increase intracellular drug delivery.20 In particular, polymeric NP-based delivery systems with pH-responsive moieties (e.g., tertiary amine groups, imidazoles, and some amino acids) that act as “proton-sponge” in acidic conditions have been designed for intracellular drug release.21 However, the “proton-sponge” effect alone could not enhance endosomal release into the cytoplasm efficiently enough.21,22 Accordingly, new advanced © XXXX American Chemical Society

methods and strategies may allow therapeutics with intracellular targets to work more effectively through endosomal disruption. Various other methods using external interventions, including light, ultrasound, magnetic fields and shock waves, have been used as extracorporeal and spatiotemporal regulators of therapeutic delivery to overcome biological barriers and facilitate intracellular accumulation.23 Light irradiation is safe and easy to use, and lasers have been demonstrated to enhance the intracellular delivery of therapeutic molecules by photochemically triggered endolysosomal membrane disruption.24,25 This technique is termed photochemical internalization (PCI), and it is a progressive strategy to improve photodynamic therapy (PDT). These techniques use a photosensitizer (PS), a light source and oxygen molecules to initiate a photochemical reaction that produces singlet oxygen (1O2).25,26 Because PCI has enormous potential for light-induced intracellular drug delivery, it has been studied extensively and verified in practical and laboratory use in recent decades.18,26−28 However, PSs and therapeutic molecules are difficult to direct to a target region simultaneously via systemic administration. The light intensity also suffers from interference and is attenuated significantly while passing through skin and tissue.29 Therefore, new Received: August 25, 2014 Revised: September 23, 2014

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Scheme 1. Schematic Representation of Doxorubicin-Loaded pH-Responsive Hyaluronic Acid Nanoparticles (DOX@PHANs)a

a

First, DOX@PHANs are administered systemically through intravenous injection. In the tumor site, DOX@PHANs are received into cells via CD44 receptor-mediated endocytosis and accumulate in endolysosomes. DOX is then released from DOX@PHANs due to the protonation of diisopropylethylamine (DIP) groups, which causes a hydrophobic-to-hydrophilic transition in the core. Upon laser irradiation, Ce6 generates singlet oxygen that induces the disruption of endolysosomal membranes and cytosolic release of DOX. Synthesis of Poly(β-benzyl L-aspartic acid) (PBLA). β-Benzyl-Laspartic acid N-carboxyanhydride (BLA-NCA) was synthesized by the Fuchs-Farthing method using triphosgene. Briefly, BLA (3 g, 13.44 mmol) was suspended in THF (50 mL) containing triphosgene (3 g, 10.11 mmol) and stirred at 60 °C for 2 h. The crude mixture was filtered twice. The product was precipitated by the addition of hexane (900 mL) and collected by filtration and drying under vacuum to obtain BLA-NCA as a white powder. PBLA was synthesized by the ring-opening polymerization of BLANCA initiated by the terminal amino group of n-butylamine. The nbutylamine (30 μL, 0.41 mmol) was dissolved in DCM (50 mL). A solution of BLA-NCA (3.07 g, 12.32 mmol) in DMF (20 mL) was added to the solution of butylamine, and the reaction mixture was stirred for 48 h at room temperature. The crude mixture was filtered and concentrated on a rotary evaporator under vacuum at 60 °C for 30 min. The resulting solution was dialyzed against distilled water using a dialysis membrane (Spectra/Por; mol wt cutoff 3500) for 2 days. Lyophilization afforded PBLA as a white powder. The degree of polymerization (DP) of the BLA units was calculated to be 24 from 1H NMR measurements. NMR spectra were recorded in dimethyl sulfoxide-d6 (DMSO-d6) at room temperature using a 300 MHz NMR spectrometer (Bruker, Germany). Synthesis of Poly((2-diisopropylaminoethyl)aspartimide) (PDIPASP). PBLA (200 mg, 37.38 μmol) was dissolved in DMSO (10 mL). DIP (1.93 mg, 14.95 mM) was added to the solution, and the reaction mixture was stirred for 12 h. The resulting solution was dialyzed against 0.01 M hydrochloric acid solution (replaced three times) and distilled water (replaced three times). The hydrochloride salt of PDIPASP was obtained as a white powder after lyophilization. 1 H NMR spectra were recorded in DMSO-d6 at 25 °C using a Bruker300 MHz NMR Spectrometer (Bruker, Germany). Synthesis of Acetylated Hyaluronic Acid (AcHA). Hyaluronic acid (0.5 g, 86.21 μmol) was suspended in 20.0 mL of formamide and then dissolved by 50 min of vigorous stirring at 50 °C. Pyridine (1.5 mL, 18.62 mmol) was added and dissolved by 1 h of vigorous stirring at room temperature. Acetic anhydride (1.2 mL, 12.72 mmol) was then added and dissolved via vigorous stirring. This mixture was stirred for 48 h at room temperature. The resulting solution was dialyzed against distilled water using a dialysis membrane (Spectra/Por; mol wt cutoff 3500) for 2 days. Lyophilization afforded AcHA as a white powder. The synthesized AcHA was identified via 1H NMR (Bruker, Germany). Synthesis of Acetylated Hyaluronic Acid-graf t-Poly((2diisopropylaminoethyl)aspartimide) (AcHA-g-PDIPASP). PDIPASP was attached to the carboxyl groups of hyaluronic acid via a conventional carbodiimide reaction. Separate solutions of PDIPASP

combined delivery systems to localize simultaneously PSs with therapeutics and spatiotemporal PCI that will respond to attenuated low light fluence are needed to overcome these limitations. Keeping these concerns in mind, we have established a photochemically triggered cytosolic drug delivery system combined with tumor-targeting pH-responsive hyaluronic acid nanoparticles (PHANs) and anticancer therapeutics for systemic in vivo application, as illustrated in Scheme 1. HA is a well-known, natural polysaccharide that can be used as an active tumor-targeting moiety due to its specific binding to various CD44 (HA receptor) molecules that are overexpressed in cancer cells.30,31 In our design, an acetylated hyaluronic acid (AcHA) backbone was conjugated with a tertiary amine group containing polypeptidic pH-responsive moiety and chlorin e6 (Ce6) as a photochemical agent; these PHANs encapsulated the anticancer drug doxorubicin (DOX@PHANs) by selfassembly and then released it in acidic intracellular organelles (e.g., endosomes and lysosomes, ∼pH 5.0) to enhance cytosolic drug delivery and therapeutic efficacy in both in vitro and in vivo models.

2. MATERIALS AND METHODS Materials. Oligo hyaluronic acid (MW 5800 Da) was kindly provided by the Bioland Company (Cheonan, Korea). β-Benzyl-Laspartic acid (BLA), bis(trichloromethyl)-carbonate (triphosgene), nbutylamine, 2-(diisopropylamino)ethylamine (DIP), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dichloromethane (DCM), 1,3-dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), 4-dimethylaminopyridine (DMAP), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), 9,10-dimethylanthracene (DMA), doxorubicin hydrochloride (DOX·HCl), and Hoechst 33342 were purchased from Sigma-Aldrich. Tetrahydrofuran (THF), formamide, acetic anhydride, and pyridine were purchased from Wako. Chlorin e6 (Ce6) was purchased from Frontier Scientific, Inc. (UT, U.S.A.). 4′,6-Diamidino-2-phenylindole (DAPI) and Lysotracker Green DND-26 were purchased from Molecular Probes, Inc. (OR, U.S.A.). The dialysis membranes were obtained from Spectrum Laboratories Inc. (CA, U.S.A.). RPMI1640 and DMEM medium, fetal bovine serum (FBS), antibiotics (penicillin/streptomycin), and Dulbecco’s phosphate buffered saline (DPBS) were obtained from Gibco BRL (Invitrogen Corp., CA, U.S.A.). All of the other chemicals and solvents were analytical grade. B

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(0.2 g, 32.25 μmol) and a mixture of AcHA (0.2 g, 34.48 μmol), NHS (1.2 mol equiv of PDIPASP), and DCC (1.2 mol equiv of PDIPASP) in DMSO (5 mL) were prepared, and the solutions were stirred thoroughly for 3 h prior to the condensation reaction. The two solutions were mixed and stirred at room temperature. After 48 h, the reaction solution was filtered to remove insoluble byproducts (e.g., dicyclohexylurea), and the filtrate was dialyzed using a dialysis membrane (Spectra/Por; mol wt cutoff 3500) against distilled water for 2 days. The final product was lyophilized and verified by 1H NMR. 1 H NMR spectra were recorded in DMSO-d6 at room temperature using a 300 MHz NMR Spectrometer (Bruker, Germany). Synthesis of Acetylated Hyaluronic Acid-graf t-Poly((2diisopropylaminoethyl)aspartimide)-graft-Chlorin e6) (AcHAg-PDIPASP-g-Ce6). Ce6 was attached to the hydroxyl groups of hyaluronic acid via a conventional carbodiimide reaction. Separate solutions of AcHA-g-PDIPASP (0.2 g, 16.67 μmol) and a mixture of Ce6 (20.0 mg, 33.51 μmol), DMAP (1.2 mol equiv of Ce6) and DCC (1.2 mol equiv of Ce6) in DMSO (5 mL) were prepared, and the solutions were stirred thoroughly for 3 h prior to the reaction. The two solutions were mixed and stirred at room temperature. After 24 h, the reaction solution was filtered, and the filtrate was dialyzed using a dialysis membrane (Spectra/Por; mol wt cutoff 3500) against distilled water for 2 days. The final product was lyophilized and verified by 1H NMR. 1H NMR spectra were recorded in DMSO-d6 at room temperature using a 300 MHz NMR Spectrometer (Bruker, Germany). The Ce6 content of AcHA-g-PDIPASP-g-Ce6 was measured using ultraviolet−visible (UV−vis) spectrometry at 663 nm (UV-2450; Shimadzu, Japan). Preparation and Characterization of Doxorubicin-Loaded pH-Responsive Hyaluronic Acid Nanoparticles (DOX@PHANs). DOX@PHANs were prepared via the dialysis method. AcHA-gPDIPASP-g-Ce6 (10 mg) and 1 mg of DOX were dissolved in DMSO (2 mL). The solutions were then dialyzed for 12 h against pH 7.4, 0.01 M PBS buffer (1 L). The solution was filtered through a 0.45 μm filter to remove the precipitated material. The particle size and zeta-potential of the resulting DOX@PHANs were determined using dynamic light scattering (DLS; ZetasizerNano ZS, Malvern Instruments Ltd., U.K.). DLS was performed at 25 °C in pH 7.4, 0.01 M PBS buffer with the sampling time and analysis set to automatic. The sample concentration was maintained at 0.2 g L−1. The morphology of DOX@PHANs was observed with transmission electron microscopy (TEM; JEM1010; Jeol, Japan). To observe in a dried state, sample solutions were adjusted to various pH values (pH 7.4 and 5.0). Samples for TEM analysis were prepared by depositing a drop of the sample solution (10 μL, 0.2 g L−1) onto carbon-coated copper grids and dried at room temperature. The critical aggregation concentration (CAC) of AcHA-gPDIPASP-g-Ce6 in phosphate-citrate buffer (pH 5.0, 10 mM) or PBS buffer (pH 7.4, 10 mM) was measured at 25 °C on a Shimadzu RF-5301PC fluorescence spectrometer (Japan) with Hoechst 33342 as a fluorescent probe as previously reported.14 The final concentration of Hoechst 33342 was 7.0 × 10−4 M in each sample solution.The fluorescence was measured at an excitation of 355 nm and an emission of 457 nm, and the slit widths were ex = 3 nm and em = 3 nm, respectively. To determine singlet oxygen generation (SOG) by the DOX@ PHANs, 9,10-dimethylanthracene (DMA) was introduced at a concentration of 20 mM in phosphate-citrate buffer (pH 5.0) or PBS buffer (pH 7.4). SOG was induced by irradiation at a laser intensity of 1.2 J cm−2 (6.0 mW cm−2, 200 s) using a 670 nm laser source (fiber coupled laser system, LaserLab, Korea). DMA fluorescence was determined using an excitation wavelength of 360 nm and an emission wavelength of 450 nm after irradiation to determine the extent of SOG. The decrease in the fluorescence intensity of DMA as a result of the photosensitization reaction was monitored. SOG was evaluated by observing the DMA fluorescence decrease compared to each untreated buffer using a spectrofluorophotometer (RF-5301; Shimadzu, Japan). The in vitro release profile of DOX@PHANs was measured by a fluorescence multiplate reader (TecanGenios, NC, U.S.A.). In brief, a

solution of DOX@PHANs (2 mL) was pipetted into a dialysis membrane and introduced into 10 mL of phosphate-citrate buffer (pH 5.0) or PBS (pH 7.4). The mixture was then stirred at 50 rpm and 37 °C. At a predetermined time (0.5, 1.5, 3.5, 6, 12, 24 h), aliquots (10 mL) of the medium were removed and exchanged with fresh buffer. The concentration of DOX in the buffer was determined from a standard curve with DMSO/buffer 9:1 (correlation coefficient R2 = 0.994). Due to the light sensitivity of DOX, all above tests were conducted in the dark. Cell Culture and Incubation Conditions. HCT-116 (human colon cancer), CV-1 (monkey normal fibroblast), and CT-26 (murine colon cancer) cells were obtained from the Korean Cell Line Bank (HCT-116; KCLB No. 10247, CV-1; KCLB No. 10070, CT-26; KCLB No. 80009) and cultured in RPMI1640 (HCT-116 and CV-1) or DMEM (CT-26) supplemented with 10% heat inactivated FBS and penicillin/streptomycin (100 U/mL and 100 μg/mL, respectively), which is referred to as complement medium (CM) in this study. The cells were cultured at 37 °C in a humidified atmosphere with 5% CO2 and subcultured in new media every 2−3 days. Untreated cells were irradiated or kept in the dark and used as reference standards. In Vitro Cellular Uptake and Competitive Inhibition Study. To verify the cellular uptake of DOX@PHANs and competitive inhibition with free HA, HCT-116 cells were seeded in 12-well cell culture plates at a density of 5 × 105 cells per well and incubated for 12 h at 37 °C in 5% CO2. The medium was then removed, and the cells were incubated with DOX@PHANs (0.1 μg mL−1 DOX and 0.1 μg mL−1 Ce6) alone or DOX@PHANs (0.1 μg mL−1 DOX and 0.1 μg mL−1 Ce6) with free HA (10 mg mL−1) for 1 h. The cells were rinsed, harvested, and resuspended with DPBS. Cellular uptake was quantitatively analyzed using flow cytometry (Beckman, San Jose, CA, U.S.A.). For each sample, 10000 cells (gated events) were counted, and DOX and Ce6 fluorescence was detected with logarithmic settings (FL3 (DOX), Em = 620 nm; and FL4 (Ce6), Em = 675 nm). Each experiment was analyzed statistically using the CXP analysis program (Beckman, San Jose, CA, U.S.A.). To observe the cellular uptake of DOX@PHANs by confocal laser scanning microcopy (CLSM), HCT-116 (1 × 105 cells per well in a 12-well plate) and CV-1 (1 × 105 cells per well in a 12-well plate) cells were treated with DOX@PHANs (0.1 μg mL−1 DOX and 0.1 μg mL−1 Ce6) alone or DOX@PHANs (0.1 μg mL−1 DOX and 0.1 μg mL−1 Ce6) with free HA (10 mg mL−1) for 1 h. The cells were then washed twice with DPBS, fixed with 4% paraformaldehyde, and stained with DAPI. The cells were mounted in mounting medium (Dako, Glostrup, Denmark) and visualized using a confocal laser scanning microscope (LSM 710 Meta; Carl Zeiss, Germany). Fluorescence images were analyzed using the LSM image browser software (Zen series, Carl Zeiss, Germany). Fluorescence intensity was the normalized values by the number of cells. Photochemically Triggered Endolysosomal Escape. HCT116 cells were seeded in 35 mm covered, glass-bottom sterile culture dishes at a density of 1 × 105 cells per well and incubated overnight at 37 °C in 5% CO2. The medium was removed after 12 h, and cells were incubated with DOX@PHANs (0.5 μg mL−1 DOX and 0.5 μg mL−1 Ce6) for 2 h. During incubation, the cells were treated with Hoechst 33342 to stain the nuclei, and the endolysosomes were labeled with Lysotracker Green DND-26 for 1 h. After incubation, the medium was removed, and the cells were rinsed twice with DPBS. After irradiation of 0.6 or 1.2 J cm−2 (6.0 mW cm−2, 100 or 200 s) with a 670 nm laser source (fiber coupled laser system, LaserLab, Korea), the cells were incubated in SF medium with 1% penicillin/streptomycin at 37 °C in a 5% CO2 atmosphere in a CLSM live cell system. The endolysosomal release of DOX and Ce6 was observed on a CLSM (LSM 710; Zeiss, Germany) with a live cell imaging system. Intracellular Distribution of DOX. HCT-116 cells were seeded in 35 mm sterile culture dishes at a density of 1 × 105 cells per well and incubated overnight at 37 °C in 5% CO2. The medium was removed after 12 h, and cells were incubated with free DOX or DOX@PHANs (0.5 μg mL−1 DOX and 0.5 μg mL−1 Ce6) for 4 h. After incubation, the wells were rinsed with DPBS to remove free DOX or DOX@PHANs that had not been internalized. Culture C

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Figure 1. (a) 1H NMR (300 MHz) spectra of AcHA-g-PDIPASP-g-Ce6 at pD 7.4 and 5.0 in D2O. DCl was used to adjust the solution’s pD in the 1 H NMR measurements. (b) Singlet oxygen generation of AcHA-g-PDIPASP-g-Ce6 at pH 7.4 and pH 5.0 under 670 nm laser irradiation of 1.2 J cm−2 (6 mW cm−2, 200 s). (c) Size distribution of the DOX@PHANs at pH 7.4 and 5.0. (d) TEM photographs of DOX@PHANs at pH 7.4 and 5.0. Magnification is 60000× or 150000×. (e) In vitro quantitative DOX release profile from DOX@PHANs in phosphate-citrate buffer (pH 5.0) and PBS buffer (pH 7.4; n = 3). medium (100 μL) was added to the wells, and each well was irradiated using the 670 nm laser source (0.6 or 1.2 J cm−2; 6.0 mW cm−2, 100 or 200 s, fiber coupled laser system, LaserLab, Korea). The cells were incubated for a further 24 h. The cells were then washed twice with DPBS, fixed with 4% paraformaldehyde and stained with DAPI. The cells were mounted in mounting medium (Dako, Glostrup, Denmark) and visualized using a confocal laser scanning microscope (LSM 710

Meta; Carl Zeiss, Germany). Fluorescence images were analyzed using the LSM image browser software (Zen series, Carl Zeiss, Germany). Colocalization efficiency is relative number of colocalizing pixels between DOX and DAPI signals as compared to the total number of pixels. Bright pixels contribute more than faint pixels. In Vitro Cytotoxicity. To assess cytotoxicity, HCT-116 cells (1 × 105 cells per well) were added to the wells of a black 96-well plate in D

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Figure 2. Cellular uptake of DOX@PHANs. (a) Flow cytometric quantification of HCT-116 cancer cells. The cells were treated with DOX@ PHANs in the presence or absence of free HA (10 mg mL−1). (b) CLSM images of HCT-116 cancer cells and CV-1 normal cells. The cells were treated with DOX@PHANs in the presence or absence of free HA (10 mg mL−1). In each panel, the cell nuclei stained with DAPI are blue, DOX is red, and Ce6 is cyan. Original magnification is 40×. The scale bars are 20 μm. (c) Mean fluorescence intensity of DOX and Ce6 in CLSM images (n = 3). 100 μL of complement medium and incubated overnight. Free DOX (0.1 μg mL−1), PHANs (Ce6 only, 0.1 μg mL−1), or DOX@PHANs (DOX and Ce6, 0.1 μg mL−1 each) were added to each well in 100 μL of SF medium, and the plates were returned to the incubator for 4 h. After incubation, the wells were rinsed with DPBS to remove free DOX and PHANs that had not been internalized. Complement medium (100 μL) was added to the wells, and each well was irradiated using the 670 nm laser source (0.6 or 1.2 J cm−2; 6.0 mW cm−2, 100 or 200 s, fiber coupled laser system, LaserLab, Korea). The cells were incubated for a further 48 h. Cell viability was assessed using the MTT assay. The resulting formazan crystals were dissolved in DMSO (100 μL) and transferred to a new plate. Absorbance intensity was measured at 570 nm using a microplate reader (Bio-Tek, VT, U.S.A.).

The LIVE/DEAD Viability/Cytotoxicity Assay Kit (Molecular Probes, U.S.A.) was also used to assess viability. The dyes in this kit are 2 μM calcein AM, which stains live cells green, and 4 μM EthD-1, which stains dead cells red. HCT-116 cells were seeded in 35 mm cell culture dishes at a density of 5 × 105 cells per well. Cell cultures were incubated overnight at 37 °C in 5% CO2. After 12 h, the medium was removed, and the cells were incubated in SF medium containing free DOX (0.1 μg mL−1), PHANs (Ce6 only, 0.1 μg mL−1), or DOX@ PHANs (DOX and Ce6, 0.1 μg mL−1 each) for 4 h. The medium was removed, and the cells were rinsed twice with DPBS. Laser irradiation (0.6 or 1.2 J cm−2; 6.0 mW cm−2, 100 or 200 s) was performed with a 670 nm laser source (fiber coupled laser system, LaserLab, Korea). The cells were then incubated in complement medium for 48 h. Cell E

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Figure 3. Photochemically triggered endolysosomal escape behavior. Photographs captured by confocal microscopy with live cell imaging system and related fluorescence intensity histograms (right side line plots, respectively) of DOX@PHANs-treated HCT-116 cells with laser irradiation of 0.6 or 1.2 J cm−2 (6 mW cm−2, 100 or 200 s). The white arrows are the x-axis of each histogram. In each panel, Lysotracker (endolysosome) is green, the cell nuclei stained with Hoechst 33342 are blue, DOX is red, and Ce6 is cyan. Original magnification is 40×. The scale bars are 20 μm. densities of 100 J cm−2 (100 mW cm−2, 17 min) 12 h postinjection. Tumor sizes and body weights were measured at each selected time point. To investigate histology, mice were sacrificed, and tumors were collected and fixed for 24 h in 4% paraformaldehyde. After deparaffinization, the tissue sections (5 μm) were stained with hematoxylin/eosin (H&E). The immunohistochemical study was performed using the terminal deoxynucleotidyltransferase-mediated nick-end labeling (TUNEL) assay (Promega Corp., WI, U.S.A.) with modifications. The mice were sacrificed, and tumors were collected and fixed for 24 h in 4% paraformaldehyde. After deparaffinization, the sections (5 μm) were washed twice with PBS and treated with 0.2% Triton X-100 for 10 min at room temperature. The samples were washed twice and incubated with equilibration buffer for 10 min at room temperature. The equilibration buffer was drained, and a reaction buffer containing equilibration buffer, nucleotide mix, and TdT enzyme was added to the tissue sections. Samples were incubated in a dark, humidified atmosphere at 37 °C for 1 h. The samples were then washed for 5 min each to remove unincorporated fluorescein-TdT. The samples were stained with DAPI and mounted. TUNEL analysis was conducted using a CLSM (LSM 710 Meta; Zeiss, Germany). Statistical Analysis. Data are expressed as the means ± standard deviation (SD). Differences between the values were assessed using Student’s t test.

viability was observed by fluorescence microscopy (Zeiss, Germany). Fluorescence intensity was measured by Zen 2011. Animal Models. CT-26 cells were implanted into 6-week-old BALB/c nude mice. Briefly, the cells (5 × 104) in 100 μL of serum-free DMEM were injected subcutaneously. Tumor volume was calculated using the following equation: volume = 0.5 × L × W2, where “W” and “L” are the width and length of the tumor, respectively. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the College of Pharmacy, 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. In Vivo NIRF Imaging of DOX@PHANs in Tumor-Bearing Mice. The tumor model was established as described above. When the tumor volume reached approximately 50−100 mm3 15 days after cell inoculation, DOX@PHANs (dose: 2.0 mg kg−1 DOX and 2.0 mg kg−1 Ce6) in PBS buffer (150 mM, pH 7.4) were injected intravenously into the tumor-bearing nude mice through the tail vein. A 12-bit CCD camera (Image Station 4000 MM; Kodak, Rochester, NY, U.S.A.) prepared with a special C-mount lens and a long wave emission filter (600−700 nm; Omega Optical, Brattleboro, VT, U.S.A.) was used to capture live fluorescence images of the nude mice. In Vivo Tumor Growth Inhibition. The tumor model was established as described above. When the tumor volume was approximately 50−100 mm3 15 days after cell inoculation, the CT26 bearing nude mice were randomly divided into four groups (n = 5). Solutions of PBS, free DOX (2.0 mg kg−1), PHANs (Ce6 only, 2.0 mg kg−1), and DOX@PHANs (DOX and Ce6, 2.0 mg kg−1 each) in 100 μL of PBS buffer (150 mM, pH 7.4) were injected via the tail vein at days 15 and 22. Tumor regions were irradiated with a laser at power

3. RESULTS AND DISCUSSION Synthesis and Characterization of pH-Responsive Copolymers and PHANs. AcHA-g-PDIPASP-g-Ce6 copolyF

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1.18 acetyl groups per 1 unit (2 glucose rings) of HA as determined by 1H NMR (Figure S1). The degree of polymerization of PBLA and the DIP substitution value of PDIPASP were 24 and >95%, respectively (Figures S2 and S3). Finally, PDIPASP and Ce6 were attached to AcHA through carbodiimide reaction. The copolymer (i.e., AcHA-g-PDIPASPg-Ce6) was further analyzed by 1H NMR (Figure S4). To confirm the pH-responsive hydrophobic-to-hydrophilic transition of AcHA-g-PDIPASP-g-Ce6, the critical aggregation concentration (CAC) of the copolymer was investigated using Hoechst 33342 as a fluorescence probe at pH 7.4 and pH 5.0 (Figure S5). The CAC at pH 7.4 (0.05 g L−1) was much lower than that at pH 5.0 (0.17 g L−1). This result was due to the different protonation degrees of PDIPASP at the two pH values; this protonation governs the balance between hydrophobicity and hydrophilicity.33 To understand further the structural pH-responsive transition of PHANs, 1H NMR analysis was conducted at the two different pDs in D2O (Figure 1a). The peaks for Ce6 were not detected at either pD, and the peak intensities associated with AcHA were weaker at pD 7.4 compared with those at pD 5.0, consistent with being trapped in the core of PHANs. Furthermore, the methyl peak (−CH3) of DIP produced a weak signal at 1.12 ppm in pD 7.4 solution, whereas it was strong, sharp, and shifted to 1.24 ppm at pD 5.0 due to the protonation of the DIP groups on the PDIPASP chains. To further characterize AcHA-g-PDIPASP-gCe6, the transmittance was estimated by UV−visible spectrophotometry at 500 nm (1 g L−1, UV2450, Shimadzu). As shown in Figure S6, the transmittance was increased at pH 5.0 (50.1 ± 3.2%T) compared to pH 7.4 (66.1 ± 5.1%T) because the solubility of copolymers was increased. Moreover, we also determined the singlet oxygen generation (SOG) of AcHA-g-PDIPASP-g-Ce6 copolymers using 9,10-dimethylanthracene (DMA) as a probe at two different pHs with an irradiation of 1.2 J cm−2 (Figure 1b).30 As expected, the copolymers at pH 5.0 revealed distinctly increased SOG up to approximately 3-fold compared to that at pH 7.4. Based on these results, the PDIPASP, acetyl moiety, and Ce6 molecules could effectively compose interlayered PHANs at physiological pH 7.4, whereas they should disassemble in the acidic lysosome with pH 5.0. The doxorubicin-loaded PHANs (DOX@PHANs) were prepared through self-assembly in PBS solution at pH 7.4. DOX was encapsulated into the interlayered core via hydrophobic interactions (DOX loading content: 14.2 ± 0.1%). The hydrodynamic size and morphology of the DOX@PHANs under two different pHs were determined by dynamic laser scattering (DLS) and transmission electron microscopy (TEM). Due to the pH-responsive PDIPASP chains, the average diameter of DOX@PHANs at pH 7.4 was 90 ± 5 nm (PDI < 0.1) with zeta potential of −6.7 ± 0.4 mV, whereas the diameter at pH 5.0 was 18 ± 5 nm (PDI < 0.7) with zeta potential of −4.3 ± 0.3 mV (Figure 1c). TEM images showed that DOX@PHANs had a uniform, approximately sub100 nm spherical shape (Figure 1d). The size and surface charge of DOX@PHANs are desirable for systemic administration and migration to tumor sites via the enhanced permeability and retention (EPR) effect.34 The pH-responsive controlled release was investigated at physiological pH 7.4 and lysosomal pH 5.0 using dialysis tubing (Figure 1e). Although the cumulative DOX release at pH 7.4 was about 20% within 24 h, indicating that DOX encapsulation in the hydrophobic interlayered core of PHANs was

Figure 4. Intracellular distribution of DOX. (a) Confocal microscopic images of HCT-116 cells incubated with DOX@PHANs. In each panel, red fluorescence is DOX, and cell nuclei stained with DAPI are blue. Original magnification is 40×. The scale bars are 50 μm. (b) Colocalization efficiency between nuclei (DAPI, blue) and DOX (red). Colocalization efficiency was measured by Zen 2011.

mer was synthesized via a multistep set of N-carboxyanhydride (NCA) ring-opening polymerization,18,32 aminolysis,14 and carbodiimide reactions (Scheme S1). HA has frequently been used as a drug carrier due to its biocompatibility, biodegradability, and tumor-targeting capacity. However, HA has the severe limitation that it is difficult to modify chemically due to its poor solubility in commonly used organic solvents. To overcome this problem, acetyl groups were introduced to the hydroxyl groups of HA to increase hydrophobicity and solubility in organic solvents. The degree of acetylation was G

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Figure 5. (a) In vitro cytotoxicity assays in HCT-116 cells treated with free DOX (0.1 μg mL−1), PHANs (0.1 μg mlL−1), and DOX@PHANs (DOX and Ce6, 0.1 μg mL−1 each), with or without laser irradiation of 0.6 or 1.2 J cm−2 (6 mW cm−2, 100 or 200 s, n = 3, *P < 0.05, **P < 0.005). (b) Live and dead assay in HCT-116 cells in the dark or with laser irradiation of 0.6 or 1.2 J cm−2. The live cells stained green, and dead cells stained red. Scale bars are 200 μm.

Figure 6. (a) In vivo near-infrared fluorescence (NIRF) images of time-dependent accumulation of DOX@PHANs in CT-26 tumor-bearing mice after systemic administration via tail vein route. (b) Quantification of fluorescence intensity of tumor regions in DOX@PHANs-treated tumorbearing mice after systemic administration via tail vein route.

live cell imaging equipment after DOX@PHAN treatment (Figure 3). The endolysosomes and nuclei were labeled with Lysotracker Green DND-26 (green fluorescence) and Hoechst 33342 (blue fluorescence), respectively. DOX@PHANs were exposed to an acidic environment in the endolysosomes, and they released DOX rapidly after receptor-mediated endocytosis. Following the spatiotemporal 670 nm laser irradiation (0.6 or 1.2 J cm−2), the Ce6 molecules of DOX@PHANs were activated and locally generated singlet oxygen, which can induce endolysosomal rupture and DOX release into the cytosol. The laser irradiation intensity of 0.6 or 1.2 J cm−2 was much less than the 10.0 J cm−2or greater dose that is generally used in PDT applications. 18,32,38 The fluorescence of endolysosomes was clearly detected and was colocalized with DOX fluorescence in CLSM images and histogram plots before laser irradiation. After 15 min of laser irradiation, the fluorescence of endolysosomes, DOX and Ce6 had diffused into other intracellular regions. Without laser irradiation, the fluorescence intensity in endolysosomes remained steady after 15 min of incubation. This result has relevance to the photochemically triggered endosolysomal escape and release of DOX@PHANs for light-induced cytosolic delivery. Validation of Nuclear Targeting of Released DOX. To determine if the DOX released from DOX@PHANs could efficiently access and damage DNA, confocal laser scanning microscopy (CLSM) studies were performed on HCT-116 cells with free DOX or DOX@PHANs irradiated with light intensities of 0, 0.6, and 1.2 J cm−2 (Figures 4 and S7). After 4 h of incubation with DOX@PHANs or free DOX, HCT-116

maintained, the cumulative DOX release at pH 5.0 was approximately 80% within 24 h, in agreement with a rapid destabilization via protonation and the hydrophobic-to-hydrophilic transition of PDIPASP. These results imply that DOX@ PHANs may quickly release DOX after being internalized in cancer cells. In Vitro Cellular Uptake of PHANs. To confirm the receptor-mediated cellular internalization of DOX@PHANs, we analyzed the intracellular Ce6 and DOX fluorescence signals using flow cytometry and confocal laser scanning microscopy (CLSM) with CD44 receptor-overexpressing cancer (HCT116) cells and normal fibroblast cells (CV-1; Figure 2).35,36 The cellular uptake was also observed with competitive inhibition using free HA in HCT-116 cells by flow cytometry(Figure 2a) and CLSM (Figure 2b,c). The HCT-116 cells treated with DOX@PHANs displayed high intracellular fluorescence. In contrast, when DOX@PHANs were treated simultaneously with free HA, both the DOX and Ce6 fluorescence were decreased due to free HA competing for binding to the CD44 cell surface receptor. In CV-1 cells, these fluorescence signals were lower than those of DOX@PHANs in HCT-116 cells. These results suggest that the interaction between the HA moiety of DOX@PHAN and cell surface receptor such as CD44 improves cellular uptake via receptor-mediated endocytosis.37 Photochemically Triggered Endolysosomal Escape. To verify the photochemical-induced endolysosome rupture to release DOX into the cytosol by laser irradiation, we evaluated the intracellular distribution of DOX and Ce6 by CLSM with H

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Figure 7. In vivo tumor therapy of a subcutaneous tumor model injected systemically with various treatments (PBS, DOX, PHANs without laser irradiation, PHANs with laser irradiation of 100 J cm−2, DOX@PHANs without laser irradiation, and PHANs with laser irradiation of 100 J cm−2. (a) Tumor growth inhibition after various treatments (dose: 2.0 mg DOX and 2.0 mg Ce6 per kg body weight, n = 5, *P < 0.05, **P < 0.005). (b) Body weight measurements of tumor-bearing mice throughout treatments. (c) Ex vivo histological analyses of tumor sections (31 days after inoculation of tumor cells). Nuclei were stained blue, and the extracellular matrix and cytoplasm were stained red in H&E staining. Green fluorescence indicates apoptotic cells; nuclei were stained with DAPI and appear blue in the TUNEL assay. Scale bars are 100 μm.

cm−2, the dead cells were increased and the cell density was decreased in PHANs-treated cells. These results indicate that photochemically triggered cytosolic drug delivery was critical for enhanced therapeutic efficacy. In Vivo Imaging in Tumor-Bearing Mice. The in vivo tumor accumulation of DOX@PHANs was monitored using noninvasive fluorescence imaging technique to determine the proper time point of laser irradiation after systemic administration of DOX@PHAN (dose: 2.0 mg kg−1 DOX and 2.0 mg kg−1 Ce6) into CT-26 (mouse colon cancer, CD44 expressing cell line)39 tumor-bearing mice (Figure 6). The PS, Ce6, was used for fluorescence imaging.14,38 The images show the real time images of DOX@PHAN in the tumor-bearing mice for 24 h. The fluorescence intensity of Ce6 intumors gradually increased for 12 h, implying targeting and accumulation in tumors (Figure 6a). From the quantitative analysis shown in Figure6b, the considerable signal was greatest at 12 h (1.5-fold greater than before injection) but was weakened after 24 h due to the disassembly of DOX@PHAN after cellular uptake and clearance, which could be almost free from the unintended toxicity residual PS after administration.38 This tumor-targeting property of DOX@PHANs may be due to a combination of an EPR effect and the receptor-mediated endocytosis into the tumor cells. These results indicate that CD44-mediated active tumor-targeting property as well as EPR effect of DOX@PHANs can lead to enhanced tumor accumulation. In Vivo Antitumor Efficacy Study. To further verify the antitumor efficacy of our photochemically triggered cytosolic drug delivery system, we conducted an in vivo tumor growth inhibition study with DOX@PHANs for 31 days after the subcutaneous inoculation of CT-26 cells. Fourteen days

cells were irradiated at a predetermined intensity using a 670 nm laser source (fiber coupled laser system, LaserLab, Korea), and CLSM images were captured 24 h after irradiation. The DOX (red) fluorescence increased and colocalized with DAPI stained nuclei (blue fluorescence) with increasing irradiation intensity (Figure 4a). We specifically quantified the colocalization of DOX with nuclei to confirm the movement of DOX to its target site (Figure 4b). The colocalization efficiencies of the free DOX treated and nonirradiated groups were 37.8 and 35.1%, respectively. In contrast, the colocalization efficiency was significantly increased up to 56.7 and 78.8% upon laser irradiation of 0.6 and 1.2 J cm−2, respectively. These results correspond with the photochemically triggered intracellular release of DOX@PHANs via the light-induced disruption of endolysosomal membranes. In Vitro Cytotoxicity. The therapeutic efficacy of DOX@ PHANs was demonstrated using an in vitro cell cytotoxicity test (MTT assay) in HCT-116 cells (Figure 5a). In the absence of laser irradiation, cell viability in the presence of free DOX, PHANs, or DOX@PHANs was greater than 90%. However, the cell viability in the presence of DOX@PHANs (DOX and Ce6, 0.1 μg mL−1 each) was significantly decreased to 74.9 ± 4.6 and 42.9 ± 2.6% by laser irradiation of 0.6 and 1.2 J cm−2, respectively; PHANs (Ce6, 0.1 μgml−1) slightly affected viability to 97.5 ± 8.3 and 81.6 ± 0.5% in the presence of 0.6 and 1.2 J cm−2, respectively. A similar phenomenon was observed in the fluorescent staining of live and dead cells using calcein-AM (green fluorescent dye) and ethidium homodimer (red fluorescent dye; Figure 5b). This live and dead assay revealed that the red fluorescence (dead cells) increased to a greater extent in DOX@PHANs-treated cells than in PHANstreated cells irradiated with 0.6 J cm−2. Upon irradiation of 1.2 J I

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ACKNOWLEDGMENTS This work was supported by the Strategic Research though the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP; No. 2011-0028726).

postinoculation, the tumor-bearing mice were randomly divided into six groups of five mice per group. Formulations were administered intravenously via the tail vein at 15 and 22 days. The mice were then irradiated with a laser intensity of 100 J cm−2 (100 mW cm−2, 17 min, 670 nm fiber coupled laser system, LaserLab, Korea), which successfully induced lightmediated endolysosomal disruption of PS at 16 and 23 days.18,29,40 As shown in Figure 7a,b, nonirradiated groups (PBS, free DOX, PHANs, and DOX@PHANs) had no significant effects. In contrast, the tumor sizes in mice given DOX@PHANs and exposed to laser irradiation were significantly decreased with no critical changes in body weight compared to the other groups; PHANs with laser irradiation also had some effect on tumor sizes. In situ histological and immunohistochemical analyses after treatments showed that the tumor cells of the group that received DOX@PHANs and laser irradiation had reduced H&E staining and had the strongest green fluorescence in the TUNEL assay, indicating apoptosis (Figure 7c).

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ASSOCIATED CONTENT

* Supporting Information S

Additional scheme (synthetic routes), 1H NMR spetra, critical aggregation concentration data, transmittance data, and additional confocal laser scanning microscopy data. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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4. CONCLUSIONS In summary, we have designed and developed a photochemically triggered cytosolic delivery system for anticancer drugs by combining tumor-targetable (CD44 receptor mediated), pHresponsive hyaluronic acid nanoparticles with light-induced endosomal escape. The DOX@PHANs are composed of a PS and a pH-responsive moiety, PDIPASP, conjugated with acetylated hyaluronic acid (AcHA-g-PDIPASP-g-Ce6) copolymer to allow for spatiotemporally controlled cytosolic drug release. The characterization studies confirmed that the pHresponse activities of AcHA-g-PDIPASP-g-Ce6 and DOX@ PHANs governed the hydrophobic-to-hydrophilic transition of PDIPASP through the protonation of the tertiary amine groups in the DIP units. After cellular uptake via receptor-mediated endocytosis due to hyaluronic acid interaction with the CD44 receptor and DOX@PHAN disruption by DIP protonation in acidic endolysosomes, low-intensity laser irradiation (compared with laser intensity of PDT applications) stimulated the free PS to produce reactive singlet oxygen, which released DOX into the cytosol of cancer cells. In vitro cell and in vivo animal studies demonstrated that the treatment of DOX@PHANs with laser irradiation has powerful therapeutic efficacy regardless of low laser dose. This study led to a significant improvement in antitumor efficacy by incorporating lightinduced photochemical triggering in spatiotemporally controlled manner. In addition, this promising platform may be optimal for overcoming biological barriers in cytosolic drug delivery applications.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +82-2-2164-4865. Tel.: +82-2-2164-4832. Notes

The authors declare no competing financial interest. J

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K

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