Enhanced Photodynamic Therapy and Effective Elimination of Cancer

Jul 25, 2014 - Photodynamic therapy is a potentially curative treatment for various types .... Nanomaterials in Targeting Cancer Stem Cells for Cancer...
0 downloads 0 Views 446KB Size
Download PDF
Article pubs.acs.org/molecularpharmaceutics

Enhanced Photodynamic Therapy and Effective Elimination of Cancer Stem Cells Using Surfactant−Polymer Nanoparticles Marina Usacheva,†,§ Suresh Kumar Swaminathan,†,§ Ameya R. Kirtane,† and Jayanth Panyam*,†,‡ †

Department of Pharmaceutics, ‡Masonic Cancer Center, University of Minnesota, 308 Harvard Street SE, Minneapolis, Minnesota 55455, United States ABSTRACT: Photodynamic therapy is a potentially curative treatment for various types of cancer. It involves energy transfer from an excited photosensitizer to surrounding oxygen molecules to produce cytotoxic singlet oxygen species, a process termed as type II reaction. The efficiency of photodynamic therapy is greatly reduced because of the reduced levels of oxygen, often found in tumor microenvironments that also house cancer stem cells, a subpopulation of tumor cells that are characterized by enhanced tumorigenicity and resistance to conventional therapies. We show here that encapsulation of a photosensitizer, methylene blue, in alginate−Aerosol OT nanoparticles leads to an increased production of reactive oxygen species (ROS) under both normoxic and hypoxic conditions. ROS generation was found to depend on the interaction of the cationic photosensitizer with the anionic alginate polymer. Dye−polymer interaction was characterized by formation of methylene blue dimers, potentially enabling electron transfer and a type I photochemical reaction that is less sensitive to environmental oxygen concentration. We also find that nanoparticle encapsulated methylene blue has the capacity to eliminate cancer stem cells under hypoxic conditions, an important goal of current cancer therapy. KEYWORDS: photodynamic therapy, methylene blue, type I reaction, cancer stem cells, nanoparticles

■

INTRODUCTION Photodynamic therapy (PDT) involves light-induced activation of a photosensitizer to generate reactive species that can cause cellular damage.1 Upon absorption of light, the photosensitizer is activated from its ground state to a transient singlet excited state, which can then undergo intersystem crossing to convert to a long-lived triplet state. In the triplet state, the photosensitizer can transfer electrons to surrounding substrate molecules (such as solvent, biomolecules, or oxygen) via type I reaction or transfer energy to oxygen molecules via type II reaction. This leads to the generation of a suite of cytotoxic reactive species.1−3 Consequently, PDT has the potential to be a curative, noninvasive treatment modality for many solid tumors including inoperable obstructing esophageal cancer, microinvasive endobronchial nonsmall cell lung cancer, head and neck cancer, cervical cancer, and precancerous lesions in patients with Barrett esophagus.4,5 Although both type I and II reactions occur simultaneously, most photosensitizers elicit their effects through the type II reaction.6 Because type II reaction involves energy transfer to oxygen molecules, the presence of molecular oxygen in the surrounding microenvironment is critical to the success of PDT. Paradoxically, tumors are inherently hypoxic, owing to poor vascular architecture and the presence of solid stress on the tumor blood vessels.7−9 Additionally, PDT leads to the destruction of tumor blood vessels and rapidly exhausts the limited levels of oxygen within the tumor.10,11 Hence, PDT is often considered “self-limiting”.12 This leads to the selective © XXXX American Chemical Society

survival and enrichment of drug-resistant cancer stem cells (CSCs) that are typically harbored in the hypoxic areas.13 The resultant tumor is typically more aggressive and difficult to treat.14,15 Hence, a major limitation of PDT is the loss of efficacy at low concentrations of oxygen. Type I reactions can generate radical species by transferring an electron or H atom to molecules other than oxygen. As type I reactions are less sensitive to local oxygen concentrations, altering the mechanism of radical generation from type II to type I can potentially overcome an important limitation of PDT.2 Previous studies have shown that a closed environment that ensures proximity of photosensitizer molecules, electrostatic interaction between the photosensitizer and the substrate, and reduced oxygen concentration can promote electron transfer and hence type I chemistry.16 To create these conditions and promote type I chemistry, we encapsulated a widely used photosensitizer, methylene blue (MB), in a polymer−surfactant nanoparticle (NP) system, composed of sodium alginate and docusate sodium (Aerosol OT; AOT) (Figure 1A).17−19 We hypothesized that encapsulation of MB in the NP interior will allow electrostatic interaction of the cationic MB molecules with the anionic polymer and surfactant molecules, facilitating charge transfer and the formation of free Received: May 15, 2014 Revised: July 10, 2014 Accepted: July 25, 2014

A

dx.doi.org/10.1021/mp5003619 | Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

Article

radicals.20 Additionally, we hypothesized that encapsulation in NPs will result in high local concentrations of MB molecules and thereby promote the formation of dye dimers in ground and excited states (Figure 1B). Through a series of mechanistic studies, we determined the effect of nanoencapsulating MB on formation of electron transfer complexes within the NP matrix and generation of reactive oxygen species (ROS) under normoxic as well as hypoxic environments. In addition, using soft agar colony formation and mammosphere assays, we determined the ability of MB NP-mediated PDT to eliminate CSCs under hypoxic conditions. Our results show that encapsulation of MB in NPs enables improved ROS generation over a wide range of physiologically relevant oxygen concentrations and successful elimination of CSCs even under hypoxic conditions.

To determine MB loading, NPs were dispersed in methanol (1 mg/mL). MB was extracted from NPs in dark for 1 h, and MB concentration in the extract was determined by measuring its absorbance at 652 nm using a Cary-100 UV−vis spectrophotometer. ROS Generation Ex Vitro. ROS measurements were performed as described previously.21 A mixture of MB (8 μM; free or encapsulated in NPs) and APF (10 μM) in phosphate buffered saline (0.15 mM, pH 7.4) was exposed to light (2700 mJ/cm2; 665 nm) under either normoxia or hypoxia. For normoxic irradiation, MB was incubated with APF under ambient conditions. For hypoxic irradiation, MB was incubated with APF in glass tubes sealed with hermetic rubber stoppers. The tubes were then purged with a mixture containing oxygen-free nitrogen and 5% CO2 for different time intervals using a dispenser at a flow rate of 6 L/min. At this point, the oxygen content in the suspension medium was evaluated using the Winkler method.22 APF is converted to fluorescein in the presence of ROS. Formation of fluorescein was quantified by monitoring fluorescence at 485/528 nm using a fluorescence plate reader (FLX800, Bio-Tek Instruments, Winooski, VT). To measure singlet oxygen species (1O2), the above experiments were repeated using SOSGR23 instead of APF. In the presence of 1O2, SOSGR is converted to a fluorescent product, which was quantified by measuring fluorescence at 485/528 nm. Nonlight exposed groups served as negative controls. The generation of superoxide anion (O2−•) was estimated using nitro blue tetrazolium (NBT) assay. Briefly, an aqueous solution of NBT (3 μM) was added to MB solution or NPs (8 μM) before irradiation with light under normoxia or hypoxia. The mixture was then diluted with pyridine and its spectra were recorded using a spectrophotometer. The formation of O2−• species was indicated by the presence of new absorption bands at 520 and 560−570 nm in MB spectra. Cell Culture. MCF-7 and 4T1 cell lines were cultured in RPMI-1640 medium. SKBR3 and MDA-MB-231 cells were cultured in McCoy’s 5 A medium and MEM medium containing Earle’s salts, respectively. Each of these growth media were supplemented with 10% v/v FBS and 1% v/v penicillin−streptomycin. The cells were cultured at 37 °C and 5% CO2 in a humidified incubator. Cytotoxicity Studies. Cells were trypsinized, counted, and then transferred to glass tubes. They were treated with either MB solution or MB NPs at an equimolar dye concentration for 1 h to allow uptake of photosensitizer into the cells. Untreated cells were used as controls. For normoxic irradiation, the cells were incubated with MB under ambient conditions. For hypoxic irradiation, glass tubes containing cells were sealed with hermetic rubber stoppers and were then purged with a mixture containing oxygen-free nitrogen and 5% CO2 for 20 min using a dispenser at a flow rate of 6 L/min. At this point, the oxygen content in the suspension medium was evaluated using the Winkler method22 and was routinely found to be about 50 μM. The cells were then irradiated using an Osram halogen lamp integrated into LumaCare LC-122 device (Newport Beach, CA) together with filter systems to cut off light shorter than 600 nm. The effective fluence rate of emitted light was determined by an Orion PD energy meter and was found to be 6 mW/cm2. After irradiation, the cells were transferred to 96-well plates and cultured under routine cell culture conditions for 48 h before measuring cell viability using MTS assay kit.

■

MATERIALS AND METHODS Materials. MB, sodium alginate, poly(vinyl alcohol), and cell culture grade agar were purchased from Sigma-Aldrich (St. Louis, MO). In addition, 3′-(p-Aminophenyl)fluorescein (APF), singlet oxygen sensor green reagent (SOSGR), and insulin were purchased from Invitrogen (Carlsbad, CA). Agarose was purchased from ISC Bio Express (Kaysville, UT). McCoys 5A medium (without phenol red) and AOT were purchased from Fisher Scientific (Chicago, IL). Epidermal growth factor and fibroblast growth factor were obtained from Peprotech (Rocky Hill, NJ). Aldefluor kit was obtained from Stem cell technologies (Vancouver, Canada). All cell lines used in the study were purchased from the American Type Culture Collection. AQueous Non-Radioactive Cell Proliferation Assay (MTS) kit was purchased from Promega (Madison, WI). All other cell culture media and reagents were purchased from Mediatech (Manassas, VA), Lonza (Walkersville, MD), or Invitrogen (Carlsband, CA). Methods. Preparation of MB-Loaded Alginate NPs. MB NPs were synthesized as described before.17−19 Briefly, aqueous solutions of MB (1 mL, 5 mg/mL) and sodium alginate (1 mL, 10 mg/mL) were mixed and sonicated (Sonicator 3000, Misonix, Farmingdale, NY) over an ice bath for 5 min. One milliliter of this mixture was emulsified in 2 mL of 2.5% w/v solution of AOT in chloroform by sonication at 18−21 W for 5 min. The water-in-oil emulsion was further emulsified into an aqueous solution of 2% w/v poly(vinyl alcohol) by sonication over an ice bath for 5 min at 18−21 W to obtain a water-in-oilin-water emulsion. To this final emulsion, 5 mL of an aqueous solution of 60% w/v calcium chloride was gradually added. Chloroform was evaporated by stirring the emulsion overnight under ambient conditions and then under vacuum for 2 h. NPs were washed four times with deionized water by ultracentrifugation (35 000 rpm for 30 min at 4 °C, Beckman, Palo Alto, CA) to remove excess poly(vinyl alcohol) and unencapsulated MB. After the final wash, the pellet was resuspended in deionized water and centrifuged (1000 rpm for 10 min) to remove any large aggregates. The supernatant was then lyophilized using Freezone 4.5 freeze-dry system (Labconco, Kansas City, MO). Characterization of Nanoparticles. Particle size and zeta potential were measured using a Delsa Nano C particle size analyzer (Beckman Coulter Inc., Fullerton, CA). One milligram of NPs was suspended in 1 mL of deionized water, sonicated, and then used for the analysis. The particle size and geometry of NPs was further characterized using the Tecnai G2 Spirit BioTWIN transmission electron microscopy (TEM). B

dx.doi.org/10.1021/mp5003619 | Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

Article

Figure 1. Characterization of MB NPs. (A) Schematic showing the constituents of the MB NPs (B) Light excited MB yields 1O2 by type II photoreactions (pink panel) and O2−• by type I reaction (red). ISC-intersystem crossing. (C) MB NPs were characterized for their size, zeta potential by dynamic light scattering, and dye loading by measuring UV absorbance. The results are represented as mean ± SEM of at least three different batches of the formulations. (D) Transmission electron micrograph (TEM) of MB NPs confirms the spherical shape of MBNPs. Scale bar: 500 nm (left image), 50 nm (right image).

Mammosphere Assay. Mammospheres were generated using a published protocol,24 with some modifications. MCF7 cells treated with PDT were used to generate primary mammospheres. Total number of viable cells remaining after the treatment was estimated, and ∼7500 viable cells from each group were seeded into 6-well ultralow adhesion plates and cultured in DMEM-F12 containing 1% v/v penicillin− streptomycin, 0.4% w/v BSA, insulin (5 μg/mL), recombinant h-EGF (20 ng/mL), and recombinant h-FGF (10 ng/mL). Media was replaced at frequent intervals and the mammospheres were counted on day 8 under an inverted microscope. Primary mammospheres were collected by centrifugation, dissociated with trypsin-EDTA, and then pipetted vigorously to obtain single cells. These were replated and cultured further for 7−14 days to form secondary mammospheres. Soft Agar Colony Formation Assay. Six-well plates were coated with 1 mL of 0.5% w/v agar containing MEM growth medium to form a base layer. Cells subjected to PDT treatment were suspended in 1 mL of growth media containing 0.35% w/ v agarose and then seeded on top of the base layer. The plates were maintained at 37 °C in a humidified incubator and media was changed once a week. Ten thousand cells from each group were seeded in triplicate in separate wells for the colony formation. On day 13 after plating, colonies formed were counted under an inverted microscope. The number of colonies formed was counted in 15 random fields for each sample, and the average number of colonies per treatment group was calculated. Aldefluor Assay. Three days after PDT treatment, SKBR3 cells were trypsinized and stained using the Aldefluor assay kit as per the manufacturer’s instructions. Briefly, cells were suspended in an assay buffer containing aldehyde dehydrogenase (ALDH) substrate-BODIPY-aminoacetaldehyde (BAAA) in

the presence or absence of ALDH inhibitor diethylaminobenzaldehyde (DEAB) at 37 °C for 40 min. The cells were centrifuged, resuspended in assay buffer, and analyzed by flow cytometry. Inhibitor-treated tubes were used to control for background fluorescence.

■

RESULTS Characterization of MB NPs. The average hydrodynamic diameter of MB NPs was found to be 275 ± 30 nm (Figure 1C). TEM studies indicated that NPs had a spherical morphology and were sub-100 nm in size (Figure 1D). The apparent discrepancy in the nanoparticle size as measured by the two techniques could be related to the physical state of the particles. Dynamic light scattering measures particle size in aqueous dispersions, in which particles can aggregate and swell. TEM imaging, on the other hand, is performed on dry particles that tend to be smaller in size. Further, TEM provides numberaverage diameter, whereas light scattering provides intensity average hydrodynamic diameter, which tends to be significantly higher than number-average particle size. Electrophoretic light scattering measurements indicated that NPs had a net negative surface charge of −19 mV. The negative zeta potential is likely due to the anionic sulfonate groups present in AOT and the carboxyl groups present in alginate. MB loading in NPs was 7.0 ± 0.3% w/w. Ex Vitro ROS Production. Total ROS production following photoactivation of MB was measured using fluorescence-based APF assay. As this assay is insensitive to 1 O2, we also used SOSGR to monitor 1O2 generation. We have previously shown that the photoactivation of MB NPs under atmospheric oxygen pressure generates more ROS and 1O2 than free MB.18 Because the tumor microenvironment is hypoxic, we now investigated the ability of MB NPs and free C

dx.doi.org/10.1021/mp5003619 | Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

Article

MB to generate ROS under low concentrations of molecular oxygen. The photoactivation of MB NPs produced higher fluorescence in both assays (Figure 2) than free MB solution at

Figure 3. Effect of excipients on the ROS production by MB. Contribution of AOT, sodium alginate, and CaCl2 toward the production of ROS was investigated. MB taken alone or in combination with sodium alginate and AOT was photoactivated under ambient conditions or hypoxia (50 μM oxygen) in the presence or absence of CaCl2. The combination of MB with sodium alginate resulted in higher ROS production than free MB under normoxia and hypoxia. Addition of AOT did not significantly affect the ROS yield. The addition of CaCl2 to the MB−alginate mixture decreased the yield of ROS. Data are presented as mean ± SD (n = 6−18). * P < 0.05, vs MB (O2); # P < 0.05, vs MB (N2); § P < 0.05, vs MB (CaCl2/ O2); §§ P < 0.05, vs MB (CaCl2/ N2) by one-way ANOVA with Dunnett’s post-test.

Figure 2. Ex vitro ROS and 1O2 production by MB NPs. Free MB or MB NPs were photoactivated under different concentrations of molecular oxygen. ROS and 1O2 were quantified by fluorescence-based assays performed with APF and SOSGR, respectively. The oxygen content in the reaction tubes was regulated by purging the tubes with a mixture containing 95% nitrogen and 5% CO2 for different time intervals. The photoactivation of MB NPs produced higher fluorescence in both assays than free MB solution at all oxygen concentrations tested. Error bars indicate SD.

all oxygen concentrations tested, indicating its capacity to yield increased ROS and 1O2 even at low oxygen concentrations. It should be noted that environmental conditions may have an impact on the fluorescence associated with SOSGR.25 Thus, comparisons across groups (free MB vs MB NPs, for example) should be interpreted with caution. However, it is reasonable to compare 1O2 generation as a function of oxygen concentration for each group. To further understand the reason for superior ROS yield obtained with MB NPs, we studied the effect of the excipients used in the fabrication of NPs  AOT, sodium alginate, and CaCl2  on ROS generation by free MB (Figure 3). The combination of MB with alginate resulted in higher ROS production than free MB under normoxia and hypoxia. Addition of AOT did not significantly affect the ROS yield. The addition of CaCl2 to the MB−alginate mixture decreased the yield of ROS. Similar results were obtained when we studied the effect of excipients on the production of 1O2. We reasoned that the superior ROS yield of MB−alginate combination is a result of the formation of metachromatic complex between cationic MB molecules and anionic polymer, sodium alginate, followed by the appearance of MB dimers and higher aggregates. In other words, the formation of dimeric species such as (MB+)2 would result from an increased local concentration of MB. We tested this hypothesis by performing a visible spectral analysis of MB in the presence of the individual NP components (Figure 4A, B). As the concentration of alginate increased, the spectrum of MB showed progressive depression in the monomer band at 665 nm, an increase in the ratio of dimer (612 nm) to monomer (665 nm) absorption, and the appearance of new maximum at 590 nm corresponding to higher order MB aggregates. These results are in agreement with previous studies26,27 that indicated an increased ability of MB to form dimers when bound to carboxylate groups of sodium alginate. When this dye−polymer

interaction was disrupted by the addition of increasing amounts of CaCl2, the initial spectrum of MB was gradually restored (Figure 4B). Furthermore, these data indicate that at the concentration (72 μM) used in the formulation of MB NPs, CaCl2 has negligible effect on MB dimerization. MB dimers have a propensity to undergo type I photochemical reaction and produce O2−•.28 Thus, in the presence of alginate, MB would be expected to yield elevated levels of O2−•. We tested this by using the NBT assay,29,30 which specifically detects O2−• but not other ROS. Reduction of NBT by O2−• forms mono and diformazan (λmax of 520 nm and 560−570 nm, respectively), which could be identified in the absorption spectra.30−32 In the presence of NBT, irradiation of MB alone or along with the components of NPs resulted in the appearance of new bands around 520 and 570 nm under normal and low pressure of oxygen (Figure 5). Presence of NP constituents resulted in an increased O2−• production. In particular, significantly high levels of O2−• were observed with MB in the presence of alginate under hypoxia. These results establish that the alginate polymer in MB NPs enables the increased generation of O2−• by MB. Cytotoxicity of MB and MB NPs in Breast Cancer Cell Lines. The increased ROS generation by MB NPs under normoxic and hypoxic conditions suggested that MB NPmediated PDT would be effective in killing tumor cells. Hence, we investigated the cytotoxicity of MB NP-mediated PDT in a panel of breast cancer cell lines (Figure 6A). Both MB NPs and MB in combination with light resulted in a significant cytotoxicity when compared with untreated controls. However, MB NPs in combination with light resulted in greater tumor cell kill than free dye in all the cell lines tested. Enhanced cytotoxicity with MB NPs as compared to that with the free dye may be a combined effect of increased ROS production, D

dx.doi.org/10.1021/mp5003619 | Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

Article

Figure 4. Excipients in the MB NPs formulation affects MB dimerization. (A) Spectral profile of MB alone or in combination with sodium alginate was recorded. With an increase in the concentration of alginate, the spectrum of MB shows a progressive depression in the monomer band at 665 nm, an increase in the ratio of dimer (612 nm) to monomer (665 nm), and the appearance of a new maximum at 590 nm corresponding to higher order MB aggregates. (B) Spectral profile of MB in combination with sodium alginate was recorded after addition of increasing amounts of CaCl2. At high doses, CaCl2 disrupted the MB - alginate interaction to restore the initial spectrum of MB. However, at the concentration (72 μM) used in the formulation of MB NPs, CaCl2 has minimal effect on MB dimerization.

formation. Representative images of the mammospheres obtained are shown in Figure 7A. Photoactivation of MB and MB NPs significantly decreased mammosphere formation under normoxia (to 16% with MB and 2% with MB NPs relative to control) and hypoxia (to 76% with MB and 38% with MB NPs relative to control). As is clear from the above data, MB NPs were significantly more effective in decreasing the mammosphere formation than the free drug under both hypoxia and normoxia (Figure 7B). Mammospheres contain stem cells, which are capable of yielding daughter spheres on serial passage. This is considered to be an indirect marker of stem cell self-renewal.34,35 We investigated the effect of PDT on secondary mammosphere formation. PDT of MCF-7 cells carried out under normoxia was efficient in killing a significant portion of the stem cell population and hence generated very few primary mammospheres, which were insufficient for secondary mammosphere assays. Hence, only primary mammospheres obtained from cells subjected to PDT under hypoxia were investigated. When compared to that with free MB, PDT with MB NPs effectively reduced the formation of secondary mammospheres, indicating that MB NP-mediated PDT strongly interfered with stem cell renewal (Figure 7C). CSCs are also characterized by their ability to form colonies in soft agar.36 MB and MB NP-mediated PDT decreased the number of colonies grown in soft agar in comparison to untreated controls under normoxia (to 8% with MB and 1% with MB NPs, relative to control) and hypoxia (to 65% with MB and 48% with MB NPs, relative to control) (Figure 8A). Another characteristic feature of breast CSCs is their high level of ALDH activity.37,38 Because SKBR3 cells were previously documented to contain a subset of cells with high ALDH activity,39 these cells were used to investigate the effect of MB mediated PDT on ALDH positive cells (Figure 8B). PDT with MB NPs was more effective than that with free MB. PDT decreased the ALDH positive cells under normoxia (to 82% with MB and 74% with MB NPs relative to control) and hypoxia (to 78% with MB and 42% with MB NPs, relative to control).

Figure 5. Ex vitro production of O2−• by MB in combination with sodium alginate. MB alone or along with excipients of MB NPs (MB +Tot) was photoactivated (L+) under ambient conditions or hypoxia. MB or MB+ Tot samples not excited with light (L−) served as additional controls. The samples were mixed with NBT and then visible spectra was recorded to investigate the O2−• generation. The reduction of NBT by O2−• will yield mono and diformazan (λmax of 520 nm and 560−570 nm, respectively). Irradiation of MB along with the components of NPs (MB+Tot) resulted in the appearance of new bands around 520 and 570 nm under normal (MB+Tot, O2, L+) and low pressure of oxygen (MB+Tot, N2, L+). In particular, a significantly high levels of O2−• were observed with MB in the presence of alginate under hypoxia when compared with irradiation of MB alone (MB, N2, L+ or MB, O2, L+).

enhanced cellular delivery and altered subcellular localization of the photosensitizer.19 Control studies indicate that MB or MB NPs were not cytotoxic in the absence of light (data not shown). We further validated these results by comparing the cytotoxicity of MB NPs and MB under normoxia and hypoxia. MB NPs were significantly more cytotoxic than free MB under both normoxia as well as hypoxia (Figure 6B). These results are in agreement with our previous results.17−19 Effect of MB NP-Mediated PDT on CSCs. An important property of breast CSCs is their ability to grow in anchorage independent culture conditions as mammospheres.24,33 We investigated the effect of MB-mediated PDT on mammosphere E

dx.doi.org/10.1021/mp5003619 | Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

Article

Figure 6. Efficacy of MB NP mediated photodynamic therapy in breast cancer cells. (A) Different cell lines were treated with MB (equimolar concentrations of free dye or NPs) under ambient conditions and then irradiated with light. (B) PDT was performed on MCF-7 cells under normoxia and hypoxia. The cells were cultured for 48 h, and then cell viability was measured using MTS assay kit. PDT done with MB NPs was more cytotoxic in the tested cell lines. Data are presented as mean ± SD (n = 3). § P < 0.05, vs control (MDAMB231); ‡ P < 0.05, vs control (4T1); • P < 0.05, vs control (SKBR3); * P < 0.05, vs control (MCF7, normoxia); # P < 0.05, vs control (MCF7, hypoxia) by one-way ANOVA with Dunnett’s post-test.

Figure 7. Efficacy of photodynamic therapy done with MB NPs against cancer stem cells. (A) Representative images of mammospheres obtained in MCF-7 cells subjected to MB mediated PDT. (B) MB NP-mediated PDT done under normoxia and hypoxia significantly reduced mammosphere formation. (C) Primary mammospheres derived from cells subjected to PDT under hypoxia were collected, trypsinized, and then cultured to generate secondary mammospheres. MB NP treatment efficiently reduced secondary mammosphere formation. Data are presented as mean ± SD (n = 3). * P < 0.05, vs control (normoxia); # P < 0.05, vs control (hypoxia); • P < 0.05, vs control secondary mammospheres by one-way ANOVA with Dunnett’s post-test.

■

Blood perfusion to the tumor is erratic45 and results in regions within tumors that are hypoxic. Hypoxia not only nurtures but also protects CSCs. For example, hypoxia leads to elevated levels of hypoxia inducible factor 2A (HIF2A), which mediates expression of Oct 4, Nanog, and c-myc. The upregulation of these factors supports the growth of CSCs.46 Additionally, reduced oxygen concentration in the tumor niche renders conventional therapies ineffective, thereby protecting the stem cell population.7 Hence, therapies that are effective in hypoxic microenvironments can potentially reduce the development of drug resistance and prevent tumor recurrence.

DISCUSSION

Several reports have pointed to the presence of a small subpopulation of cells within the tumor that can potentially give rise to an entire tumor.40,41 These cells, termed CSCs, are characterized by properties such as self-renewal, asymmetric cell division, and differentiation.42,43 Owing to several detoxification pathways active within these cells, the elimination of CSCs is a key challenge for cancer therapy.44 Paradoxically, most cancer therapies eliminate nonstem cell populations and enrich the CSC population within a tumor.7,14 F

dx.doi.org/10.1021/mp5003619 | Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

Article

amino)ethyl methacrylate) micelles but not in an electrondeficient poly(lactide) micelle. In this study, we used a polymer−surfactant NP system to alter the photochemistry of MB. We chose a matrix-type NP device over a micellar system because the former can potentially allow greater interaction of the photosensitizer with the matrix. This would lead to a greater interaction of photosensitizer molecules with one another followed by a more efficient electron transfer. The use of alginate provides an additional advantage. Alginate polymers are widely used in the food industry and are known for their high swelling capacity.56 High local concentration of water in the vicinity of the photosensitizer may also promote electron transfer and production of hydroxyl radical.16 The yield of 1O2 (a measure of type II reaction) for both NPs and free dye declined with decreasing oxygen concentration. Although the overall yield of total ROS decreased under hypoxia, MB NPs generated consistently high levels of total ROS even under hypoxic conditions. This provided initial evidence that the nanoencapsulated MB could potentially operate through type I mechanism, which is less sensitive to oxygen concentrations. In agreement with these results, under hypoxic conditions, free MB completely lost its activity, whereas MB NPs still showed a 40% reduction in cell viability. These results suggest that MB NPs cause cell death under hypoxic conditions via type I reaction. This is in agreement with our previous in vivo results,17 where we found necrotic cell death, a sign of type I reactions.2,28,57 CSCs are equipped with potent detoxification mechanisms including superoxide dismutases, catalases, and glutathione peroxidases that are capable of scavenging ROS.44 CSCs have also been shown to upregulate autophagy-related proteins and mediate resistance to PDT.58 Despite these resistance mechanisms, PDT has shown considerable efficacy in eliminating CSCs. For example, 5-aminolevulinic-acid-mediated PDT has been shown to decrease the relative abundance of aldefluor positive cells and CD44+ cells (both indicators of CSCs).59 However, these results were obtained under normoxic conditions. Hence, we determined the susceptibility of CSCs to MB NP-mediated PDT under both normoxic and hypoxic conditions. We used mammosphere formation and soft agar colony formation assays to determine the effect of treatments on the CSC population. Unlike most tumor cells, CSCs have the unique ability to grow in semisolid media in an anchorage independent manner. Hence the number of colonies formed in this assay can be directly correlated to the relative abundance of CSCs. In both the mammosphere and soft agar assays, we found that both MB and MB NPs effectively reduced the number of spheres and colonies under normal pressure of oxygen. However, only MB NPs were effective in disrupting the formation of mammospheres as well as soft agar colonies under hypoxic conditions. These data suggest that although MBmediated PDT is inherently effective against CSCs, the effectiveness decreases under hypoxia likely because of a decrease in 1O2 generation. By generating sufficient ROS even under hypoxia, MB NPs enable effective eradication of CSCs. Efficacy of MB NPs against CSCs under hypoxic conditions was confirmed using the aldefluor assay. We also probed the mechanism of radical generation by MB NPs. The APF probe used in our studies is particularly sensitive to the presence of hydroxyl radicals (OH•),21 and our results show that their production is significantly enhanced by the presence of alginate. This suggests increased electron transfer

Figure 8. Efficacy of photodynamic therapy done with MB NPs against cancer stem cells. PDT completed with MB NPs significantly reduced (A) soft-agar colony formation in MCF 7 cells and (B) aldefluor-bright cells in SKBR3 cells. PDT with MB NPs performed under hypoxia was effective in both these assays. Data are presented as average ± SD (n = 3). * P < 0.05, vs control (normoxia); # P < 0.05, vs control (hypoxia); • P < 0.05, vs control (normoxia); ‡ P < 0.05, vs control (hypoxia) by one-way ANOVA with Dunnett’s post-test.

PDT often involves the transfer of energy from an excited photosensitizer to surrounding oxygen molecules to produce cytotoxic singlet oxygen species through type II reaction. Although this mechanism can be very useful in the welloxygenated parts of the tumor, it loses efficacy in the hypoxic regions.11 This limitation of PDT has been previously identified, and several attempts have been made to address it. Several studies have shown that using hyperbaric oxygen can increase the performance of PDT in cancer patients.47−49 The underlying rationale of this approach is that higher levels of oxygen in blood can improve tumor oxygen concentration. An alternate approach is the use of low fluence rates.12,50−52 The lower rate of oxygen consumption by the photosensitizer allows sufficient time for restocking tumor oxygen supply and prevents rapid oxygen depletion. In both these cases, parts of the tumor that are adequately perfused likely benefit. However, tumor regions lacking blood vessels may not benefit at all.53 Switching the mechanism of radical generation from an oxygen-dependent type II to an only slightly oxygen-dependent type I reaction is an attractive strategy to improve effectiveness of PDT. Type I chemistry can be exploited by using chemical moieties that have the ability to self-switch to type I reactions at low levels of oxygen.2 For example, 5-ethylamino-9-diethyl aminobenzo[a]phenothiazinium chloride (EtNBS) displays significant cell kill under both normoxic and hypoxic conditions.54 Evans et al. tested the efficacy of EtNBS in a multicellular spheroid model of ovarian cancer. The authors found that PDT at high fluence rates showed significant cytotoxicity, indicating that the cell kill mechanism was not limited by the presence/diffusion of oxygen to the core of the spheroids.54 The goal of the current studies was to investigate nanoencapsulation as a means to elicit the type I photoreaction. Vakrat-Haglili et al. showed that the microenvironment of the photosensitizer may play an important role in determining the mechanism of PDT.55 The authors found that confining Pdbacteriopheophorbide in liposomes or micelles can increase the probability of type I reaction. Similarly, Ding et al. showed that appropriate interaction with the material of the NP formulation is also essential for switching from type II to type I chemistry.20 Type I chemistry resulted only when electron-deficient 5,10,15,20-tetrakis(meso-hydroxyphenyl)porphyrin (mTHPP) was encapsulated in an electron rich poly(2-(diisopropylG

dx.doi.org/10.1021/mp5003619 | Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

Article

meric micelles for enhanced photodynamic therapy. Adv. Drug Delivery Rev. 2009, 61 (4), 327−338. (4) Agostinis, P.; Berg, K.; Cengel, K. A.; Foster, T. H.; Girotti, A. W.; Gollnick, S. O.; Hahn, S. M.; Hamblin, M. R.; Juzeniene, A.; Kessel, D.; Korbelik, M.; Moan, J.; Mroz, P.; Nowis, D.; Piette, J.; Wilson, B. C.; Golab, J. Photodynamic therapy of cancer: an update. CA Cancer J. Clin. 2011, 61 (4), 250−281. (5) Brown, S. B.; Brown, E. A.; Walker, I. The present and future role of photodynamic therapy in cancer treatment. Lancet Oncol. 2004, 5 (8), 497−508. (6) Weishaupt, K. R.; Gomer, C. J.; Dougherty, T. J. Identification of singlet oxygen as the cytotoxic agent in photoinactivation of a murine tumor. Cancer Res. 1976, 36 (7 PT 1), 2326−2329. (7) Kirtane, A. R.; Kalscheuer, S. M.; Panyam, J. Exploiting nanotechnology to overcome tumor drug resistance: Challenges and opportunities. Adv. Drug Delivery Rev. 2013, 65 (13−14), 1731−1747. (8) Provenzano, P. P.; Cuevas, C.; Chang, A. E.; Goel, V. K.; Von Hoff, D. D.; Hingorani, S. R. Enzymatic targeting of the stroma ablates physical barriers to treatment of pancreatic ductal adenocarcinoma. Cancer Cell 2012, 21 (3), 418−429. (9) Jain, R. K. Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy. Science 2005, 307 (5706), 58−62. (10) Henderson, B. W.; Fingar, V. H. Relationship of tumor hypoxia and response to photodynamic treatment in an experimental mouse tumor. Cancer Res. 1987, 47 (12), 3110−3114. (11) Dolmans, D. E.; Fukumura, D.; Jain, R. K. Photodynamic therapy for cancer. Nat. Rev. Cancer 2003, 3 (5), 380−387. (12) Sitnik, T. M.; Hampton, J. A.; Henderson, B. W. Reduction of tumour oxygenation during and after photodynamic therapy in vivo: effects of fluence rate. Br. J. Cancer 1998, 77 (9), 1386−1394. (13) Keith, B.; Simon, M. C. Hypoxia-inducible factors, stem cells, and cancer. Cell 2007, 129 (3), 465−472. (14) Gupta, P. B.; Onder, T. T.; Jiang, G.; Tao, K.; Kuperwasser, C.; Weinberg, R. A.; Lander, E. S. Identification of selective inhibitors of cancer stem cells by high-throughput screening. Cell 2009, 138 (4), 645−659. (15) Creighton, C. J.; Li, X.; Landis, M.; Dixon, J. M.; Neumeister, V. M.; Sjolund, A.; Rimm, D. L.; Wong, H.; Rodriguez, A.; Herschkowitz, J. I.; Fan, C.; Zhang, X.; He, X.; Pavlick, A.; Gutierrez, M. C.; Renshaw, L.; Larionov, A. A.; Faratian, D.; Hilsenbeck, S. G.; Perou, C. M.; Lewis, M. T.; Rosen, J. M.; Chang, J. C. Residual breast cancers after conventional therapy display mesenchymal as well as tumor-initiating features. Proc. Natl. Acad. Sci. U.S.A. 2009, 106 (33), 13820−13825. (16) Junqueira, H. C.; Severino, D.; Dias, L. G.; Gugliotti, M. S.; Baptista, M. S. Modulation of methylene blue photochemical properties based on adsorption at aqueous micelle interfaces. Phys. Chem. Chem. Phys. 2002, 4 (11), 2320−2328. (17) Khdair, A.; Chen, D.; Patil, Y.; Ma, L.; Dou, Q. P.; Shekhar, M. P.; Panyam, J. Nanoparticle-mediated combination chemotherapy and photodynamic therapy overcomes tumor drug resistance. J. Controlled Release 2010, 141 (2), 137−144. (18) Khdair, A.; Gerard, B.; Handa, H.; Mao, G.; Shekhar, M. P. V.; Panyam, J. Surfactant-polymer nanoparticles enhance the effectiveness of anticancer photodynamic therapy. Mol. Pharmaceutics 2008, 5 (5), 795−807. (19) Khdair, A.; Handa, H.; Mao, G.; Panyam, J. Nanoparticlemediated combination chemotherapy and photodynamic therapy overcomes tumor drug resistance in vitro. Eur. J. Pharm. Biopharm. 2009, 71 (2), 214−222. (20) Ding, H.; Yu, H.; Dong, Y.; Tian, R.; Huang, G.; Boothman, D. A.; Sumer, B. D.; Gao, J. Photoactivation switch from type II to type I reactions by electron-rich micelles for improved photodynamic therapy of cancer cells under hypoxia. J. Controlled Release 2011, 156 (3), 276− 280. (21) Setsukinai, K.; Urano, Y.; Kakinuma, K.; Majima, H. J.; Nagano, T. Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species. J. Biol. Chem. 2003, 278 (5), 3170−3175.

to water molecules in the presence of the polymer. Moreover, the production of O2−• is also enhanced in the presence of the matrix-forming alginate polymer. It is interesting to note that, under hypoxic conditions, the presence of alginate does not have any influence on the production of 1O2. Electrostatic interaction and trapping of MB within the polymer generate MB dimers. These electrostatic interactions likely occur between the carboxylate groups in the polymer and the imine nitrogen of MB. Addition of the divalent calcium to the polymer leads to the cross-linking of polymer chains via the carboxylate groups. This displaces MB and decreases dimer formation. However, these results are observed only at very high levels of the calcium salt and do not affect ROS production at the concentration we use during NP preparation. Taken together, our results show a proximity effect caused by the alginate polymer promotes interaction of the dye with solvent molecules as well as with itself. This effect, in turn, enhances ROS production via a type I mechanism, making the formulation effective under hypoxic conditions. These studies may provide an important framework for future development of effective formulations for PDT. In summary, we present a NP formulation of MB that demonstrates the potential of to switch photochemistry of photosensitizer from type II to type I mechanism under hypoxia. This formulation enabled increased ROS production and cytotoxicity in tumor cells under hypoxic conditions. Further, PDT with MB NPs resulted in efficient eradication of CSCs. These results suggest that approaches that improve ROS production under hypoxia can help improve the anticancer effectiveness of PDT.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: 612-626-2125. Tel.: 612624-0951. Author Contributions §

M.U. and S.K.S. contributed equally to this work.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS TEM was carried out in the Characterization Facility, University of Minnesota, which receives partial support from NSF through the MRSEC program.

■

LIST OF ABBREVIATIONS photodynamic therapy, PDT; cancer stem cells, CSCs; methylene blue, MB; nanoparticles, NPs; aerosol OT, AOT; reactive oxygen species, ROS; 3′-(p-aminophenyl)fluorescein, APF; singlet oxygen sensor green reagent, SOSGR; BODIPYaminoacetaldehyde, BAAA; transmission electron microscopy, TEM; nitroblue tetrazolium, NBT; diethylaminobenzaldehyde, DEAB; aldehyde dehydrogenase, ALDH

■

REFERENCES

(1) Henderson, B. W.; Dougherty, T. J. How does photodynamic therapy work? Photochem. Photobiol. 1992, 55 (1), 145−157. (2) Ochsner, M. Photophysical and photobiological processes in the photodynamic therapy of tumours. J. Photochem. Photobiol., B 1997, 39 (1), 1−18. (3) Nishiyama, N.; Morimoto, Y.; Jang, W. D.; Kataoka, K. Design and development of dendrimer photosensitizer-incorporated polyH

dx.doi.org/10.1021/mp5003619 | Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

Article

(22) Carpenter, J. H. The accuracy of the Winklet method for dissolved oxygen analysis. Limnol. Oceanogr. 1965, 10 (1), 135−140. (23) Gollmer, A.; Arnbjerg, J.; Blaikie, F. H.; Pedersen, B. W.; Breitenbach, T.; Daasbjerg, K.; Glasius, M.; Ogilby, P. R. Singlet Oxygen Sensor Green(R): photochemical behavior in solution and in a mammalian cell. Photochem. Photobiol. 2011, 87 (3), 671−9. (24) Ponti, D.; Costa, A.; Zaffaroni, N.; Pratesi, G.; Petrangolini, G.; Coradini, D.; Pilotti, S.; Pierotti, M. A.; Daidone, M. G. Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/ progenitor cell properties. Cancer Res. 2005, 65 (13), 5506−5511. (25) Kim, S.; Fujitsuka, M.; Majima, T. Photochemistry of Singlet Oxygen Sensor Green. J. Phys. Chem. B 2013, 117 (45), 13985−13992. (26) Michaelis, L.; Granick, S. Metachromasy of Basic Dyestuffs. J. Am. Chem. Soc. 1945, 67 (7), 1212−1219. (27) Seely, G. R. Binding of methylene blue to alginate: Effect of acid treatment on metachromasy. Carbohydr. Polym. 1983, 3 (2), 109−127. (28) Severino, D.; Junqueira, H. C.; Gugliotti, M.; Gabrielli, D. S.; Baptista, M. S. Influence of negatively charged interfaces on the ground and excited state properties of methylene blue. Photochem. Photobiol. 2003, 77 (5), 459−468. (29) Chekulayeva, L. V.; Shevchuk, I. N.; Chekulayev, V. A.; Ilmarinen, K. Hydrogen peroxide, superoxide, and hydroxyl radicals are involved in the phototoxic action of hematoporphyrin derivative against tumor cells. J. Environ. Pathol. Toxicol. Oncol. 2006, 25 (1−2), 51−77. (30) Bielski, B. H. J.; Shiue, G. G.; Bajuk, S. Reduction of nitro blue tetrazolium by CO2− and O2− radicals. J. Phys. Chem. 1980, 84 (8), 830−833. (31) Kovács, A.; Baranyai, M.; Wojnárovits, L.; Moussa, A.; Othman, I.; McLaughlin, W. L. Aqueous-ethanol nitro blue tetrazolium solutions for high dose dosimetry. Radiat. Phys. Chem. 1999, 55 (5), 799−803. (32) Butcher, R. G. The measurement in tissue sections of the two formazans derived from nitroblue tetrazolium in dehydrogenase reactions. Histochem. J. 1978, 10 (6), 739−44. (33) Kakarala, M.; Brenner, D. E.; Korkaya, H.; Cheng, C.; Tazi, K.; Ginestier, C.; Liu, S.; Dontu, G.; Wicha, M. S. Targeting breast stem cells with the cancer preventive compounds curcumin and piperine. Breast Cancer Res. Treat. 2010, 122 (3), 777−785. (34) Rota, L. M.; Lazzarino, D. A.; Ziegler, A. N.; LeRoith, D.; Wood, T. L. Determining mammosphere-forming potential: application of the limiting dilution analysis. J. Mammary Gland Biol. Neoplasia 2012, 17 (2), 119−23. (35) Shaw, F. L.; Harrison, H.; Spence, K.; Ablett, M. P.; Simoes, B. M.; Farnie, G.; Clarke, R. B. A detailed mammosphere assay protocol for the quantification of breast stem cell activity. J. Mammary Gland Biol. Neoplasia 2012, 17 (2), 111−117. (36) Visvader, J. E.; Lindeman, G. J. Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat. Rev. Cancer 2008, 8 (10), 755−768. (37) Charafe-Jauffret, E.; Ginestier, C.; Iovino, F.; Tarpin, C.; Diebel, M.; Esterni, B.; Houvenaeghel, G.; Extra, J. M.; Bertucci, F.; Jacquemier, J.; Xerri, L.; Dontu, G.; Stassi, G.; Xiao, Y.; Barsky, S. H.; Birnbaum, D.; Viens, P.; Wicha, M. S. Aldehyde dehydrogenase 1positive cancer stem cells mediate metastasis and poor clinical outcome in inflammatory breast cancer. Clin. Cancer Res. 2010, 16 (1), 45−55. (38) Ginestier, C.; Hur, M. H.; Charafe-Jauffret, E.; Monville, F.; Dutcher, J.; Brown, M.; Jacquemier, J.; Viens, P.; Kleer, C. G.; Liu, S.; Schott, A.; Hayes, D.; Birnbaum, D.; Wicha, M. S.; Dontu, G. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 2007, 1 (5), 555− 567. (39) Marcato, P.; Dean, C. A.; Pan, D.; Araslanova, R.; Gillis, M.; Joshi, M.; Helyer, L.; Pan, L.; Leidal, A.; Gujar, S.; Giacomantonio, C. A.; Lee, P. W. Aldehyde dehydrogenase activity of breast cancer stem cells is primarily due to isoform ALDH1A3 and its expression is predictive of metastasis. Stem Cells 2011, 29 (1), 32−45.

(40) Prince, M. E.; Sivanandan, R.; Kaczorowski, A.; Wolf, G. T.; Kaplan, M. J.; Dalerba, P.; Weissman, I. L.; Clarke, M. F.; Ailles, L. E. Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proc. Natl. Acad. Sci. U. S. A. 2007, 104 (3), 973−8. (41) Singh, S. K.; Clarke, I. D.; Terasaki, M.; Bonn, V. E.; Hawkins, C.; Squire, J.; Dirks, P. B. Identification of a cancer stem cell in human brain tumors. Cancer Res. 2003, 63 (18), 5821−5828. (42) Wicha, M. S.; Liu, S.; Dontu, G. Cancer stem cells: an old idea–a paradigm shift. Cancer Res. 2006, 66 (4), 1883−1890 discussion 1895−1896. (43) Clarke, M. F.; Dick, J. E.; Dirks, P. B.; Eaves, C. J.; Jamieson, C. H.; Jones, D. L.; Visvader, J.; Weissman, I. L.; Wahl, G. M. Cancer stem cells–perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res. 2006, 66 (19), 9339− 9344. (44) Trachootham, D.; Alexandre, J.; Huang, P. Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nat. Rev. Drug Discovery 2009, 8 (7), 579−591. (45) Stylianopoulos, T.; Jain, R. K. Combining two strategies to improve perfusion and drug delivery in solid tumors. Proc. Nat. Acad. Sci. U.S.A. 2013, 110 (46), 18632−18637. (46) Heddleston, J. M.; Li, Z.; McLendon, R. E.; Hjelmeland, A. B.; Rich, J. N. The hypoxic microenvironment maintains glioblastoma stem cells and promotes reprogramming towards a cancer stem cell phenotype. Cell Cycle 2009, 8 (20), 3274−84. (47) Maier, A.; Anegg, U.; Fell, B.; Rehak, P.; Ratzenhofer, B.; Tomaselli, F.; Sankin, O.; Pinter, H.; Smolle-Juttner, F. M.; Friehs, G. B. Hyperbaric oxygen and photodynamic therapy in the treatment of advanced carcinoma of the cardia and the esophagus. Lasers Surg. Med. 2000, 26 (3), 308−315. (48) Maier, A.; Anegg, U.; Tomaselli, F.; Rehak, P.; Sankin, O.; Fell, B.; Renner, H.; Pinter, H.; Smolle-Juttner, F. M.; Friehs, G. B. Does hyperbaric oxygen enhance the effect of photodynamic therapy in patients with advanced esophageal carcinoma? A clinical pilot study. Endoscopy 2000, 32 (1), 42−48. (49) Maier, A.; Tomaselli, F.; Anegg, U.; Rehak, P.; Fell, B.; Luznik, S.; Pinter, H.; Smolle-Juttner, F. M. Combined photodynamic therapy and hyperbaric oxygenation in carcinoma of the esophagus and the esophago-gastric junction. Eur. J. Cardiothorac. Surg. 2000, 18 (6), 649−654 discussion 654−655. (50) Kawauchi, S.; Morimoto, Y.; Sato, S.; Arai, T.; Seguchi, K.; Asanuma, H.; Kikuchi, M. Differences between cytotoxicity in photodynamic therapy using a pulsed laser and a continuous wave laser: study of oxygen consumption and photobleaching. Lasers Med. Sci. 2004, 18 (4), 179−83. (51) Miyamoto, Y.; Umebayashi, Y.; Nishisaka, T. Comparison of phototoxicity mechanism between pulsed and continuous wave irradiation in photodynamic therapy. J. Photochem. Photobiol., B 1999, 53 (1−3), 53−59. (52) Henderson, B. W.; Gollnick, S. O.; Snyder, J. W.; Busch, T. M.; Kousis, P. C.; Cheney, R. T.; Morgan, J. Choice of oxygen-conserving treatment regimen determines the inflammatory response and outcome of photodynamic therapy of tumors. Cancer Res. 2004, 64 (6), 2120−2126. (53) Foster, T. H.; Murant, R. S.; Bryant, R. G.; Knox, R. S.; Gibson, S. L.; Hilf, R. Oxygen consumption and diffusion effects in photodynamic therapy. Radiat. Res. 1991, 126 (3), 296−303. (54) Evans, C. L.; Abu-Yousif, A. O.; Park, Y. J.; Klein, O. J.; Celli, J. P.; Rizvi, I.; Zheng, X.; Hasan, T. Killing hypoxic cell populations in a 3D tumor model with EtNBS-PDT. PLoS One 2011, 6 (8), e23434. (55) Vakrat-Haglili, Y.; Weiner, L.; Brumfeld, V.; Brandis, A.; Salomon, Y.; McLlroy, B.; Wilson, B. C.; Pawlak, A.; Rozanowska, M.; Sarna, T.; Scherz, A. The microenvironment effect on the generation of reactive oxygen species by Pd-bacteriopheophorbide. J. Am. Chem. Soc. 2005, 127 (17), 6487−6497. (56) Hoffman, A. S. Hydrogels for biomedical applications. Adv. Drug Delivery Rev. 2002, 54 (1), 3−12. I

dx.doi.org/10.1021/mp5003619 | Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

Article

(57) Foote, C. S. Mechanisms of photosensitized oxidation. There are several different types of photosensitized oxidation which may be important in biological systems. Science 1968, 162 (3857), 963−970. (58) Wei, M. F.; Chen, M. W.; Chen, K. C.; Lou, P. J.; Lin, S. Y.; Hung, S. C.; Hsiao, M.; Yao, C. J.; Shieh, M. J. Autophagy promotes resistance to photodynamic therapy-induced apoptosis selectively in colorectal cancer stem-like cells. Autophagy 2014, 10 (7), 1179−1192. (59) Yu, C. H.; Yu, C. C. Photodynamic therapy with 5aminolevulinic acid (ALA) impairs tumor initiating and chemoresistance property in head and neck cancer-derived cancer stem cells. PLoS One 2014, 9 (1), e87129.

J

dx.doi.org/10.1021/mp5003619 | Mol. Pharmaceutics XXXX, XXX, XXX−XXX