A Multifunctional Theranostic Platform Based on Phthalocyanine

(1, 10) These photosensitizers generally have large conjugated domains resulting .... The final concentration of Cremophor EL in working solutions was...
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A Multifunctional Theranostic Platform Based on PhthalocyanineLoaded Dendrimer for Image-Guided Drug Delivery and Photodynamic Therapy Olena Taratula, Canan Schumann, Michael A. Naleway, Addison J. Pang, Kaitlyn J. Chon, and Oleh Taratula* Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, Oregon 97331, United States S Supporting Information *

ABSTRACT: Owing to the outstanding near-infrared (NIR) optical properties, phthalocyanines (Pc) have promising potential as theranostic agents for fluorescence image-guided drug delivery and noninvasive treatment of deep tumors by photodynamic therapy (PDT). Nevertheless, clinical application of phthalocyanines is substantially limited by poor water solubility, aggregation and insufficient selectivity for cancer cells. To address these issues, we have developed a novel dendrimer-based theranostic platform for tumor-targeted delivery of phthalocyanines. The preparation procedure involved the modification of the Pc molecule with a hydrophobic linker, which significantly enhances physical encapsulation of the hydrophobic drug into a generation 4 polypropylenimine (PPI G4) dendrimer. In order to improve biocompatibility and tumor-targeted delivery, the surface of the resulting Pc−PPIG4 complexes was additionally modified with poly(ethylene glycol) (PEG) and luteinizing hormone-releasing hormone (LHRH) peptide, respectively. The developed nanocarriers have an average diameter of 62.3 nm and narrow size distribution with a polydispersity index of 0.100. The drug encapsulation efficiency was 20% w/w, and the synthesized phthalocyanine derivative entrapped in the dendrimer-based nanocarrier exhibits a distinct NIR absorption (700 nm) and fluorescence emission (710 and 815 nm), required for an efficient PDT and fluorescence imaging. It was demonstrated that subcellular localization in vitro and organ distribution in vivo of the developed nanocarrier can be determined based on the intrinsic fluorescence properties of encapsulated phthalocyanine, validating its role as an imaging agent. The imaging experiments revealed that the LHRH targeted nanocarrier is capable of an efficient internalization into cancer cells as well as tumor accumulation when intravenously administered into mice. Finally, the prepared formulation exhibited low dark cytotoxicity (IC50 = 28 μg/mL) while light irradiation of the cancer cells transfected with the developed theranostic agents resulted in significant PDT effects (IC50 = 0.9 μg/mL) through excessive generation of toxic reactive oxygen species. Thus, the obtained results demonstrated significant potential of the designed dendrimer-based nanocarrier as an efficient NIR theranostic agent. KEYWORDS: photodynamic therapy, phthalocyanines, dendrimer, cancer therapy, drug delivery, theranostics

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INTRODUCTION

sensitizers) which, upon exposure to light of specific wavelength, damage cancer cells by producing reactive oxygen species (ROS).5,6 In addition to the therapeutic effect, the lightinduced excitation of photosensitizers can also result in fluorescence emission, allowing photoactive drugs to act as

Novel modalities for simultaneous therapy and diagnostics, known as theranostics, have gained significant interest for realtime biomedical imaging and site-specific treatment of the various diseases, including cancer.1−3 Photodynamic therapy (PDT), a clinical therapeutic modality, has significant potential to become an efficient theranostic approach for noninvasive treatment of cancer tumors and visualization of malignant tissue by fluorescence imaging.1,2,4 The general procedure for PDT involves administration of nontoxic photoactive drugs (photo© XXXX American Chemical Society

Received: July 9, 2013 Revised: August 20, 2013 Accepted: August 25, 2013

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both therapeutic and imaging agents.2,4,7 Thus, following the accumulation of photosensitizer in a tumor monitored by fluorescence imaging after systemic administration, PDT can be precisely applied onto a detected region by selectively illuminating the cancer tissue with light of an appropriate wavelength, while leaving normal organs untouched.1,2,4 Despite the above-mentioned potential of PDT for combined imaging and treatment, one of the major challenges in clinical application is the limited tissue penetration of visible light used for activation of conventional photoactive drugs.8,9 Therefore, there has been increased interest in the development and application of PDT agents, which can be activated by light in the region of 700−900 nm, known as the “near infrared (NIR) optical window”.1,10 Body tissue is fairly transparent in this spectral window, and thus NIR light can be used for activation of photosensitizers accumulated in deep-seated cancer tumors, without causing phototoxicity to normal tissue. Moreover, fluorescence imaging in the “NIR optical window” holds much promise due to minimal tissue autofluorescence and light scattering.11 Phthalocyanines (Pc) and their derivatives have significant potential as theranostic agents due to strong absorption of the far-red and NIR light.1,10 These photosensitizers generally have large conjugated domains resulting in strong energy absorption and thus high fluorescence quantum yield, needed for efficient PDT and fluorescence imaging. However, due to their planar structures, phthalocyanines have a tendency to aggregate in aqueous medium through π−π stacking and hydrophobic interactions, resulting in the self-quenching effect of their excited state.10 The described behavior of phthalocyanines significantly decreases the photodynamic effect and imaging abilities after their systemic administration. Furthermore, clinical application of phthalocyanines suffers considerably from their poor water solubility and limited selectivity for cancer cells.1,10 Various strategies have been widely explored to enhance water solubility, overcome aggregation and improve limited tumor selectivity of phthalocyanines.10,12 Besides the use of toxic Cremophor EL based emulsions,13 the main conventional approaches are focused on encapsulation of phthalocyanines into micelles,14 liposomes,15 and nanoparticles,16,17 as well as their chemical conjugation with hydrophilic polymers and tumor-targeted moieties.18 Most of these strategies suffer from poor drug loading and increased self-aggregation of the drug in the entrapped state. The application of dendrimers for the delivery of different anticancer drugs, including photosensitizers, has also attracted increasing interest in the past decade.19−23 Dendrimers are particularly well-suited for the delivery of theranostic agents because of their high water solubility and significant encapsulation ability for hydrophobic drugs.19,22,23 The internal cavities of dendrimer structures allow a high drug loading efficiency and prevention of drug selfaggregation.20,24 Moreover, in comparison to nanoparticles and polymers, these macromolecules are characterized by their monodisperse size and well-defined morphology, which lead to consistent batch-to-batch anticancer activity of dendrimerbased drug delivery systems.19,20 Loading of hydrophobic molecules into dendrimers is typically achieved by simple mixing of the polymer and drug solutions during which the hydrophobic agent associates with the nonpolar core of the dendrimer through hydrophobic interactions.20,24 Despite these unique properties, there are no current reports on the development of delivery systems based on encapsulation of

hydrophobic phthalocyanines into nonpolar interior of the dendrimer. As an alternative, a series of dendrimers built around a phthalocyanine core have been chemically synthesized.14,21 Thus, Kataoka et al. prepared phthalocyaninecentered ionic dendrimers capable of forming polyion micelles through electrostatic interactions with oppositely charged block copolymers. 21 The study showed that the developed formulation was effective in inducing efficient rapid cell death in vitro and significantly enhanced in vivo antitumor efficacy. However, one of the disadvantages of such an approach is a long and tedious multistep synthetic process required for preparation and purification of the phthalocyanine-core dendrimer. Moreover, the developed strategy allows embedding of only one Pc molecule per dendrimer. As a result, the low drug loading efficiency significantly increases the number of dendrimers required for the delivery of essential Pc doses to the cancer site, and thereby enhances undesirable side effects typically associated with the inherent toxicity of the carriers.25 It is expected that the development of a simple approach based on noncovalent encapsulation of phthalocyanine into a dendrimer can overcome the above-mentioned drawbacks including low drug loading efficiency. For instance, Kojima et al. demonstrated a possibility for loading up to 20 molecules of hydrophobic photosensitizer, protoporphyrin IX, per PEGylated-polypropylenimine (PPI) dendrimer.24 The work presented here addresses the development of an effective approach for the preparation of a novel theranostic nanocarrier comprising a silicon-based phthalocyanine and amine terminated PPI dendrimer. It is of note that this is the first example of encapsulation of hydrophobic phthalocyanine molecules by PPI dendrimers providing a novel base for the development of effective drug delivery systems. The specific structure of PPI dendrimer, having a number of hydrophobic pockets, offers a possibility to encapsulate and separate the Pc molecules and thus decrease their aggregation and enhance water solubility.24,26,27 The development of the phthalocyaninebased theranostic agent (Pc−LHRH) was achieved by the initial encapsulation of monosubstituted phthalocyanine (PcSi(OH)(mob)) into PPI dendrimer followed by the modification of the dendrimer surface with polyethylene glycol (PEG) and luteinizing hormone-releasing hormone (LHRH) peptide to improve its biocompatibility and tumor cell selectivity. Without the need to release the drug from the carrier the prepared formulation can act as a theranostic agent, generating effective fluorescence emission and ROS upon laser activation to detect and injure tumor cells. Thus, the designed nanocarrier is capable of preventing the leakage of Pc into the blood circulation without compromising the efficacy of PDT. The preparation and characterization of Pc−LHRH as well as its in vitro photodynamic activities and in vivo imaging capabilities are reported herein.

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MATERIALS AND METHODS Materials. Silicon-based phthalocyanine (PcSi(OH)2) and methyl 4-chloro-4-oxobutyrate (mob) were purchased from Sigma-Aldrich. α-Maleimide-ω-N-hydroxysuccinimide ester poly(ethylene glycol) (MAL-PEG-NHS, 5000 Da) was obtained from NOF Corporation (White Plains, NY). A synthetic analogue of LHRH, Lys6-des-Gly10-Pro9-ethylamide (Gln-His-Trp-Ser-Tyr-DLys(DCys)-Leu-Arg-Pro-NH-Et) peptide, was synthesized by Amersham Peptide Co. (Sunnyvale, CA). Trinitrobenzenesulfonic acid (TNBSA) and bicinchoninic acid (BCA) protein assay kit were obtained from Pierce B

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Figure 1. (A) Synthesis of mono- (PcSi(OH)(mob) 2) and disubstituted (PcSi(mob)2 3) derivatives of the silicon phthalocyanine (PcSi(OH)2 1). (B) Schematic representation of tumor targeted theranostic platform based on phthalocyanine-loaded dendrimer (Pc−LHRH).

m, 4H). 13C NMR (CDCl3) δ(ppm): 171.3, 165.3, 155.3, 149.9, 135.7, 133.3, 129.3, 123.6, 120.1, 50.6, 36.2, 31.9, 27.3, 22.6. HRMS (m/z): [M]+ calculated for C58H62N8O8Si, 1026.4460; found, 1026.4425. Pc Loading into PPI Dendrimer. Pc encapsulation was achieved by simply mixing 2 mM solution of PPI G4 in methanol (1 mL) with 11 mM solution of an appropriate phthalocyanine (PcSi(OH)2 1, PcSi(OH)(mob) 2, or PcSi(mob)2 3) in THF (1.5 mL) for 24 h. After overnight stirring at room temperature, organic solvents were evaporated and resulting solids were dissolved in water and purified using size exclusion chromatography (a 1 cm × 18 cm Sephadex G50 column). The dendrimer-based complexes of PcSi(OH)2 1, PcSi(OH)(mob) 2, and PcSi(mob)2 3 will be further mentioned as PcSi(OH)2−PPIG4, PcSi(OH)(mob)−PPIG4, and PcSi(mob)2−PPIG4, respectively. To determine drug loading efficiency for each individual phthalocyanine, the obtained solutions were freeze-dried to remove water, followed by weighing of the obtained pellets and redissolving in THF. The amount of Pc encapsulated into the PPI G4 was quantified based on UV−visible absorption spectra of Pc−PPIG4 samples in THF, with prominent Q-bands appearing at 672 nm, 692 nm and 679 nm for PcSi(OH)2−PPIG4, PcSi(OH)(mob)−PPIG4, and PcSi(mob)2−PPIG4, respectively (UV-1800 spectrophotometer, Shimadzu, Carlsbad, CA). The standard curves were generated by measuring drug absorption intensity at the abovementioned wavelengths in the standard samples containing different concentrations of Pc. Drug loading efficiency is expressed as the percentage of Pc weight encapsulated into PPI G4 over the weight of the Pc−PPIG4 complexes. Modification of PcSi(OH)(mob)−PPIG4 with PEG and LHRH. The previously published procedure was employed to modify the PcSi(OH)(mob)−PPIG4 complex with PEG and

(Rockford, IL). Polypropylenimine dendrimer generation 4 (PPI G4) was obtained from SyMO-Chem (Eindhoven, The Netherlands). All other chemicals were of analytical grade and were purchased from VWR International, Sigma-Aldrich and Fisher Scientific Inc. as indicated in the Supporting Information. Preparation and Characterization of the Theranostic Platform. Synthesis of Mono- (PcSi(OH)(mob)) and Disubstituted (PcSi(mob)2) Derivatives of PcSi(OH)2. Methyl 4chloro-4-oxobutyrate (0.03 mL, 0.251 mmol, 2 equiv) was added to PcSi(OH)2 (100.0 mg, 0.125 mmol, 1 equiv) and DMAP (75.5 mg, 0.618 mmol) dissolved in the mixture of solvents 2-picoline (10 mL) and CH2Cl2 (5 mL) (Figure 1A). The resulting suspension was stirred for 24 h at 40 °C. The reaction mixture was cooled to room temperature followed by standard workup procedure using dichloromethane for extraction. The mono- (PcSi(OH)(mob) 2) and disubstituted (PcSi(mob)2 3) derivatives of PcSi(OH)2 1 were purified and separat ed by silica gel column ch rom at og raphy (MeOH:CH2Cl2, 0.5:99.5, v/v) to yield 35.1 mg (0.038 mmol, yield: 61.6%) of 2 and 19.2 mg (0.019 mmol, yield: 30%) of 3 as dark aqua green solids. Typically 5−10% of starting material, PcSi(OH)2 1, is recovered from the chromatography column. PcSi(OH)(mob) 2: TLC (silica gel, MeOH:CH2Cl2, 1:99, v/v), Rf(2) = 0.79. 1H NMR (CDCl3) δ(ppm): 9.63−9.73 (m, 8H), 8.46 (m, 4H), 3.37 (s, 3H), 1.83 (s, 36H), 0.33 (m, 2H), −0.37 (m, 2H). 13C NMR (CDCl3) δ(ppm): 173.1, 165.3, 155.5, 150.1, 135.5, 133.1, 129.5, 123.6, 120.1, 51.7, 37.0, 31.9, 27.7, 24.9. HRMS (m/z): [M − OH]+ calculated for C53H55N8O4Si, 895.4110; found, 895.4151. PcSi(mob)2 3: TLC (silica gel, MeOH:CH2Cl2, 1:99, v/v), Rf(3) = 0.87. 1H NMR (CDCl3) δ(ppm): 9.57−9.73 (m, 8H), 8.44 (m, 4H), 2.85 (s, 6H), 1.83 (s, 36H), 0.31 (m, 4H), −0.37 C

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LHRH.28,29 Briefly, the NHS groups on the distal end of heterobifunctional 5 kDa PEG polymer (MAL-PEG-NHS) was reacted with primary amines on the surfaces of PPI G4 dendrimer in 50 mM PBS buffer (pH 7.4), at primary amines to PEG molar ratio of 1:5 (Figure 1B). The reaction was carried out for 1 h at room temperature under shaking, following the addition of LHRH peptide and stirring overnight. The molar ratio of MAL-PEG-NHS to LHRH peptide in the reaction mixture was 1:1. The peptide was covalently conjugated to the distal end of PEG layer through the maleimide groups on the PEG and the thiol groups in LHRH (Figure 1B). After 12 h, the modified complexes were purified by using size exclusion chromatography (a 1 cm × 18 cm Sephadex G75 column). The concentration of amino groups available on the PPI G4 surface for PEGylation as well as the decrease in their concentration after the surface modification was determined by a modified spectrophotometric TNBSA assay as previously described.29 The presence of LHRH peptide on the surface of final complex Pc-(OH)(mob)-PPIG4-PEG-LHRH (Pc−LHRH) was performed using the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL).28,29 Size and Zeta Potential Measurements. The hydrodynamic size, polydispersity index (PDI) and zeta potential of the prepared complexes were measured by Malvern ZetaSizer NanoSeries (Malvern Instruments, U.K.) according to the manufacturer’s instructions. Samples were diluted with 50 mM PBS buffer (pH 7.4) to yield a final Pc concentration of 1.0 μg/ mL. The intensity of the He−Ne laser (633 nm) was measured at an angle of 173°. All measurements were performed at 25 °C after pre-equilibration for 2 min, and each parameter was measured in triplicate. PcSi(OH)2-Cremophor EL formulation. Stock solution of PcSi(OH)2-Cremophor EL formulation was prepared by dissolving 5 mg of PcSi(OH)2 in 10 mL of the 1% v/v of Cremophor EL aqueous solution via sonication until the drug was completely dissolved. Next, the drug suspension was diluted with cell culture medium up to the desired concentration followed by filtration through a 0.45 μm filter. Final Pc concentration in Cremophor EL emulsion was estimated upon dilution in THF by measuring the absorbance at 672 nm. The final concentration of Cremophor EL in working solutions was ∼0.1%. Dye Loading. Loading capacity of phthalocyanine (Pc) in the PPI G4 dendrimer and PPIG4-PEG-LHRH complex was calculated as follows:

content in the delivery system at different time points was quantified based on UV−visible absorption spectra of samples in PBS buffer, with a prominent Pc peak appearing around 700 nm (UV-1800 spectrophotometer, Shimadzu, Carlsbad, CA). Following the same procedure, drug release was also studied in 50% human plasma in PBS for Pc−LHRH. The percentage of drug release at different time points was calculated as follows: drug release (%) = [Pc]R /[Pc]T × 100

where [Pc]R is the amount of Pc released at collection time and [Pc]T is the total amount of Pc that was encapsulated in the delivery system. In Vitro Study. Cell Lines. The LHRH receptor positive A2780/AD multidrug resistant human ovarian carcinoma and the LHRH receptor negative SKOV-3 human ovarian carcinoma cell lines were obtained from T. C. Hamilton (Fox Chase Cancer Center, Philadelphia, PA) and ATCC (Manassas, VA), respectively. Previous reports demonstrated that A2780/ AD cell line overexpresses LHRH receptors at the highest levels in comparison to other well established cancer cell lines such as MCF-7 breast and PC-3 prostate cancer cell lines.30 Therefore, A2780/AD allows us to experimentally assess two important features of the developed delivery system simultaneously: (1) receptor targeted delivery and (2) significant efficacy against drug resistant cancer cells. In addition, control SKOV-3 cell line (LHRH-negative) was employed in the study to demonstrate the important role of LHRH peptide as a targeting agent. Cells were cultured in RPMI 1640 medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (VWR, Visalia, CA) and 1.2 mL/100 mL penicillin−streptomycin (Sigma, St. Louis, MO). Cells were grown at 37 °C in a humidified atmosphere of 5% CO2 (v/v) in air. All experiments were performed on cells in the exponential growth phase and between passages 2 and 6. Cytotoxicity Study. The dark cellular cytotoxicity of all studied formulations was assessed under subdued lighting using a modified Calcein AM cell viability assay (Fisher Scientific Inc.). Cancer cells were seeded into 96-well microtiter plates at a density of 10 × 103 cells/well and allowed to grow for 24 h at 37 °C. Then the culture medium was discarded, and the cells were treated for 24 h with 100 μL of medium containing different concentrations (1, 1.7, 3.5, 7.0, 14.0, and 28.0 μg/mL) of Pc−LHRH, PcSi(OH)(mob)−PPIG4, and PcSi(OH)2 in Cremophor EL. A2780/AD cells only in fresh medium were used as a control. After treatment, the cells were rinsed with Dulbecco’s phosphate-buffered saline (DPBS) buffer and incubated for 1 h with 200 μL of freshly prepared Calcein AM solution (10 μM in DPBS buffer) in the dark. Fluorescence was measured using a multiwell plate reader (Synergy HT, BioTek Instruments, Winooski, VT) with 485 nm excitation and 528 nm emission filters. On the basis of these measurements, cellular viability was calculated for each concentration of the formulation tested. The relative cell viability (%) was expressed as a percentage relative to the untreated control cells. The 50% inhibitory concentration (IC50) was determined as the drug concentration that resulted in a 50% reduction in cell viability. Confocal Microscopy. Prior to the visualization, A2780/AD cells were plated in a 35 mm cell culture treated Petri dish (MatTek Corporation, Ashland, MA) at a density of 100 × 103 cells/well and cultured for 24 h. The medium was then replaced by a suspension of Pc−LHRH in culture medium at a phthalocyanine concentration of 5 μg/mL and incubated for 12 h. Before imaging, the Pc-containing medium was removed

LC (%) = (W1/W2) × 100

where LC (%) is the loading capacity, W1 is the weight of Pc measured in lyophilized powder, and W2 is the weight of drugfree PPIG4 or PPIG4-PEG-LHRH complex in the lyophilized powder. Drug Release. The drug release profile of PcSi(OH)(mob) from the final complex Pc−LHRH was evaluated in PBS at pH 7.4 and pH 5.5 containing 10 mM of reduced glutathione. The drug loaded delivery system was dissolved in PBS buffer and placed in a Float-A-Lyzer dialysis tubes (molecular weight cutoff of 50 kDa). The dialysis tubes were immersed in 15 mL of the appropriate medium and incubated at a constant temperature of 37 °C. At fixed time intervals, 200 μL of the samples were withdrawn from the dialysis tubes to record the absorbance of Pc at 700 nm as described above. After each absorption measurement, the samples were returned to the appropriate dialysis tubes for further incubation. The Pc D

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were used: (1) Pc−LHRH without light treatment (maintained in dark), (2) cells only in fresh medium, (3) cell only treated with light, and (4) medium only (background control). At the end of each treatment, all samples were returned to the cell culture incubator for a further 24 h post PDT, and then assayed for viability using Calcein AM as described above. Finally, the PDT effect of Pc−LHRH complex at a concentration of 3.5 μg/mL and the corresponding dark toxicity were further verified by Live/Dead Cell Viability Assay Kit (Life Technologies) according to the manufacturer’s protocol. After treatment, the cells were washed with DPBS and then incubated with 100 μL of Live/Dead staining solution containing Calcein AM (green fluorescence) and ethidium homodimer-1 labeled nucleic acids (red fluorescence) for 30 min at room temperature. After incubation, the appropriate images were taken using a fluorescence microscope (Leica Microsystems Inc., Buffalo Grove, IL). ROS Measurements. Intracellular ROS levels were determined by using the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) assay as previously described.33 Briefly, A2780/ AD cells were seeded in 96-well microtiter plates at a density of 10 × 103 cells/well followed by incubation with medium containing different concentrations (3.5, 1.7, and 1.0 μg/mL) of Pc−LHRH for 24 h. After incubation, the cells were rinsed with warm DPBS and 100 μL of 10 μM DCFH-DA was added under subdued light conditions. The test samples were exposed to 120 mW/cm2 laser diode light of 670 nm for 5 min (36 J/cm2). Pc−LHRH of the same concentrations (3.5, 1.7, and 1.0 μg/ mL) without light treatment was used as a dark control in addition to the cell only control. Fluorescence was measured using a multiwell plate reader with 485 nm excitation and 528 nm emission filters. In Vivo Study. In Vivo Image-Guided Drug Delivery. An animal model of human ovarian carcinoma xenografts was established as previously described.34 Briefly, A2780/AD multidrug resistant human ovarian carcinoma cells (5 × 106) were subcutaneously injected into the flanks of female athymic nu/nu mice. A group of seven mice have been used for this study. When the tumors reached a size of about 100 mm3 (15− 20 days after transplantation), the mice were treated intravenously with tumor-targeted Pc−LHRH complexes. The fluorescent images were recorded using a Li-Cor Pearl Animal Imaging System at 10 h after iv injection. Afterward, the organs of injected mice were harvested for ex vivo imaging to visualize tissue distribution of the developed complexes. The distribution of fluorescence in different organs was analyzed using the manufacturer’s software. Statistical Analysis. Data were analyzed using descriptive statistics, single-factor analysis of variance (ANOVA), and presented as mean values ± standard deviation (SD) from three to eight independent measurements. The comparison among groups was performed by the independent sample Student’s ttest. The differences were considered significant at a level of P < 0.05.

and the cells were rinsed with warm DPBS before fresh medium was added. To visualize the subcellular distribution, nuclei of A2780/AD cells were stained with either 4′,6diamidino-2-phenylindole (DAPI) or MitoTracker Green FM for 30 min at 37 °C as previously described.24,31 Cellular internalization was analyzed by a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss Inc., Germany). Flow Cytometry. Cellular internalization efficiency of the LHRH-targeted (Pc−LHRH) and nontargeted (Pc−PEG) nanocarriers was quantitatively assessed using flow cytometry analysis (BD Accuri C6 Flow Cytometer, Inc., Ann Arbor, MI). A2780/AD and SKOV-3 cells were treated with Pc−PEG and Pc−LHRH for 24 h at a drug concentration of 9 μg/mL. Untreated cells served as a negative control for background fluorescence. After treatment, cells were washed three times with DPBS, trypsinized and collected for flow cytometry analysis. Hemolysis Assay. Hemolysis experiments were performed according to previous reports.32 EDTA stabilized human blood samples were freshly obtained from Innovative Research (Novi, MI). First, 3 mL of blood was centrifuged at 1600 rpm for 5 min, and blood plasma and the surface layer were removed. The remaining red blood cell (RBC) pellet was washed five times with 6 mL of PBS solution, and RBCs were diluted in 25 mL of PBS solution. Then, 0.8 mL of Pc−LHRH solutions in PBS at different concentrations was added to 0.2 mL of RBC suspension. Also, positive and negative control samples were prepared by adding 0.8 mL of water and PBS, respectively to 0.2 mL of RBC solution. Then, the samples were incubated at room temperature for 4 h. The samples were slightly shaken once every 30 min. After 4 h, the samples were centrifuged at 1600 rpm for 3 min and 100 μL of the supernatant was transferred to a 96-well plate. Absorbance of hemoglobin in supernatants was measured with a microplate reader at 540 nm. Hemolysis percentages of the RBCs were calculated using the following equation: % hemolysis = (Abs − Abs0)/(Abs100 − Abs0) × 100

Where Abs is the absorbance of the sample, Abs0 is the absorbance of the negative control, and Abs100 is the absorbance of the positive control. Percent hemolysis values were calculated from three separate trials. Student’s t test was applied to all three data sets, and the difference between them was accepted to be statistically significant when p < 0.05. PDT Treatment. All manipulations prior to PDT were carried out under subdued lighting. A2780/AD cancer cells were seeded into 96-well microtiter plates at a density of 10 × 103 cells/well and allowed to grow for 24 h at 37 °C. Then cells were treated in the dark for 12 h with 100 μL of medium containing different concentrations (7, 3.5, 1.7, 1.0, and 0.6 μg/ mL) of Pc−LHRH. The drug-containing medium was then removed and the cells were rinsed with warm DPBS before fresh medium was added. Test samples were immediately exposed to 120 mW/cm2 laser diode (QPhotonics, LLC, Ann Arbor, MI) light of 670 nm for 5 min (36 J/cm2). Furthermore, to evaluate the influence of laser irradiation time on phototherapeutic effect of the developed nanocarrier, A2780/ AD cancer cells were treated with Pc−LHRH at a concentration of 0.6 μg/mL and irradiated with laser diode light for 5, 10, and 15 min, respectively. In addition, the efficacy of Pc−LHRH at a concentration of 1 μg/mL and exposure time of 5 min was compared to that of PcSi(OH)2 in Cremophor EL under the same experimental conditions. The next controls

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RESULTS AND DISCUSSION Synthesis and Characterization of Phthalocyanine Derivatives. Owing to their molecular structure, the majority of unsubstituted phthalocyanines, including PcSi(OH)2, are insoluble in water, and their partial solubility in most organic solvents makes it difficult to obtain Pc-encapsulated dendrimers with an adequate drug loading efficiency. The low solubility in common organic solvents can be overcome by the introduction E

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All three compounds resulted in strong absorption spectra of nonaggregated phthalocyanines, showing characteristic Qbands at 672−692 nm, together with two vibronic bands 610−620 nm and 640−660 nm.37 Importantly, the additional axial mob linkers in 2 and 3 induced a red shift of characteristic Q-band moving it closer to the NIR region desired for an efficient PDT and fluorescence imaging. The most pronounced effect is observed for monosubstituted Pc 2 where the Q-band is red-shifted (692 nm) by 20 nm compared to the Q-band of parent PcSi(OH)2 1 (672 nm) (Figure 2A). Monochromatic irradiation of the phthalocyanine compounds in THF solution at room temperature resulted in the observation of a strong emission band at 677−697 nm (Figure S1 in the Supporting Information). The fluorescence emission band for 2 and 3 was shifted by ∼20 nm toward the red region of the spectrum in comparison to the parent PcSi(OH)2 1 (Figure S1 in the Supporting Information), which is in good agreement with the absorption data (Figure 2A). The fluorescence quantum yields for PcSi(OH)(mob) 2 and PcSi(mob)2 3 (ΦF = 0.52 and 0.48, respectively) were comparable with that of the unsubstituted PcSi(OH)2 1 (ΦF = 0.50) in THF. Thus, the detected photophysical properties make the modified phthalocyanines good candidates for PDT and NIR in vivo imaging. Preparation and Characterization of Tumor Targeted Theranostic Nanocarrier. The cosolvent evaporation method was developed for an efficient encapsulation of phthalocyanine derivatives within hydrophobic interior of amine terminated PPI dendrimers. Due to the fact that drug encapsulation ability, as well as the cytotoxicity of dendrimers, increased with increasing dendrimer generation,25,40 PPI G4 was chosen as an optimal compromise between the drug loading efficiency and cellular toxicity compared to other generations of PPI dendrimers. In order to select the appropriate solvent mixture for the drug encapsulation process, we took advantage of the fact that amine terminated PPI G4 is highly soluble in both water and methanol.25,29 On the other hand, modification of PcSi(OH)2 molecules with mob linkers significantly improved their solubility in THF. To achieve encapsulation of PcSi(OH)2 1, PcSi(OH)(mob) 2 and PcSi(mob)2 3 into PPI G4 molecules, methanol solution of PPI G4 dendrimer was mixed with each individual phthalocyanine dissolved in THF at a 1/10 molar ratio. After overnight stirring at room temperature, organic solvents were evaporated and the resulting complexes were easily dissolved in water and purified by size exclusion chromatography. The highest drug loading efficiency of 20% w/w was detected for the monosubstituted derivative (PcSi(OH)(mob) 2) in PPI G4 whereas these values were 0.5 and 10% w/w for unmodified (PcSi(OH)2 1) and disubstituted (PcSi(mob)2 3) phthalocyanines, respectively. According to the calculations based on the detected concentrations of the dendrimer and phthalocyanines in the final complex, it was determined that one PPI G4 molecule encapsulated approximately 4 and 2 molecules of PcSi(OH)(mob) 2 and PcSi(mob)2 3, respectively. Taking into consideration the highest drug encapsulation efficiency (20% w/w) and the most red-shifted NIR absorption (692 nm) of monosubstituted phthalocyanine, PcSi(OH)(mob) 2 was selected for preparation of the final tumor-targeted theranostic nanocarrier (Figure 1B). The size and surface charge are the two major physiochemical parameters in the development of nanosized drug delivery systems. Excessive positive surface charge promotes nanocarrier aggregation in the bloodstream due to

of appropriate substituents to the Pc structure, which prevent their strong intermolecular interactions.35 The presence of two axial hydroxyl groups around the silicon center in the PcSi(OH)2 structure provides an opportunity to tune chemical and photophysical properties of commercially available PcSi(OH)2 by varying the nature and number of linkers conjugated to the drug molecules.36,37 In order to achieve sufficient encapsulation of photosensitizers into PPI dendrimers, commercially available PcSi(OH)2 1, poorly soluble in both aqueous and organic solutions, was modified with the 4methoxy-4-oxobutanoic acid (mob) linkers to produce monoand disubstituted derivatives PcSi(OH)(mob) 2 and PcSi(mob)2 3 (Figure 1A). In accordance with our expectations, the introduced mob linkers significantly increased solubility of PcSi(OH)2 in organic solvents such as THF, simultaneously causing steric hindrance to suppress drug aggregation, which usually leads to the formation of nonfluorescent dimers of Pc.38,39 The mono- and disubstituted derivatives PcSi(OH)(mob) 2 and PcSi(mob) 2 3 were synthesized from commercially available PcSi(OH)2 1 by reaction with 2 equiv of mob in the presence of DMAP/2-picoline/CH2Cl2 (Figure 1A). The reaction proceeded relatively slowly and after stirring for 24 h at 40 °C resulted in the mixture of PcSi(OH)(mob) and PcSi(mob)2 with ∼60 and 30% yields, respectively. PcSi(OH)(mob) and PcSi(mob)2 were separated and purified by column chromatography, and their structures were confirmed by 1H and 13C NMR and HRMS analyses (Supporting Information). For effective PDT treatment and fluorescence imaging of deep-seated tumors, phthalocyanines should exhibit strong absorption in the NIR wavelength range, where light has its maximum depth of penetration in tissue.1,10 In order to evaluate the influence of chemically conjugated mob linkers on the optical properties of the parent PcSi(OH)2, the absorption spectra of 1−3 were recorded in THF solutions (Figure 2A).

Figure 2. (A) Absorption spectra of PcSi(OH)2 1 (black), PcSi(OH)(mob) 2 (red), and PcSi(mob)2 3 (blue) in THF solution. (B) Size distribution of LHRH targeted theranostic nanocarriers (Pc− LHRH) measured by dynamic light scattering. F

dx.doi.org/10.1021/mp400397t | Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

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overnight conjugation step with PEG and LHRH, but is instead due to the method of calculation of % w/w drug loading. Since drug loading capacity is expressed as the percentage of Pc weight encapsulated into the delivery system over the weight of the delivery system (PPI G4 or PPIG4-PEG-LHRH), the observed change corresponds to the increase in weight of the modified delivery system (Pc-PPIG4-PEG-LHRH) in comparison to the nonmodified PPI G4 dendrimer delivery system. In general, drug delivery systems that are 10−100 nm in size are considered to be optimal for systemic delivery whereas particles >100 nm and 10 nm) and recognition by macrophage cells (