Article pubs.acs.org/molecularpharmaceutics

Tat/HA2 Peptides Conjugated [email protected] as a Photosensitizer Carrier for Near Infrared Triggered Photodynamic Therapy Shefang Ye,† Ning Kang,† Min Chen,† Caiding Wang,† Tianxiao Wang,† Yarun Wang,† Yongliang Liu,† Donghui Li,‡ and Lei Ren*,†,§ †

Research Center of Biomedical Engineering, Department of Biomaterials, College of Materials, ‡Medical College, and §State Key Laboratory for Physical Chemistry of Solid Surfaces, Xiamen University, Xiamen 361005, PR China S Supporting Information *

ABSTRACT: To achieve an efficiency of intracellular photosensitizers (PSs) delivery and efficacy of photodynamic therapy, we have developed a novel class of PS formulation for encapsulating sulfonated aluminum phthalocyanine (AlPcS4) by taking advantage of the membrane-disruptive peptides Tat/HA2 and the photothermally triggered delivery system using [email protected] The coordinated effects of cell penetrating peptide Tat and fusogenic peptide HA2 could enhance the efficient cellular internalization and endo/ lysosome escape of PSs delivery systems. Singlet oxygen generation was inhibited due to the reaction between loaded AlPcS4 and Au nanorods, which indicated that the AlPcS4loaded, [email protected] delivery system might be nonphototoxic in the circulatory system. However, this PSs-loaded nanosystem became highly phototoxic as it underwent the near-infrared irradiation by using the combined lights of 808 and 680 nm. Upon irradiation, the Tat/HA2 conjugated [email protected] elicited an active photodynamic response against the cancer cells, leading to effective cells killing via mitochondria-associated apoptotic pathway. This study also demonstrated improved PDT therapeutic efficacy after intravenous administration of Tat/[email protected] and the subsequent lights irradiations in tumor-bearing mice. We describe here a strategy for enhanced photodynamic eradication of solid tumors by endo/ lysosomal escape and highlight the great promise of peptide-based nanocarriers used for cancer therapy. KEYWORDS: gold nanorods, Tat/HA2 peptide, endo/lysosome escape, photodynamic therapy

1. INTRODUCTION Photodynamic therapy (PDT) is a well-established therapeutic modality for the treatment of tumors and involves photochemical reactions mediated by the interaction of photosensitizers (PSs) with specific light and oxygen. Each individual component is harmless by itself, but acting in concert they can trigger the generation of reactive oxygen species (ROS) such as singlet oxygen (1O2), which can result in cell injury and cell death.1 However, the efficiency of PDT is limited by shallow light penetration into tissue and the poor selectivity of currently available PSs, which causes undesirable side effects on adjacent normal tissues and prolonged skin photosensitization.1,2 To overcome these limitations, various drug delivery systems, such as liposomes,3 polymer conjugates,4 polymeric nanoparticles,5 silica nanoparticles,6 and photosensitizer−antibody conjugates,7 are being actively explored for controlled release of PSs to achieve a higher local concentration and reduce the systemic toxicity. Near infrared (NIR, 700−950 nm), also known as optical window, offers many distinct advantages over other types of light for triggering the drug release in biological systems due to their ability to penetrate deep into blood and tissue.8 Recently, the strong absorption in the NIR region of gold nanostructures (shells,9 rods,10,11 and cages12) enables them to have a wide range of bioapplications such as biochemical sensing, © 2015 American Chemical Society

biomedical diagnostics, and therapeutics. Gold nanoparticles could absorb light efficiently in the visible spectral region due to their coherent oscillations of conduction band electrons, exhibiting strong resonance in the visible-light range.13 In addition, photoexcitation of metal nanostructures might trigger the formation of a heated electron gas that cools rapidly within 1 ps by electron−lattice energy exchange in metal nanoparticles, followed by heat transfer to the surrounding medium on the time scale of 100 ps, resulting in localized heating. Such fast energy conversion and dissipation can be readily obtained by using light radiation with a frequency overlapping with the surface plasmon resonance (SPR) absorption band of the nanoparticle.14 On the other hand, mitochondria are involved in diverse physiologic and pathologic processes that are crucial for cell survival and death.15 Tumor cells are metabolically active and highly energy-dependent, so mitochondrial dysfunction contributes to the plethora of cellular processes involved in tumorigenesis.16 Previous studies have shown that the intracellular localization of PSs plays a crucial role in the outcome of Received: Revised: Accepted: Published: 2444

February 24, 2015 May 26, 2015 June 1, 2015 June 1, 2015 DOI: 10.1021/acs.molpharmaceut.5b00161 Mol. Pharmaceutics 2015, 12, 2444−2458

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Molecular Pharmaceutics PDT.17 In fact, some PSs such as protoporphyrin IX (PpIX) and phthalocyanine appear to preferentially accumulate in mitochondria.18,19 However, the mitochondria seems not to be the primary cellular target for nanocarrier-mediated PDT system.2 Recently, the use of gold nanostructures-based drug carriers, which can preferentially and effectively accumulate in solid tumors through enhanced permeation and retention (EPR) effect, has attracted extensive attention.20 In general, PSs-loaded nanocarriers are internalized by cells localized in endosome/lysosome compartments, the target site of photodamage.21 However, intracellular components such as the mitochondria are also considered one of the most important subcellular sites of photodamage. Therefore, the development of nanocarriers for efficient intracellular delivery of PSs with endosomal/lysosomal escape capability might lead to improved PDT efficacy. One approach to increase intracellular delivery of nanoparticles involves the use of cell penetrating peptides (CPPs) such as a Tat peptide adopted from the HIV-1 transactivator of transcription (TAT) protein.22,23 However, Tat peptidemediated endocytosis of nanocarriers is generally confined to the endosome/lysosome.23 In this regard, efficient delivery of the PSs into the cytosol thus requires disruption of the membrane of endoso/lysosomes as the pH in the endosome decreases from 7.0 to 5.0. Recently, membrane-disruptive peptides (e.g., influenza virus hemagglutinin-2, HA2) have drawn particular attention due to their ability to simulate viral mechanisms of endo/lysosomal escape.24 We have shown that the synergistic effect of Tat and fusogenic peptide HA2 could improve efficient cellular internalization, endolysosomal escape, and nuclear entry of nanoparticles.23 In this study, we attempted to establish a concept that takes advantage of the membrane-disruptive peptides Tat/HA2 and the photothermally triggered PSs delivery system based on [email protected] nanogels. Among PSs, the phthalocyanines have gained increasing attention because of their merits: (1) ease of synthesis; (2) efficient absorption of NIR light (λmax = ca. 680 nm); (3) resistance to photochemical degradation; (4) long-lived in the photoexcited triplet state; and (5) low dark toxicity.25 Although the water-soluble phthalocyanine derivative sulfonated aluminum phthalocyanine (AlPcS4) has the appropriate photobiological features for PDT, the poor uptake efficiency by cell as well as the selective PSs delivery into a target lesion still faces a great challenge. With the different aspect ratios that resulted in SPR, gold nanorods (AuNRs) exhibit unique absorption bands in NIR spectral region, thus conducive to higher photothermal conversion efficiency. This property allows AuNRs to be used in NIR-trigged PDT.10,11 Herein, we hypothesized that the integration of Tat/HA2 peptides onto AuNRs could induce more ROS formation and improve photocytotoxicity efficiency through a mitochondriamediated apoptosis signaling pathway and further enhancing PDT efficacy in tumor-bearing models in mice.

sobenzofuran (DPBF), neutral red (NR), Hoechst33258, BclxL, Bax, β-actin, and fluorogenic substrates Ac-LEHD-AFC, AcVETD-AMC, and Ac-DEVD-AFC were supplied by SigmaAldrich (St. Louis, MO, USA). Trypsin, 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT), and Annexin VFITC/propidium iodide (PI) apoptosis detection kit were purchased from Amresco (Solon, OH, USA). Cathepsin-B activity assay kit and lactate dehydrogenase (LDH) activity assay kit were purchased from BioVision (CA, USA). Sulfosuccinimidyl 6-[3′(2-pyridyldithio)-propionamido] hexanoate (SPDP) was purchased from Thermo Fisher Scientific (America). NpyS-activated C-terminal Cys containing peptides (Tat, KYGRRRQRRKKRGC; HA2, GDIMGEWGNEIFGAIAGFLGC) were provided by Chinese Peptide Co. (China). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin−streptomycin were obtained from Hyclon (America). LysoTracker Green, 2′,7′dichlorofluorescin diacetate (DCFH-DA), 4′,6-diamidino-2phenylindole (DAPI), and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-arbocyanineiodide (JC-1) were obtained from Molecular Probe (Eugene, OR, USA). MitoTracker Green FM and Histostain-Plus kits were obtained from Invitrogen (Carlsbad, CA, USA). Dead End fluorometric TUNEL system was purchased from Promega (Madison, WI, USA). All the antibodies used in this study were purchased from Santa Cruz Biotechnology (Cruz, CA). 2.2. Instruments. Spectrofluorophotometer (Hitachi F7,000, Japan) was used to record fluorescence spectra. Excitation wavelength for AlPcS4 was 633 nm, and the emission spectra were recorded between 650 and 800 nm. UV−visible spectrophotometer (Varian Cary 50, America) was used to record the absorption spectrum. Atomic force microscopy (AFM, Bruker AXS, America) was used to observe the formation of the nanoparticles and to measure their diameters, and images were collected in the force-minimized height-mode under solution using a Nanoscope MultiMode VIII. Data were recorded with 512 lines per scan direction with a scan rate of 1−2 Hz. For the particle sizing histograms, the average diameter of the particles in a particular area scanned by the AFM was determined by measuring at the individual diameters of at least 100 randomly selected particles in that area. A microplate reader (Infinite 200 PRO, Tecan, Switzerland) was used for the MTT assay. Flow cytometry was performed with an EPICS XL-MCL (Beckman Coulter) equipped with an argon laser (488 nm emission). In all experiments, 104 events were collected in each experiment gate. Fluorescence images were observed with laser confocal scanning microscope (CLSM; FluoView FV1000, Olympus Japan). To avoid bleedthrough between the two fluorescent channels, sequential rather than simultaneous acquisition was used. 2.3. Preparation of Tat/HA2 Peptides Conjugated [email protected] AuNRs were prepared by using a seeding growth method as described previously.26 In a typical synthesis of [email protected] core−shell nanoparticles, acrylamide (AAm, 0.2 mmol) was first added to 25.0 mL of as-prepared AuNRs (1.6 × 10−6 μmol) in the deionized water and stirred at 25 °C for 12 h. [email protected] NPs were then collected by centrifugation at 10 000 rpm and redispersed in 5.0 mL of water, followed by the addition of an aqueous solution (15.0 mL) containing AAm (0.2 mmol), N-isopropylacrylamide (NIPAAm, 1.8 mmol), SDS (86.7 μmol), and BIS (13.0 μmol). The mixture was then heated to 75 °C with stirring and maintained under vacuum. After 1 h of equilibration, the

2. MATERIALS AND METHODS 2.1. Materials. N-isopropylacrylamide (NIPAAm) was purchased from TCI (Tokyo, Japan) and purified by recrystallization from n-hexane. Sodium dodecyl sulfate (SDS), acrylamide (AAm), ammonium persulfate (APS), sodium citrate (C6 H 5 Na 3 O 7 ·2H 2 O), N,N-methylenebis(acrylamide) (BIS), ascorbic acid (AA), chloroauric acid (HAuCl4), cetyltrimethylammonium bromide (CTAB), sodium borohydride (NaBH4), silver nitrate (AgNO3), 1,3-diphenyli2445

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mL, and the overall optical density of the solution was kept below 1.5. The absorbance is related to concentration by the Beer−Lambert law. The mixture was irradiated by 680 nm LED light and the combined light (808 nm laser and 680 nm LED light), respectively. The experiment was repeated three times for each sample. DPBF degradation at 415 nm was monitored in deionized water by an UV−vis spectrophotometer, and ΦΔ values for free and loaded AlPcS4 were obtained by the relative method using ZnPc as the reference (eqs 2 and 3):30

polymerization was initiated by the addition of APS (109.6 μmol). The reaction was allowed to proceed for 4 h at 75 °C and terminated by exposing to air. The resulting [email protected] pNIPAAm was then collected by repeated centrifugation at 10 000 rpm for further investigation. The introduction of thiol groups onto [email protected] was achieved by the reaction of SPDP with amino groups on the surface of [email protected], following the manufacturer’s protocol. Immobilization of Tat/HA2 peptides onto [email protected] pNIPAAm was achieved by the reaction of the introduced thiol groups with NpyS-activated C-terminal cysteine of peptides (Tat/HA2) at a ratio of peptides/[email protected] = 1.58 μmol mg−1 and a molar ratio of Tat/HA2 = 1.27 2.4. Loading and Release of AlPcS4. AlPcS4 was synthesized and purified according to the methods described previously.28 The general procedure for the formation of AlPcS4-loaded [email protected] was exemplified as follows. During the emulsion−polymerization, AlPcS4 (2.0 mg) was added to a stirred reaction solution (10.0 mL) containing NIPAAm (1.8 mmol), AAm (0.2 mmol), APS (109.6 μmol), BIS (13.0 μmol), SDS (86.7 μmol), and AuNRs (1.6 × 10−6 μmol). The hydrophilicity of pNIPAAm enables AlPcS4 to be easily dispersed within the matrix. Free AlPcS4 was removed by dialysis against PBS solution (10 mM, pH7.4) for 24 h using a dialysis membrane (MWCO 10 000) in the dark. The obtained AlPcS4-loaded [email protected] ([email protected]) was kept under 4 °C in the dark. NIR-triggered drug release was carried out in PBS (10 mM, pH 7.4) at 37 °C. An optical-fiber coupled power-tunable diode laser with wavelengths at 808 nm (maximal power = 2 W, HiTech Optoelectronics Co., Beijing, China) or a light-emitting diode (LED) device consisting of 32 red LEDs (λ = 680 ± 10 nm, powered by 10 W) were employed, respectively. For PDT experiments, 808 nm laser irradiation was performed with a mean intensity of 400 mW cm−2 for 15 min (360 J/cm2), whereas 680 nm LED light irradiation was carried out with a mean intensity of 10 mW cm−2 for 40 min (24 J/cm2), respectively. For NIR-triggered AlPcS4 release test, 5.0 mL of the purified [email protected] (1 mg mL−1) was loaded into a dialysis tube (MWCO 10 000). The tube was immersed in a transparent vial filled with 4.0 mL of PBS (pH 7.4, 10.0 mM) during release experiments. At desired time intervals, 100 μL of the release media was sampled for analysis. The amount of AlPcS4 released in the supernatant following centrifugation at 15 000 rpm for 10 min was assayed in deionized water by UV− vis absorbance measurement. All measurements were conducted in triplicate. The drug release efficiency was calculated by eq 1: Release efficiency (%) =

Wt × 100 W1

ΦSΔ = ΦΔR

κ SIaRT κ R IaST

Ia = I0(1 − e−2.3A)

(2) (3)

where superscript S and R indicate the sample and reference compound, respectively. κ is the DPBF photo bleaching rate, Ia is defined as the total amount of light absorbed by the sensitizers. A is the corresponding absorbance at irradiation wavelength of 680 nm. ΦΔ for reference compound, ZnPc in DMSO, is 0.67. 2.6. Cellular Uptake and Endo/Lysosome Escape. For studying cellular uptake, human cervical carcinoma cell line (HeLa) (ATCC CCL-2; ATCC, USA) were seeded onto overslips in six-well plates at at a density of 1 × 105 cells per well and incubated for 24 h. After they reached approximately 80% confluence, the cells were then treated with either free AlPcS4 at 0.9 μg mL−1 or [email protected] and Tat/[email protected] at 100 μg mL−1 (normalized to free AlPcS4 concentration) for 12 or 24 h. The fluorescence images of AlPcS4 in cells were captured with a CLSM equipped with a matched pinhole and a 590 nm long-pass filter in a detection channel, and the fluorescence intensity of AlPcS4 in cells was assayed using a flow cytometer. Since the increase in nanoparticles uptake may results in an increase in cellular granularity, light scattering measurements were used to quantify this increase with flow cytometry as described previously.31 To monitor intracellular trafficking, the cells were stained with either Lysotracker Green DND-26 or MitoTracker Green FM according to the supplier’s instructions and then visualized using a CLSM. The colocalization analysis was processed by ImageJ software 1.45f (NIH, Bethesda, MD) on the merged images.21 To assess lysosomal damage by [email protected] conjugated with or without Tat/HA2 peptides, a fluorescence-based assay kit was used to determine the content of the lysosomal cathepsin B activity according to the manufacturer’s instructions. Activities of cathepsin B in samples were measured by fluorescence spectrophotometer (Hitachi 650−60, Tokyo, Japan) with excitation and emission wavelengths set at 400 and 505 nm. Lysosomal stability was also determined using the NR retention method, which is based on the accumulation of the dye in intact lysosomes, according to the manufacturer’s protocol. Lead (50 μM) was included as positive control.32 A reduction in lysosomal stability leads to reduction in NR uptake by cells. 2.7. In Vitro PDT. For dark cytotoxicity assay, HeLa cells and A549 cells (ATCC CCL185; ATCC, USA) at a density of 1 × 105 cells per well were seeded on 96-well plates and allowed to attach overnight, and treated with free AlPcS4 at a concentration range of 0.45−5.4 μg mL−1 or AlPcS4-loaded [email protected] at a concentration range of 50−600 μg mL−1 (normalized to free AlPcS4 at the concentration range of

(1)

where Wt is the cumulative amount of AlPcS4 released from [email protected] at time point t, and W1 is the loading content of AlPcS4, respectively. 2.5. Detection of Singlet Oxygen Quantum Yield. Singlet oxygen quantum yield (ΦΔ) determinations were carried out using DPBF as a chemical quencher.29 Typically, the respective [email protected] solution containing AlPcS4 (5 × 10−6 M) was stored in a 1 cm cuvette and saturated with O2 in the dark. Next, 9× 10−8 mol of DPBF solution was added, and the mixture was stirred at room temperature. The final volume in the cell was maintained at 2 2446

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Molecular Pharmaceutics 0.45−5.4 μg mL−1). Cells treated with medium only served as a negative control group. The cells were incubated in the dark for 24 h, and the cell viability was then measured using the MTT tetrazolium reduction assay.33 The LDH leakage as a biomarker of damage to the plasma membrane and cell death was also determined according to the manufacturer’s instructions. For in vitro phototoxicity experiments, HeLa cells were seeded in 24-well plates and incubated for 24 h in the dark with different concentrations of free AlPcS4 (0.45−1.8 μg mL−1), [email protected] conjugated with or without Tat/HA2 peptides (50−200 μg mL−1). Afterward, HeLa cells were washed three times with fresh PBS and maintained in complete medium during the subsequent irradiation and post-treatment. Following drug incubation, HeLa cells were then irradiated with 808 nm laser at 400 mW cm−2 to a total fluence of 360 J/cm2 (15 min) or 680 nm LED light at 10 mW cm−2 to a total fluence of 24 J/cm2 (40 min). MTT assay was then performed 24 h after PDT. Cell viability was normalized by control group (no nanoparticle) in the dark. Apoptosis/necrosis was assayed by 24 h after PDT using an Annexin V-FITC/PI apoptosis detection kit.34 Data were analyzed by EPICS XL flow cytometer software and plotted for Annexin V-FITC and PI in a two-way dot plot. Live, early apoptotic, and late apoptotic/necrotic cells are designated as Annexin−/PI−, Annexin+/PI−, and Annexin+/PI+, respectively. Caspase activities of HeLa cells were determined 24 h after PDT by fluorometric measurement of the amounts of 7amino-4-trifluoromethyl coumarin (AFC) released from fluorogenic substrates Ac-DEVD-AFC (caspase-3 substrate), Ac-VETD-AMC (caspase-8 substrate), and Ac-LEHD-AFC (caspase-9 substrate) according to the manufacturer’s protocol (Promega, USA). Formation of ROS was monitored 24 h after PDT by the conversion of cell-permeable 2′,7′-dichlorofluorescin diacetate (DCFH-DA) to fluorescent DCFH (dichlorofluorescin), as described.35 DCFH fluorescence of the cells from each well was detected by a flow cytometer at an excitation wavelength of 488 nm. Changes in the mitochondrial membrane potential (ΔΨm) were assayed by the lipophilic cationic probe JC-1.36 JC-1 dye exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (∼529 nm) to red (∼590 nm). In living cells, JC-1 exists as a monomer at low membrane potential (depolarized membrane potential) and yields green fluorescent, and forms an orange-red fluorescent J-aggregate at high membrane potentials (hyperpolarized membrane potentials). The loss of ΔΨm was quantified 24 h after PDT by flow cytometric analysis of the decrease in the fluorescence intensity ratio of 590 nm (red)/527 nm (green). For detection of cellular proteins by Western blot analysis, HeLa cells were incubated with [email protected] conjugated with or without Tat/HA2 peptides for 24 h, and then exposed to 808 nm laser at 400 mW cm−2 for 15 min (360 J/cm2) or 680 nm LED light at 10 mW cm−2 for another 40 min (24 J/cm2). HeLa cells were harvested and lysed immediately after PDT. The cell lysates were passed through a 25-gauge needle several times to shear the DNA and were clarified from cell debris by centrifugation at 4000 rpm. The mitochondria and cytosol fractionations were obtained according to a method described previously.37 The protein concentration was determined by using a Bio-Rad protein assay kit. Cellular proteins with equivalent numbers of cells for each sample were resuspended in 20.0 μL of Laemmli electrophoresis sample buffer (10.0 mM Tris-HCl, pH 6.8, 100.0 mM

dithiothreitol, 0.1% bromophenol blue, 2.0% SDS, 10.0% glycerol), boiled for 5 min, and resolved by SDS-polyacrylamide gel electrophoresis (PAGE) on 12.0% gel. Samples were then transferred to a nitrocellulose membrane (MSI, America) overnight at 50 V in a transfer buffer (25.0 mM Tris, 20.0% methanol, 200.0 mM glycine, 0.02% SDS). The membranes were blocked with 5% skim milk in TBS for 1 h at room temperature, and then incubated with specific antibodies for 2 h at room temperature followed by a antimouse immunoglobulin G antibody or secondary goat antirabbit immunoglobulin G antibody conjugated with horseradish peroxidase (1:2000 dilution) for 1 h at room temperature. The primary antibodies used in the present study include monoclonal anti-Cathepsin B antibody (1.0 μgmL−1), polyclonal anti-Bax antibody (1.0 μg mL−1), polyclonal anti-Bad antibody (1:1000 dilution), a monoclonal anticyto c antibody (1.0 μg mL−1), and a monoclonal anti-β-actin antibody (1:2500). Protein bands were developed using an enhanced chemiluminescence (ECL) system followed by autoradiography with exposure times ranging from 30 s to 5 min. Densitometry data analysis was performed using ImageJ software 1.45f. 2.8. In Vivo PDT. Female athymic mice (7−8 weeks old), weighing 20−25 g, were housed. All the animal experiments and maintenance were approved by the Animal care and use committee of Xiamen University. HeLa cells, cultured in vitro, were harvested and resuspended in serum-free media at a density of 6 × 107 cells mL−1. The animals received one subcutaneous injection of 50 μL of cell suspension (3 × 106 cells) in the front flank region of mice. Tumor volume (V) calculation was obtained using the formula V = 1/2 × d2 × D, where d and D are the short and long diameters of the tumor, respectively. Tumor growth was monitored daily until they reached approximately 50 mm3. For in vivo imaging observation, [email protected] conjugated with or without Tat/HA2 peptides in 0.5 mL physiological saline (at dosage of 1 mg kg−1) was injected into the mouse via the tail vein. The control group was injected with physiological saline alone. In vivo optical images were taken by using a Maestro in vivo imaging system (CRi, Woburn, MA, USA; excitation, 488−505 nm; emission, 530 nm long-pass) after 1 h intravenous administration in mice. To examine the PDT effects mediated by [email protected] pNIPAAm-Pc conjugated with or without Tat/HA2 on HeLa tumors in vivo, PDT was performed on the xenograft tumor models established by implanting HeLa cells subcutaneously. One hour postinjection, the animals were exposed to 808 nm laser at 400 mW cm−2 for 15 min (360 J/cm2) and 680 nm LED light at 10 mW cm−2 for another 40 min (24 J/cm2) by closely positioning the light to the skin right above the tumor. The relative tumor volumes in each treatment were measured daily during the experimental period. The mice were sacrificed at day 12 after PDT, and the primary tumor was surgically excised, fixed, embedded in paraffin wax, sectioned, and analyzed by hematoxylin eosin (H&E) staining. For immunohistochemical (IHC) analysis of Ki-67 expression, we used the labeled-streptavidin−biotin (LAB-SA) method and the Histostain-Plus kits according to the manufacturer’s procedure. TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay was performed using a Dead End Fluorometric TUNEL System (Promega, Madison, WI, USA). The slides were observed under a fluorescent microscope (Leica DMR, Solms, Germany) with relative apoptotic 2447

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Figure 1. (A, B) Typical TEM and AFM images of [email protected]; (C) schematic synthetic routes of preparation of [email protected]; (D) UV−vis spectra of AuNRs, AlPcS4, [email protected], and [email protected]; (E) fluorescence emission spectra of AlPcS4 and [email protected] pNIPAAm-Pc. Deionized water was used as a solvent.

Figure 2. (A) Photothermoresponsive time-dependent release curves of AlPcS4 from pNIPAAm or [email protected] under irradiation with 808 nm laser (400 mW cm−2) and 680 nm LED lamp (10 mW cm−2), respectively. Error bars were based on triplicate samples. (B) Photodecomposition of DPBF by 1O2 after irradiation of the solution of free AlPcS4, [email protected] with 808 nm laser (400 mW cm−2), and 680 nm LED lamp (10 mW cm−2), respectively. Monitoring the maximum absorption of DPBF at 410 nm; error bars were based on triplicate samples.

(Figure 1A) indicated the formation of core (AuNRs)−shell (pNIPAAM) structures and still retained the morphological features of AuNRs. The dimensions of [email protected] were estimated to be 53.4 ± 3.4 nm long and 39.7 ± 3.2 nm wide with a pNIPAAM shell thickness of 10−15 nm from TEM images. However, AFM images shown in Figure 1, panel B demonstrated that the dimensions for [email protected] were estimated to be 154.0 ± 5.2 nm long and 79.7 ± 8.2 nm wide (Figure 1B). It should be noted that the actual sizes of pNIPAAM shell are larger than ones measured by TEM due to thermal dehydration and degradation of the hydrogel shell induced by electron-beam irradiation.38 The mechanism to form [email protected] might involve two steps consisting of the absorption of an AAm monolayer onto AuNR followed by subsequent in situ polymerization and

cells assessed by counting the number of TUNEL-positive cells in five randomly chosen visual fields per sample. 2.9. Statistical Analysis. All data are presented as mean ± SD of at least three experiments. Statistical analysis was performed using the Student’s unpaired t-test or one-way analysis of variance (ANOVA). A p-value of less than 0.05 was considered statistically significant.

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of [email protected] pNIPAAm-Pc. The average aspect ratio (length divided by width) of as-prepared Au NRs is 4.4 ± 0.4, as estimated by measuring more than 100 gold nanoparticles from different transmission electron microscopy (TEM) images. The representative TEM image of the hybrid [email protected] 2448

DOI: 10.1021/acs.molpharmaceut.5b00161 Mol. Pharmaceutics 2015, 12, 2444−2458

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Figure 3. continued

2449

DOI: 10.1021/acs.molpharmaceut.5b00161 Mol. Pharmaceutics 2015, 12, 2444−2458

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Figure 3. (A) CLSM images and (B, C) flow cytometry analysis of cell uptake of free AlS4Pc, [email protected], and Tat/[email protected] pNIPAAm-Pc by utilizing the intrinsic red fluorescence of the AlPcS4. (D, E) Quantitative flow cytometry analysis of cell uptake of [email protected] pNIPAAm-Pc and Tat/[email protected] by using the light SSC parameter. After 12 and 24 h of exposure, the cells were sampled for the SSC assay by flow cytometry. (F, G) CLSM analysis of the cellular uptake and sublocalization of [email protected] and Tat/[email protected] pNIPAAm-Pc (red) with (F) endo/lysosome and (G) mitochondria. (H, I) The endo/lysosome escape capacity and mitochondrial distribution of [email protected] and Tat/[email protected] calculated by the quantitative analysis of CLSM images shown in panels F and G). (J, K) Effects of [email protected] and Tat/[email protected] on lysosomal membrane stability by detecting the expression and activity of cytosolic cathepsin B. ∗, p < 0.05 compared with [email protected] group.

Figure 4. (A) In vitro dark cytotoxicity as determined by the LDH assay 24 h after exposure of the cells to either free AlPcS4 or [email protected] conjugated with or without Tat/HA2. Results are presented as the mean of three measurements ± SD. (B) In vitro photototoxicity as measured by the LDH assay. HeLa cells were incubated for 24 h in the dark with indicated concentrations of free AlPcS4, [email protected] conjugated with or without Tat/HA2 peptides, followed by exposure to 808 nm laser at 400 mW cm−2 for 15 min (360 J/cm2) or 680 nm LED light (10 mW cm−2) for another 40 min (24 J/cm2). LDH assay was then performed 24 h after PDT. Results are presented as the mean of three measurements ± SD ∗, p < 0.05 compared with untreated-control; #, p < 0.05 compared with Tat/[email protected], irradiation with 808 nm laser.

cross-linking of NIPAAm and AAm (Figure 1C). In the first step, AuNR surface was modified with self-assembled AAm monolayer through an amino−gold interaction. In the second step, AAm-protected AuNR served as a template for in situ formation of hydrogel by polymerization and cross-linking of NIPAm and AAm with BIS as cross-linker, APS as initiator, and SDS as emulsifier.39 Figure 1, panel D shows the normalized UV−vis absorption apectra of AuNRs, AlS4Pc, [email protected] pNIPAAm, and [email protected] The extinction spectrum of the obtained AuNRs exhibits two typical resonance bands of the surface plasmons with the transverse one at 516 nm and the longitudinal band at 763 nm. The coating of pNIPAAm on AuNPs can be revealed by UV−vis spectra as shown in Figure 1, panel D, indicating that the plasmon absorption peak is red-shifted from 764 nm for AuNPs to 777 nm for the hybrid [email protected] nanogels. In the case of [email protected], the two peaks at 611 and 678 nm assigned to AlPcS4 were present compared with that of [email protected], indicating successful loading of AlPcS4 in [email protected] (Figure 1D).

AlPcS4 as a second-generation photosensitizer possesses good optical properties for NIR fluorescence imaging and cancer therapy.40 The fluorescence spectra were measured with the excitation at 633 nm and plotted in Figure 1, panel E. The fluorescence emission peak for both free and encapsulated AlPcS4 was observed at ∼690 nm, but the encapsulation by [email protected] caused the blue shift of the maximum emission and a significant reduction of the emission intensity. Namely, the fluorescence of AlPcS4 was markedly quenched once it was loaded in [email protected] Singlet oxygen has been considered as the main mediator of cell death induced in PDT.41 Singlet oxygen generationin DMSO was assayed by measuring the dye-sensitized photooxidation of DPBF, that is, ΦΔ values, which were calculated by plotting the changes in absorbance of DPBF at 410 nm with the irradiation time, were 0.38 and 0.05 for free and loaded AlPcS4, respectively. Since the peak absorption band of AuNRs in the NIR range significantly overlapped with the emission band of free AlPcS4 (Figure 1D), the encapsulation of AlPcS4 into [email protected] may significantly reduce its ΦΔ and 2450

DOI: 10.1021/acs.molpharmaceut.5b00161 Mol. Pharmaceutics 2015, 12, 2444−2458

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Molecular Pharmaceutics

Figure 5. (A) Flow cytometry analysis of PDT-induced apoptosis. HeLa cells were treated with free AlPcS4, [email protected] conjugated with or without Tat/HA2 peptides for 24 h, followed by exposure to 808 nm laser at 400 mW cm−2 for 15 min (360 J/cm2) or 680 nm LED light (10 mW cm−2) for another 40 min (24 J/cm2). Apoptosis was determined 24 h after PDT by flow cytometry analysis of annexin V/PI staining. (B) The activity of caspase-3, -8, and -9 in HeLa cells after PDT. HeLa cells were treated free AlPcS4, [email protected] conjugated with or without Tat/ HA2 peptides for 24 h, followed by exposure to 808 nm laser at 400 mW cm−2 for 15 min (360 J/cm2) or 680 nm LED light (10 mW cm−2) for another 40 min (24 J/cm2). The activity of caspase-3, -8, and -9 was assayed 24 h after PDT using commercial kits. Results are presented as the mean of three measurements ± SD. ∗, p < 0.05 compared with [email protected] group.

Figure 6. Flow cytometric analysis of (A) intracellular ROS generation and (B) disruption of ΔΨm in Hela cells induced by PDT. HeLa cells were treated with [email protected] conjugated with or without Tat/HA2 peptides for 24 h, followed by exposure to 808 nm laser at 400 mW cm−2 for 15 min (360 J/cm2) or 680 nm LED light (10 mW cm−2) for another 40 min (24 J/cm2). Intracellular ROS generation and disruption of ΔΨm were evaluated 24 h after PDT by DCFH-DA and JC-1 assays, respectively.

fluorescence. This is not only due to the energy transfer from the excited AlPcS4 to AuNRs, but also due to the selfquenching effect between the encapsulated PSs. 3.2. NIR-Triggered Release of Photosensitizer. [email protected] pNIPAAm could be used as a drug carrier due to the unique scattering characteristics of AuNRs core and thermal-responsive shell of pNIPAAm under physiological conditions.42 Photothermally triggered AlPcS4 release from [email protected] was tested by irradiation with 808 nm laser at 400 mW/cm2 (approach to the LSPR of AuNRs) or 680 nm LED light at 10 mW/cm2. As shown in Figure 2, panel A, pNIPAAm-Pc

nanogels (without AuNRs) were highly transparent to NIR lights (both of 808 and 680 nm) and exhibited release of little AlPcS4 ( 0.98) (Figure 2B). The 1O2 ΦΔ of free AlPcS4 was 0.51. For AlPcS4 loaded on [email protected], a 1O2 ΦΔ = 0.05 was measured. It is not surprising to note that after exposure to 808 nm laser at 400 mW cm−2 up to 30 min, the ΦΔ of AlPcS4 could be recovered from 0.05 to 0.32. This is correlated with the fact that 74.2 ± 2.6% of the loaded AlPcS4 was released from [email protected] (Figure 2A). It is thus likely to assume that [email protected] might be nonphototoxic while in the circulatory system due to the low 1O2 ΦΔ of AlPcS4. However, this PSs-loaded nanosystem could become highly phototoxic as it undergoes 808 nm NIR 2452

DOI: 10.1021/acs.molpharmaceut.5b00161 Mol. Pharmaceutics 2015, 12, 2444−2458

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Molecular Pharmaceutics

Figure 8. (A) Schematic representation of the intracellular internalization and endo/lysosomal escape of [email protected] conjugated with or without Tat/HA2 peptides. (B) Schematic representation of mitochondria-mediated apoptotic cell death induced by Tat/HA2-conjugated [email protected] pNIPAAm-Pc, followed by irradiation with 680 nm LED light alone and the combined NIR light of 808 and 680 nm, respectively.

colocalization percentage. As indicated in Figure 3, panels G and I, Tat/[email protected] showed a higher colocalization with mitochondria than [email protected] These data are consistent with our proposed model that Tat/ [email protected] might accumulate in lysosomes prior to migrating to mitochondria, though such model needs further verification. To assess lysosomal instability by [email protected] conjugated with or without Tat/HA2 peptides, we used Western blotting analysis to study the containment of the lysosomal enzyme, cathepsin B.45 As shown in Figure 3, panels J and K, [email protected] had little effect on lysosomal membrane stability after 24 h of incubation. Interestingly, cells treated with Tat/[email protected] showed an increased level of expression and activity of cytosolic cathepsin B. Additionally, lysosomal membrane integrity was further measured by NR uptake and accumulation in lysosomes over 24 h following AlPcS4 loaded-nanoparticles treatment. As expected, treatment with Tat/[email protected] resulted in a significant decreased NR uptake compared to [email protected] (Figure S1, Supporting Information), consistent with high endo/lysosomal escape of [email protected] pNIPAAm-Pc mediated by Tat/HA2 peptides observed in Figure 3, panels F−I. 3.4. In Vitro PDT Studies on HeLa Cells. An important requirement for PSs delivery systems is low intrinsic cytotoxicity.46 The mitochondrial function of the HeLa cell was measured by the LDH and MTT assays after incubation of cells with free AlPcS4, or [email protected] conjugated with or without Tat/HA2 peptides for 24 h. As shown in Figure 4, panel A and Figure S2 of the Supporting Information, no

Tat/[email protected] uptake by cells was much higher than that of [email protected] In addition, the different cell uptake efficiency was further confirmed by the flow cytometry analysis using intrinsic fluorescence of AlPcS4 (Figure 3B,C). As shown in Figure 3, panels D and E, Tat/ [email protected] showed more intensity of SSC following treatment compared with [email protected], implying that Tat/HA2 peptides could facilitate the cellular uptake of [email protected] To track the subcellular localization of nanoparticles following their uptake, HeLa cells were stained with LysoTracker Green DND-26, a marker for secondary endo/ lysosomes, and the colocalizations of nanoparticles (red) with lysosome (green) present as yellow fluorescence in the merged fluorescence scans (Figure 3F). The endo/lysosome escape efficiency was further analyzed quantitatively by analyzing the colocalization percentage between nanoparticles and endo/ lysosomes. It was shown that the conjugation of Tat/HA2 peptide onto [email protected] could lead to 3.2-fold higher in endosome escape efficiency than that of nonconjugated ones (Figure 3H), indicating that Tat/HA2 peptides might enhance [email protected] to escape from the endo/lysosomes into the cytoplasm following their uptake. This behavior may be attributed to the ability of HA2 to disrupt the membrane and cause cell lysis at a low pH and insert itself into the endo/lysosomal membrane, resulting in swelling and rupture of endosomes and subsequent release of [email protected] To define whether the release of AlPcS4- loaded [email protected] could target mitochondria, we stained HeLa cells treated with Tat/[email protected] pNIPAAm-Pc with MitoTracker Green FM and calculated 2453

DOI: 10.1021/acs.molpharmaceut.5b00161 Mol. Pharmaceutics 2015, 12, 2444−2458

Article

Molecular Pharmaceutics

Figure 9. (A) In vivo fluorescence imaging of [email protected] conjugated with or without Tat/HA2 peptide in HeLa tumor-bearing mice 1 h after injection. The arrows indicated the tumors. (B) The PDT effects mediated by [email protected] conjugated with or without Tat/HA2 peptide in nude mice-bearing HeLa tumor xenografts. Tumor volumes in the different groups (light only, [email protected], and Tat/[email protected]) as a function of postinjection time. Tumor length and width were measured every 2 days using calipers. Values represent the mean ± SD of eight animals per group. ∗, p < 0.05 compared to the control (irradiation by light only). (C) Tumor images in the different groups (light only, [email protected], and Tat/[email protected]) 12 days after PDT.

combined light of 808 and 680 nm, there was 1.4-, 1.8-, and 2.2-fold further increases in the cell death observed, respectively. The in vitro phototoxic effects toward HeLa cells were also evaluated by MTT assay, and results correlated well with the observations by LDH assay (Figure S3, Supporting Information). In addition, an in vitro phototoxicity assay using A549 cells showed similar findings to the results obtained with HeLa cells (Figure S4, Supporting Information), indicating different tumor cells might share the same response pattern to PDT. These findings might be explained by AuNRs absorbing a SPR wavelength (808 nm) and converting it into heat. The heat then diffuses into the shell and causes shrinkage of the pNIPAAm nanogels and the release of AlPcS4. Upon illumination at 680 nm, the released AlPcS4 may transfer the photon energy to oxygen molecules, stimulating ROS generation to kill cancer cells. 3.5. Induction of Apoptosis by PDT. We expected that the current PDT could influence apoptotic pathways and therefore evaluated the induction of apoptosis in HeLa cells. After PDT, apoptosis was detected by initially staining the cells with Annexin V/PI followed by flow cytometry analysis (Figure 5A). Similar to the cell viability results obtained by the MTT and LDH assay, either free AlPcS4 or [email protected] (200 μg mL−1; normalized to AlPcS4 concentrations at 1.8 μg mL−1) showed no obvious effect on apoptotic cell death in all irradiated groups. In contrast, apoptosis and necrosis for HeLa

significant decrease in cell viability was observed when cells were exposed to either free AlPcS4 or AlPcS4 [email protected] pNIPAAm at normalized AlPcS4 concentrations as high as 2.7 μg mL−1 in the dark. However, as exposed to concentration of 5.4 μg mL−1, [email protected] and Tat/[email protected] pNIPAAm-Pc resulted in a 1.5- and 3.5-fold decrease in cell viability in HeLa cells, respectively, as compared to free AlPcS4. Nevertheless, [email protected] conjugated with or without Tat/HA2 peptides exhibited a quite low cytotoxicity at a concentration of 300 μg mL−1 (normalized to 2.7 μg mL−1 of free AlPcS4), while AuNRs had a much lower level, about 50 μg mL−1.47 Therefore, a concentration less than 200 μg mL−1 was used in the following PDT efficacy studies. For in vitro phototoxicity experiments, it is obvious from Figure 4, panel B that either free AlPcS4 or [email protected] exhibited negligible toxicity (

HA2 Peptides Conjugated [email protected] as a Photosensitizer Carrier for Near Infrared Triggered Photodynamic Therapy.

To achieve an efficiency of intracellular photosensitizers (PSs) delivery and efficacy of photodynamic therapy, we have developed a novel class of PS ...
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