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Photodiagnosis and Photodynamic Therapy (2014) xxx, xxx—xxx
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/pdpdt
Indocyanine green loaded liposome nanocarriers for photodynamic therapy using human triple negative breast cancer cells Colby S. Shemesh a, Claire W. Hardy b, David S. Yu b, Brian Fernandez a, Hailing Zhang Ph.D. a,∗ a
Drug Delivery Laboratory, Department of Pharmaceutical Sciences, College of Pharmacy, Mercer University, 3001 Mercer University Drive, Atlanta, GA 30341, United States b Department of Radiation Oncology, Emory University School of Medicine, 1365 Clifton Road Northeast, Atlanta, GA 30322, United States
KEYWORDS Indocyanine green (ICG); Photodynamic therapy (PDT); Thermosensitive liposome; Triple negative breast cancer (TNBC); DNA damage
∗
Summary Background: The goal of the current research is to evaluate the potential of photodynamic therapy (PDT) in the treatment of triple negative breast cancer (TNBC) with the development of a theranostic thermosensitive liposome platform to deliver indocyanine green (ICG) as the near-infrared (NIR) photosensitizer excited by an 808 nm diode laser. Methods: In the PDT protocol, an optimized thermosensitive liposome formulation is investigated to formulate ICG as the photosensitizer, which is exited by laser light at the wavelength of 808 nm delivered by a fiber-coupled laser system. ICG in both free solution and thermosensitive liposomal formulation were evaluated as the NIR photosensitizer and compared in the PDT treatment on a panel of triple negative breast cancer cell lines along with the nontumorigenic mammary epithelial cell line MCF-10A. In addition to cytotoxicity, and clonogenic survival assessment, the role of DNA double strand break damage was evaluated. Results: Both MTT and clonogenic assays revealed that PDT using ICG inhibited the growth of several TNBC cell lines as well as the non-tumorigenic human breast epithelial cell line MCF-10A; and the liposomal formulation of ICG did not compromise the in vitro treatment potency, though free ICG performed slightly more effective in certain cell lines, but was not statistically significant. Cell viability was dose dependent in regards to ICG concentration and irradiation energy. Interestingly, PDT using the described protocol was more potent to inhibit the growth of MDA-MB-468 and HCC-1806 cells, coinciding with the observation that these cells are more sensitive toward DNA damaging agents. In comparison, cell lines HCC-70, BT-549, and
Corresponding author. Tel.: +1 678 547 6242; fax: +1 678 547 6423. E-mail address: zhang [email protected] (H. Zhang).
http://dx.doi.org/10.1016/j.pdpdt.2014.02.001 1572-1000/© 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Shemesh CS, et al. Indocyanine green loaded liposome nanocarriers for photodynamic therapy using human triple negative breast cancer cells. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.02.001
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C.S. Shemesh et al. MCF-10A were found to have less of an inhibitory effect. Furthermore, substantial DNA double strand breaks (DSBs) were observed 30 min after the PDT treatment via a ␥-H2AX staining assay. PDT induced DNA damage has the potential to lead to mutagenicity, which may have various responses depending on the repair capabilities of the cells. Conclusion: Our results suggest that PDT using indocyanine green loaded liposomes were effective in inhibiting tumor cell growth to varying extents with higher responses observed for MDA-MB-468 and HCC-1806 cells. © 2014 Elsevier B.V. All rights reserved.
Introduction In 2012 an estimated 226,000 new breast cancer cases were expected to develop in the United States with nearly 40,000 deaths indicating breast cancer as the leading cause of cancer mortality among women [1]. As a subclass of breast cancer, triple negative breast cancers (TNBCs) are characterized by neither expressing hormone receptors (estrogen or progesterone receptors) nor overexpressing human epidermal growth factor receptor 2 (HER2) [2]. About 15% of all diagnosed breast cancers belong to this subclass. Due to the characteristic genetic profiles, selective therapeutics that target hormonal or HER2 receptors are generally ineffective to treat TNBCs. Despite initial response to chemo/radio therapy treatment, relapse and recurrence occur more often in this subclass [3]. Poor prognosis and high mortality of patients with TNBCs have provoked a wide array of research aimed at seeking more effective treatments including recently developed poly(ADP-ribose) polymerase inhibitor and epidermal growth factor receptor targeted therapy [3], but the clinical outcomes of these new treatments remain controversial [3—5]. This manuscript encompasses our attempt at developing photodynamic therapy (PDT) as a potential treatment for TNBCs. During PDT, a photosensitizer is first administered, followed by excitation of the photosensitizer by exposing the tumor area to light of a specific wavelength. Then the excited photosensitizer may undergo a type I reaction to produce radicals and/or a type II reaction to generate reactive oxygen species (ROS) [6]. Moreover, PDT is based on the type II reaction that occurs to transfer energy of the activated photosensitizer to ground state oxygen forming singlet oxygen. The singlet oxygen and other ROS may then react with a large array of biomolecules in cells, such as proteins, nucleic acids, and lipids. Previous investigations have suggested that TNBC cell lines are more susceptible to H2 O2 through oxidative DNA damage due to mutations in the breast cancer susceptibility gene 1 (BRCA1) [7]. Therefore we hypothesized that TNBCs may be effectively treated by PDT, in which ROS and radicals are produced and cause DNA damage. We were particularly interested in using indocyanine green (ICG) as a photosensitizer. As a near infrared (NIR) dye used clinically to visualize the human vasculature, ICG is excited by light around 800 nm [8], which is of a longer wavelength compared to 630 nm that is used to excite photofrin (the photosensitizer used in clinical oncology) [6]. A light with a longer wavelength is presumably capable of penetrating deeper into tissue to treat more in-depth cancers owing to its lower absorption and scattering by endogenous biomolecules and intracellular organelles
[9]. Of course limitations in the feasibility of a localized photodynamic therapy exist due to very limited effects over 10 mm within tissue. However, ICG itself is not suitable for cancer treatment because of its very short circulation halflife [10] and low in vivo photo stability [11]. We attempted to address this issue by developing thermosensitive liposomes to formulate ICG as the photosensitizer in PDT. In our previous studies, liposomal ICG using a thermosensitive formulation was successfully and preferentially delivered to the tumor, as shown in the in vivo NIR imaging [12]. In addition to targeted delivery of ICG to tumor areas due to the enhanced permeability and retention effect (EPR), we expect the thermosensitive formulation will offer the potential for controlled release. Due to the EPR phenomena, in the translation to an in vivo procedure of PDT, tumor tissue may be selectively targeted and the PDT effect may be preferentially localized to tumor tissues while avoiding damage to normal tissue. The formulation is comprised of DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), soyPC (L-␣-phosphatidylcholine), cholesterol, and DSPC-PEG 2000 (N-(carbonyl-methoxypolyethyleneglycol 2000)-1,2distearoyl-sn-glycero-3-phosphoethanolamine) with a transition temperature around 42 ◦ C. We observed excellent stability at 37 ◦ C with this formulation, yet burst release was achieved at 42 ◦ C [12]. When ICG molecules encapsulated in thermosensitive liposomes are excited, we expect they will produce an increase in temperature within the liposomes [13]. The resulting hyperthermia may then increase the permeability of bilayers leading to the release of ICG, thereby reacting with O2 to produce ROS and radicals. Thermosensitive liposomes have the advantage of offering carrier stability at normal physiological temperatures with an increased permeation of the lipid bilayer above transition temperature. These phenomena may be exploited to better facilitate release of cargo in a more targeted approach by application of a very brief, limited, and localized hyperthermia with the intention to better minimize treatment resistance. Uniform heating of a tumorous tissue may be facilitated by utilization of a collimating lens to ensure a uniform distribution and focusing of the light. Liposome ICG is ultimately excited by NIR irradiation; it is hypothesized that ROS may diffuse freely through the liposomal bilayer due to their small size. In addition, ICG may also be released by an increase in temperature in liposomes due to the NIR excitation, which could react with the oxygen in the surrounding environment of cells or tissue to produce ROS. However, the direct investigation of releasing ICG from liposomes by laser irradiation is hampered by the high reactivity of excited ICG. Therefore, our initial focus was to evaluate the inhibition effectiveness of TNBC cell lines by
Please cite this article in press as: Shemesh CS, et al. Indocyanine green loaded liposome nanocarriers for photodynamic therapy using human triple negative breast cancer cells. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.02.001
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Photodynamic therapy using human triple negative breast cancer cells photodynamic therapy when the aforementioned liposomal ICG was used as the photosensitizer. A panel of TNBC cells, including MDA-MB-468, MDA-MB-231, BT-549, HCC-1806, and HCC-70 were included in a MTT assay for cell viability evaluation post PDT treatment. Several TNBC cell lines including the non-tumorigenic epithelial cell line MCF10A were employed to evaluate the selectivity of PDT. As a subtype of breast cancer, the heterogeneity of TNBCs has been recognized due to the various genomic profiles associated with differences in TNBCs. The significance of including different TNBC cell lines is two-fold. Firstly, it helps us explore the selectivity of ICG-based PDT and further elucidate the possible mechanism. Secondly, translational and/or clinical relevance is more likely to be demonstrated by including different TNBC cell lines derived from humans. The sensitivity and selectivity of our treatment were further confirmed using a clonogenic assay to determine long-term and overall cell survival and proliferation ability after PDT treatment, which is presumably more reflective of the clinical outcome. Historically, the tumor-inhibiting effect of PDT is attributed to the damage of certain organelles, such as mitochondrial or endoplasmic reticular induced by ROS or radicals, as most photosensitizers target either the cell membrane or mitochondria [14,15]. Substantial PDT-induced DNA damage may be greatly enhanced by nuclear delivery of the photosensitizer [16]. Exploration of the cellular localization of photosensitizer may shed some light on the underlying mechanism of PDT to treat cancer. Our approach was to utilize a ␥-H2AX assay to evaluate whether part of ICG might be localized in the nucleus after internalization. This is particularly relevant for the treatment of TNBCs, because they are more susceptible to DNA damage. Due to the highly targeted selectivity of treatment, potential carcinogenesis and/or mutagenesis caused by the PDT-induced DNA damage may be minimized.
Materials and methods Materials 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and L-␣-phosphatidylcholine (soy-PC) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). N-(carbonyl-methoxypolyethyleneglycol 2000)-1,2distearoyl-sn-glycero-3-phosphoethanolamine (DSPE—PEG 2000) was purchased from NOF Corporation (Tokyo, Japan). Cholesterol was purchased from Alfa Aesar (Ward Hill, MA, USA). Indocyanine green, dimethylsulfoxide (DMSO >99.9% reagent grade), formic acid, cholera toxin, hydrocortisone, insulin, MTT, crystal violet, sodium and ammonium acetate, glutaraldehyde, paraformaldehyde, bovine serum albumin, poly-L-lycine and Sephadex G-75 were obtained from Sigma—Aldrich (St. Louis, MO, USA). Acetonitrile and fetal bovine serum were acquired from Fisher Scientific (Hanover Park, IL, USA). RPMI 1640, DMEM, DMEM-F12, phosphate buffered saline, horse serum, Alexa Fluor 488 and H2AX antibody were purchased from Life Technologies (Grand Island, NY, USA). Penicillin streptomycin (PS) was purchased from Atlanta Biologicals (Lawrenceville, GA, USA) and
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epidermal growth factor was obtained from Peprotech (Rocky Hill, NJ, USA).
Tissue culture Because TNBCs are a very heterogeneous subset of breast cancer a variety of cells were included in the evaluation panel. Cell types included MDA-MB-468, HCC-1806, and HCC-70, which are grouped into the basal type basal like subgroup, as well as cell lines MDA-MB-231 and BT-549 which are sub-grouped as basal like normal type. Normal mammary epithelial cell line MCF-10A was also used to assess the treatment selectivity. These cell lines have different genotypes and phenotypes and usually exhibit varying prognosis. These cells were selected to comprise the various subset of the TNBC group as a whole to allow for a more variable outcome. Human TNBC cell lines including MDA-MB-468, MDA-MB231, BT-549, HCC-1806, HCC-70 and mammary epithelial cell line MCF-10A were graciously donated from Dr. Ruth O’Regan’s research group of the Winship Cancer Institute at Emory University. Cell lines including MDA-MB-231, BT-549, HCC-1806, and HCC-70 were cultured in RPMI 1640 and supplemented with 100 units/mL of penicillin and 100 g/mL streptomycin in addition to 10% fetal bovine serum (FBS). Cell line MDA-MB-468 was cultured with DMEM with the same supplement. Cell line MCF-10A was cultured in DMEM-F12 and supplemented with 5% horse serum, 20 ng/mL of epidermal growth factor, 0.5 mg/mL of hydrocortisone, 100 ng/mL of cholera toxin, 10 g/mL of insulin, 100 units/mL of penicillin, and 100 g/mL streptomycin. Growth media was changed every three days and cell confluence and morphology were continuously monitored.
Preparation and characterization of liposomal ICG Liposomal ICG was formulated by a standard thin film/extrusion method, and the thermosensitive formulation was comprised of DPPC:SoyPC:Chol:DSPE-PEG 2000 at a molar ratio of 100:50:30:0.5, with a mass of total lipids weighing approximately 20 mg. Formulation components were weighed into a 25 mL round bottom flask and dissolved in ∼2 mL chloroform/methanol (2:1). Then the solvent was evaporated using rotary evaporator at 37 ◦ C in order to form a uniform lipid thin film, and an in-house vacuum was used overnight to remove the residual solvent. The resulted thin film was hydrated by 1.03 × 104 M ICG aqueous solution that was briefly pre-heated to 60 ◦ C. After spinning the flask at 110 rpm by a rotary evaporator at 60 ◦ C for 60 min, the resulting vesicle suspension was extruded 19 times through an Avanti mini-extruder equipped with a 100 nm polycarbonate membrane (Avanti Polar Lipids, Alabaster, AL, USA) and maintained at 65 ◦ C. Un-encapsulated ICG was isolated by standard size exclusion chromatography using Sephadex G75. The particle size distribution of liposome solution was estimated by using a Nano Zetasizer (Malvern Instruments, Malvern, United Kingdom), and liposomal ICG was quantified by a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI—MS—MS) assay. The assays were performed using an Agilent 1200 series binary pump liquid chromatographic system interfaced to an Agilent 6410B
Please cite this article in press as: Shemesh CS, et al. Indocyanine green loaded liposome nanocarriers for photodynamic therapy using human triple negative breast cancer cells. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.02.001
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triple quadrupole mass spectrometer (Agilent, Santa Clara, CA, USA). As described previously, IR-820 was selected as a structural analog to ICG and used as a suitable internal standard [17]. Chromatographic separation was performed using an Agilent Zorbax Eclipse Plus (100 mm × 2.1 mm, 3.5 m) C18 column, and the column compartment was maintained at 25 ◦ C. The injection volume was 15 L. The mobile phase consisted of 10 mM ammonium acetate solution at pH 3.0 (adjusted by formic acid) and acetonitrile in 50:50 (v/v), and the flow rate was held at 0.45 mL/min. Samples were run using an isocratic elution. Ionization was achieved by electrospray operating in positive ion mode, and source conditions were optimized as follows: drying gas (N2 ) temperature 350 ◦ C and flow rate 12 L/min, nebulizer set at 40 psi, and capillary voltage set to 3500 V. Analysis was carried out in multiple reaction monitoring (MRM) mode with a dwell time of 100 ms and electron multiplier voltage of +400 V. For ICG, MRM transition of m/z 753→330 Da was monitored with a fragmentor voltage of 200 V and collision energy 37 V; IR-820 was monitored by MRM transition of m/z 827→330 Da with a fragmentor voltage of 185 V and collision energy of 40 V. The retention time for ICG and IR-820 was 1.5 and 3.6 min, respectively, and the total run time was 5.0 min. The limit of detection was 5.0 ng/mL and the limit of quantitation was 10.0 ng/mL. Samples were analyzed with a calibration curve between 50 and 2000 ng/mL. Internal standard was added to liposome samples whereby liposomes were lysed using a solution of 20% Triton X-100, vortexed briefly, and dried down. Samples were reconstituted and diluted in mobile phase followed by filtration using a 13 mm diameter and 0.2 m pore size Millex-LG polytetrafluoroethylene (PTFE) syringe driven filter unit (Millipore Corporation, Cork, Ireland). The procedure was validated in regards to precision and accuracy. Precision was calculated by the percent coefficient of variation (CV%) of the replicate procedure assayed at 100 and 1,000 ng/mL: the within-run CV% were 2.5% and 1.8%, respectively, while the betweenrun CV% were 4.9% and 5.2%. Accuracy was calculated as the absolute value of percent error at both 100 and 1000 ng/mL with values of 3.6% and 3.1% obtained.
Photodynamic therapy ICG solutions for cell culture were prepared by diluting ICG stock solution (in DMSO) with fresh cell culture growth media under sterile conditions after thawing the stock solution to room temperature. The concentration of DMSO was kept under 0.1%. In the photodynamic therapy care was taken to maintain cells under dark conditions when possible by covering plates with aluminum foil during brief transport and irradiating in a dark environment. Firstly, cells were incubated with ICG either in free solution or in liposomal formulation (ICG concentration ranging from 15 to 37.5 M) in the dark for 16 or 20 h, then cell media was replaced with 1× PBS to ensure uniform and maximum laser penetration. A fiber-coupled laser system with a wavelength of 808 nm (Edmunds Optics, Barrington, NJ, USA) was used to excite ICG. The laser system was calibrated in terms of power output ranging between 0 and 450 mW. Laser irradiation was delivered via a fiber (0.5 m in length and 100 m in diameter) coupled with a laser diode. A uniform
distribution of light was maintained in all experiments, which was capable of illuminating the entire cell containing wells of interest. The size of laser light remained the same for the same assay but varied in different assays in order to fully cover the cells. The diameter of the beam (spot size) varied slightly for certain procedures depending on the type of plate used (96 well or 6 well) but remained constant within batch. Temperature effects were monitored to be negligible and were very consistent. The different size of light was achieved by adjusting the distance of the fiber optic above the plate. The irradiation time was 3 min for all the assays. The light does were expressed by irradiation energy per unit area (J/cm2 ), which is calculated by multiplying the energy output per area (W/cm2 ) with irradiation time (180 s). The light dose ranging from 1 to 14 J/cm2 was evaluated. During the irradiation, the temperature of the cell media was periodically monitored. Upon the completion of the irradiation, PBS was removed and replaced with growth media, and then cells were returned to the dark in a humidified incubator for different time intervals for various assays.
MTT assay Cell viability after PDT treatment was investigated by a MTT assay. Briefly: 8,000 cells per well were seeded into 96 well plates in a humidified incubator overnight. The following day media was removed, and ICG, either in solution or liposomal formulation, at various concentrations was added to the appropriate wells; controls were processed in the same manner but with fresh media. The cell plates were immediately returned to the incubator for 16 or 20 h to facilitate cell internalization of the photosensitizer. The media was then replaced with 1× PBS. PDT or laser alone control groups were irradiated with laser at 808 nm at different light doses for 3 min, whereby 1× PBS was removed and replaced with 100 L of appropriate growth media. Cells were then returned to the incubator. After a 24-h incubation, 10 L of a 5 mg/mL solution of 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) dissolved in PBS was added and cells were returned to the incubator. After 3 h, media was removed from all wells, and 100 L of DMSO was added to solubilize the purple formazan crystal formed by actively metabolizing cells with maintained structural integrity, and the absorbance was measured at 570 nm using a Synergy HT plate reader (Bio-Tek Instruments, Winooski, VT, USA). Cell viability was determined by comparing each treatment group to the untreated control groups. All experiments were carried out in triplicate and also repeated three independent times, and data were plotted as mean ± SD.
Clonogenic assay A clonogenic assay was performed by plating 20,000 cells per well into 6 well plates and left to incubate in a humidified incubator overnight for cell adhesion. The following day media was removed whereby 37.5 of M indocyanine green in free solution was added and cells were further incubated overnight for 16 h. The media was replaced with PBS and cells were irradiated according to PDT protocol (light dose
Please cite this article in press as: Shemesh CS, et al. Indocyanine green loaded liposome nanocarriers for photodynamic therapy using human triple negative breast cancer cells. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.02.001
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Photodynamic therapy using human triple negative breast cancer cells was 3.5 or 8 J/cm2 ). After PDT treatment, cells were immediately trypsinized, counted, diluted, and plated at 500 cells per well. Plates were returned to the incubator for 8—12 additional days to allow time for 5—10 cycles of cell divisions in order for colonies to contain 50 or more cells each. Cells were initially examined microscopically before staining to ensure colonies consisted of at least 50 cells. Then cell media was removed and cells were rinsed gently with 1× PBS, followed by the addition of 1 mL of a 6% aqueous glutaraldehyde solution containing 0.5% crystal violet. Cells were left to incubate with the adhesive and dye for 30 min, whereby plates were carefully submerged in water for stain removal. After air drying, plates were examined and colonies were scored with the percentage of colony formation calculated by comparing the number of colonies from each treatment group to that from the group without laser irradiation (untreated control). All experiments were carried out in triplicate and data were plotted as mean values ± SD.
Gamma-H2AX immunofluorescence assay Gamma-H2AX assay was performed by at first coating glass coverslips placed into 12 well plates with poly-L-lysine. MDA-MB-468 and BT-549 cells were seeded at a density of 80,000 cells per well and left to incubate overnight in a humidified incubator. The following day a 37.5 M ICG in free solution was added to the appropriate wells and left to incubate for 20 h, whereby ICG was then removed and replaced with 1× PBS. Cells were irradiated with a light dose of 3.5, 8, and 14 J/cm2 . After irradiation, PBS was replaced with fresh media and the culture plate was returned to a humidified incubator for 30 min. Media was then removed from the plate and cells were washed twice with 2 mL of ice-cold 1× PBS. Cells were fixed using 1 mL of 3%
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paraformaldehyde for 10 min, whereby cells were rinsed with ice-cold 1× PBS twice and permeabilized using 1 mL of 0.5% Triton-X-100 in PBS and left to incubate for 10 min. After rinsing twice with ice-cold 1× PBS, cells were blocked using 1 mL of 5.0% bovine serum albumin (BSA) in PBS and left to incubate for 15 min at room temperature. Cells were incubated with the primary antibody solution which contained ␥-H2AX and 1% BSA in PBS at a 1:4500 ratio for 1 h at 37 ◦ C (Abcam, Cambridge, MA, USA). After thoroughly washing cells of primary antibody, cells were incubated for an additional 30 min in a secondary antibody with a concentration of 1:500, using Alexa Fluor 488 in 1× PBS. To control for background staining, secondary antibody control samples were incubated with 1% BSA and incubated under the same conditions as the primary control. After incubation samples were rinsed with PBS three times and mounted to glass slides using mounting media with 4 ,6-diamidino-2-phenylindole (DAPI). Slides were dried and stored at 4 ◦ C. Results were analyzed under oil immersion 63× magnification, and foci were counted using an Observer Z1 fluorescent microscope with AxioVision software (Carl Zeiss, Thornwood, NY, USA). Cells exhibiting more than 6 distinct foci were scored as positive, and percentage of foci positive cells was determined by analyzing 100 healthy cells and then compared to the untreated control group. All experiments were carried out in triplicate and data were plotted as mean values ± SD.
Statistical analysis All experiments were performed in triplicate and the data were expressed as mean plus and minus the standard error of the mean. Analysis of variance (ANOVA) and Student’s t-test were used to determine the statistically significant difference among different groups when appropriate.
Figure 1 Cell viability of HCC-1806 cells assessed by a MTT assay after PDT treatment with 37.5 M ICG either in free solution or encapsulated in the thermosensitive liposomes. Cells were incubated with ICG for 16 h. Solid bars represent ICG in free solution, while cross-hatched bars represent liposomal ICG. Data were presented as the mean cell viability with standard deviations after triplicate analysis.
Please cite this article in press as: Shemesh CS, et al. Indocyanine green loaded liposome nanocarriers for photodynamic therapy using human triple negative breast cancer cells. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.02.001
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C.S. Shemesh et al. after measurements taken from three independent batches. The concentration of encapsulated ICG ranged from 446.5 to 548.4 M, determined by the optimized LC-ESI-MS-MS assay. Based on our previous research [12], liposomal ICG demonstrated excellent short term stability at 4 ◦ C for up to 4 days in regards to particle size and ICG concentration. Formulations used in this study were prepared fresh or up to 24 h prior.
Cell viability assessment after PDT treatment Figure 2 Cell viability of HCC-1806 cells assessed by a MTT assay after PDT treatment using 15, 25, and 37.5 M ICG, either in free solution or encapsulated in the thermosensitive liposomes. Cells were incubated with ICG for 16 h. Solid bars represent ICG in free solution, while cross-hatched bars represent liposomal ICG. Data were presented as the mean cell viability with standard deviations after triplicate analysis.
Results Liposomal ICG preparation and characterizations Thermosensitive liposomes that encapsulated ICG ranged from 51 to 85 nm in diameter with an average of 71 ± 10 nm
Optimization of PDT treatment protocol was carried out with HCC-1806 cells. During the brief irradiation the temperature of the PBS solution remained very close to room temperature at 22.7 ◦ C and did not rise. Initially, three doses of ICG: 15, 25, and 37.5 M, respectively, either in free solution or formulated with thermosensitive liposomes, were evaluated as the photosensitizer. Compared to the untreated control, the group that was treated with laser alone at a dose of 14 J/cm2 (the highest tested) showed 100% cell viability while 37.5 M of ICG alone, either in free solution or liposomal formulation, showed slightly decreased cell viability (Fig. 1). After the treatment, cell viability was decreased significantly but similar between groups using liposomal ICG or free ICG solution as the photosensitizer. The potency of the PDT treatment was closely correlated with ICG
Figure 3 Cell viability of MDA-MB-468 (A), HCC-1806 (B), MDA-MB-231 (C), HCC-70 (D), BT-549 (E), and MCF-10A (F) evaluated by a MTT assay after PDT treatment with 37.5 M ICG either in free solution or encapsulated in the thermosensitive liposomes. Cells were incubated with ICG for 20 h. Solid bars represent ICG in free solution, while cross-hatched bars represent liposomal ICG. Data were presented as the mean cell viability with standard deviation after triplicate analysis.
Please cite this article in press as: Shemesh CS, et al. Indocyanine green loaded liposome nanocarriers for photodynamic therapy using human triple negative breast cancer cells. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.02.001
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Photodynamic therapy using human triple negative breast cancer cells
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DNA damage evaluation by Gamma-H2AX (␥-H2AX) assay
Figure 4 Comparison of cell viability of MDA-MB-468 (dashed lines) and HCC-1806 (solid lines) with MDA-MB-231, HCC-70, BT549, and MCF-10A after PDT treatment with 37.5 M liposomal ICG and 14 J/cm2 laser irradiation. Cell viability was evaluated using a MTT assay and data were presented as the mean cell viability with standard deviation after triplicate analysis. Student t-test was performed to compare cell viability of MDA-MB-468 and HCC-1806 to that of MDA-MB-231, HCC-70, BT-549, and MCF10A; P value < 0.001 are indicated by three asterisks (***) and P value < 0.01 are indicated by two asterisks (**).
concentrations, where 37.5 M of ICG was the most effective in inhibiting the growth of HCC-1806 cells (Fig. 2). Therefore, 37.5 M of ICG was used for the subsequent experiments described in this article. The data in Figs. 1 and 2 also indicate that the efficacy of PDT depends on the irradiation energy of the laser used to excite ICG in the treatment. Upon expansion of the cytotoxicity studies to four other TNBC cell lines, a similar light dose response was observed (Fig. 3). Once again, liposomal ICG showed similar inhibitory potency compared with ICG in free solution. The results in Fig. 3 also suggested that PDT treatment inhibited the growth of different TNBC cell lines to different extents. The variable responses of treatment may be related to differing phenotypes, gene expression, molecular behavior, and DNA repair processes of these cells. After PDT treatment with a 14 J/cm2 laser dose, cell viability of MDA-MB-468 and HCC-1806 is significantly lower than that of MDA-MB-231, BT-549, and HCC-70 cell lines (P < 0.001). Furthermore, nontumorigenic mammary epithelial cell line MCF-10A is shown to be more resistant to the treatment when compared to MDA-MB-468 and HCC-1806 cell lines (P < 0.01) (Fig. 4).
Clonogenic assay assessment In our experiment, 37.5 M of ICG in free solution was used as the photosensitizer, as the MTT assay revealed that the liposomal formulation of ICG has negligible impact on the in vitro PDT treatment. Light doses of 3.5 J/cm2 and 8 J/cm2 were selected in PDT in order to observe colony formation with certain cell lines. Among the five cell lines evaluated, HCC-1806 and MDA-MB-468 showed higher sensitivity toward the treatment therefore having fewer colonies formed (Figs. 5 and 6), confirming the findings in the MTT assay experiment.
In an effort to reveal the possible role of DNA damage in PDT treatment, a ␥-H2AX assay based on immunofluorescence microscopy was conducted to examine if the treatment could induce DSBs. We specifically examined MDA-MB-468 and BT-549 cell lines due to their significantly different response to the treatment. Indeed, ␥-H2AX foci were observed 30 min after the treatment even with a low laser dose of 3.5 J/cm2 , indicating some level of DSBs induced by PDT (Fig. 7). The degree of DSBs seemed positively correlated to the dose of laser, as more ␥-H2AX foci were found with higher doses of laser. However, a similar amount of DSBs were seen for MDA-MB-468 and BT-549 (pictures of BT-549 cells not shown). This is further confirmed by the quantitative comparisons of these two cell lines as shown in Fig. 8.
Discussion Presented in this article was the exploration of liposomal ICG as the photosensitizer in PDT to treat TNBCs. ICG concentration, liposomal formulation, and laser dose were investigated in PDT on a panel of five TNBC cell lines. The results from both the MTT assay and clonogenic assay indicate PDT treatment using ICG is effective in inhibiting the growth of TNBC cell lines in vitro, and liposomal formulation of ICG did not compromise the phototoxicity, suggesting that thermosensitive liposomes could serve as the delivery platform for ICG in a PDT treatment. While a slight loss of cell viability was observed for HCC-1806 cells after a 16 h incubation with ICG was observed, this was found to be unique for this cell line as these cells were much more sensitive compared to the remaining panel. This effect may be minimized by incubation for shorter time intervals at a lower concentration while exposing to longer irradiation times. Interestingly, MDA-MB-468 and HCC-1806 cell lines are particularly sensitive to the treatment. If further classification is applied according to the gene expression profile, MDA-MB-468 and HCC-1806 belong to basal-like subtype of TNBC cell lines. This subtype of TNBCs is significantly more sensitive to DNA damage agents such as cisplatin due to the elevated cell-cycle and DNA damage response genes [18]. Similarly, PDT is more effective in inhibiting these two basallike TNBC cell lines, and this observation suggests that DNA damage might play a significant role in the PDT treatment. PDT induced DNA damage may be unique to the type of photosensitizer, based on different physiochemical properties, which may be highly dependent on the ultimate localization inside of a cell. Nuclear delivery of photosensitizer may be facilitated using high-cost peptides, nuclear localization signals, or other sophisticated delivery systems. However, most photosensitizers target either cell membranes or mitochondria. To the best of our knowledge, PDT induced DSB DNA damage by PS itself was firstly demonstrated in our work. Due to the extreme short life time and diffusion distance of ROS the intracellular damage by PDT may be limited to the subcellular location of the photosensitizer. Therefore, nuclear targeting of the photosensitizer is necessary for achieving significant DNA damage. Although we were not
Please cite this article in press as: Shemesh CS, et al. Indocyanine green loaded liposome nanocarriers for photodynamic therapy using human triple negative breast cancer cells. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.02.001
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Figure 5 A clonogenic assay was carried out with MDA-MB-468, HCC-1806, MDA-MB-231, HCC-70, BT-549, and MCF-10A cell lines after PDT treatment with 37.5 M ICG solution as the photosensitizer and laser doses was 3.5 or 8 J/cm2 . Images are representative colonies for 500 seeded cells after 8—12 days of incubation.
able to study the subcellular location of ICG, the results from the ␥-H2AX revealed obvious DSB DNA damage after the treatment, which may indicate the partial nuclear delivery of ICG.
Earlier investigations suggested that PDT mainly targeted mitochondria and endoplasmic reticulum to induce apoptosis in tumor cells [14]. However, our preliminary studies suggested that the PDT may kill cancer cells through a
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Figure 6 Quantitative comparison of percentage of colony formation among MDA-MB-468, HCC-1806, MDA-MB-231, HCC70, BT-549, and MCF-10A cell lines after PDT treatment with 37.5 M ICG solution as the photosensitizer and laser dose was 3.5 or 8 J/cm2 . Data were presented as the mean colony formation percentage with standard deviation after triplicate analysis.
different mechanism. As observed by the MTT and clonogenic assays, basal-like cell lines HCC-1806 and MDA-MB-468 are more sensitive to the treatment especially compared to mesenchymal-like cell lines BT-549 and MDA-MB-231. A similar trend was reported for DNA damage agents such as cisplatin by other investigators [18]. Hence, we speculated that PDT may kill cancer cells through DNA damage by ROS or radicals, which have shown to cause direct or indirect double-strand breaks (DSBs) among DNA [19]. DSBs are generally considered to be the most toxic DNA lesion, and a single DSB can ultimately lead to cell death if the cell is left without efficient DNA repair [20]. Followed by DSBs, histone H2AX is rapidly phosphorylated to produce ␥-H2AX, and measurement of ␥-H2AX foci with an immunocytochemical assay was utilized as a sensitive method to detect and quantify DSBs [21,22]. The results from the ␥-H2AX assay showed an increased level of ␥-H2AX foci after the PDT, suggesting that DNA damage, particularly DSBs, may be one of mechanisms that underlie the PDT’s inhibition on cancer cell growth. MDA-MB-468 and BT-549 cell lines showed similar level of ␥-H2AX foci after the treatment. The difference in cell viability after treatment found with these two cell lines may result from their different efficiency to repair DSBs in response to DNA damage induced by the PDT treatment. PDT may lead to more extensive cell death via DNA damage among basal-like TNBC cells such as MDA-MB468 and HCC-1806, due to their elevated DNA repair gene mutation. In summary, we have demonstrated that PDT using ICG as the photosensitizer can effectively inhibit the growth of several TNBC cell lines in vitro, particularly the basal-like cell lines MDA-MB-468 and HCC-1806. In addition, liposomal ICG performed similarly to free ICG solution in the in vitro PDT treatment, suggesting the potential application of the thermosensitive liposome formulation for the delivery of ICG in a PDT treatment. Although a complete understanding of this liposomal ICG formulation in the PDT treatment begs for more detailed investigations on the cellular uptake, the stability of liposomal ICG, the release characteristics of ICG,
Figure 7 Induction of ␥-H2AX foci in MDA-MB-468 cells 30 min after PDT treatment with 37.5 M ICG solution as the photosensitizer. Increased levels ␥-H2AX foci were observed with higher laser doses. A, B, C, D, and E are corresponding to laser control, ICG control, PDT treatment with 3.5, 8, and 14 J/cm2 , respectively. Very similar levels of ␥-H2AX foci were observed with the BT-549 cell line (pictures not included). DAPI, 4 ,6-diamidino-2phenylindole (fluorescent stain). Only cells exhibiting more than 6 foci were scored which were found in PDT treatment groups.
etc., a higher in vivo efficacy is predicted based on the targeted delivery of ICG to the tumor area from our previous in vivo imaging studies [12]. We also investigated DSBs on DNA after treatment by a ␥-H2AX assay, which revealed a certain degree of DNA damage 30 min after the treatment, even with a relatively low light dose. This is indicative of ROS and radicals produced during PDT, which may cause DNA damage and lead to cell death, but further investigations are necessary to validate this statement. The primary aim of this study was to investigate the effectiveness of liposomal ICG using a thermosensitive formulation as the photosensitizer in photodynamic therapy to treat TNBCs. Both MTT and clonogenic assays revealed PDT is effective at inhibiting the growth of TNBCs to different extents, which might due to the reason that TNBC is a very heterogeneous
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References
Figure 8 Quantitative comparison of percentage of ␥-H2AX foci positive cells between MDA-MB-468 and BT-549 cell lines after PDT treatment with 37.5 M ICG solution as the photosensitizer. Data were presented as the mean foci positive cells percentage with standard deviation after triplicate analysis.
type of cancer with a wide variability in receptor expression. Moreover, the thermosensitive liposome formulation does not compromise potency of the in vitro PDT treatment. DNA damage by PDT was investigated by a ␥-H2AX assay, suggesting that DNA damage could be the main event leading to the extensive cancer cell death. Thermosensitive ICG loaded liposomes would ideally be administered to a patient as a sterile parenteral formulation reconstituted in saline for intravenous administration. After i.v. administration, ICG is confined to the circulation due to its high binding affinity to plasma proteins; in addition, the half-life of free ICG in humans is about 3—5 min and it is also prone to chemical and photo-degradation. Hence the liposome vesicles are essential to protect ICG, prolong the circulation time, and eventually deliver ICG to the tumor site for subsequent illumination. This therapy has the advantage to be conducted multiple times by repeat administration and illumination in a targeted approach. We would anticipate this therapy to hold promise to be exploited as a form of personalized medicine tailored to the patient. In an ideal setting a physician may adjust the photosensitizer dosage and light energy for the patient to obtain the optimal therapeutic response. However, due to the aggressive nature of TNBC and the limitations of a localized PDT treatment, this therapy may offer potential for early-treatment of TNBC either alone or in a combination therapy.
Conflict of interest The authors report that there are no conflicts of interest regarding any aspects of this manuscript.
Acknowledgements We thank Dr. LaTonia Taliaferro-Smith and Tongrui Liu of the Winship Cancer Institute for their valuable assistance in obtaining the various TNBC cells. This research was carried out using the facilities of the Department of Pharmaceutical Sciences at Mercer University College of Pharmacy in addition to the Winship Cancer Institute located at Emory University. This work was funded in part by the Georgia Cancer Coalition’s Cancer Research Award.
[1] American Cancer Society. Cancer Facts & Figures; 2012. Available at: http://www.cancer.org/acs/groups/ content/@epidemiologysurveilance/documents/document/ acspc-031941.pdf [accessed November 2012]. [2] de-Ruijter TC, Veeck J, de-Hoon JPJ, van-Engeland M, TjanHeijnen VC. Characteristics of triple-negative breast cancer. J Cancer Res Clin Oncol 2011;137:183—92. [3] Gelmon K, Dent R, Mackey JR, Laing K, McLeod D, Verma S. Targeting triple-negative breast cancer: optimising therapeutic outcomes. Ann Oncol 2012;23:2223—34. [4] Gluz O, Liedtke C, Gottschalk N, Pusztai L, Nitz U, Harbeck N. Triple-negative breast cancer—–current status and future directions. Ann Oncol 2009;20:1913—27. [5] Pal SK, Childs BH, Pegram M. Triple negative breast cancer: unmet medical needs. Breast Cancer Res Treat 2011;125:627—36. [6] Dolmans DE, Fukumura D, Jain RK. Photodynamic therapy for cancer. Nat Rev Cancer 2003;3:380—7. [7] Alli E, Sharma VB, Sunderesakumar P, Ford JM. Defective repair of oxidative DNA damage in triple-negative breast cancer confers sensitivity to inhibition of poly(ADP-ribose) polymerase. Cancer Res 2009;69:3589—96. [8] Ghoroghchian PP, Therien MJ, Hammer DA. In vivo fluorescence imaging: a personal perspective. Wiley Interdiscip Rev Nanomed Nanobiotechnol 2009;1:156—67. [9] Hilderbrand SA, Weissleder R. Near-infrared fluorescence: application to in vivo molecular imaging. Curr Opin Chem Biol 2010;14:71—9. [10] Licha K, Riefke B, Ntziachristos V, Becker A, Chance B, Semmler W. Hydrophilic cyanine dyes as contrast agents for nearinfrared tumor imaging: synthesis, photophysical properties and spectroscopic in vivo characterization. Photochem Photobiol 2000;72:392—8. [11] Saxena V, Sadoqi M, Shao J. Degradation kinetics of indocyanine green in aqueous solution. J Pharm Sci 2003;92: 2090—7. [12] Turner DC, Moshkelani D, Shemesh CS, Luc D, Zhang H. Near-infrared image-guided delivery and controlled release using optimized thermosensitive liposomes. Pharm Res 2012;29:2092—103. [13] Zhu L, Banerjee RK, Salloum M, Bachmann A, Flower RW. Temperature distribution during ICG-dye-enhanced laser photocoagulation of feeder vessels in treatment of AMDrelated choroidal neovascularization. J Biomech Eng 2008;130: 031010. [14] Shahzidi S, Cunderlikova B, Wiedlocha A, Zhen Y, Vasovic V, Nesland JM, et al. Simultaneously targeting mitochondria and endoplasmic reticulum by photodynamic therapy induces apoptosis in human lymphoma cells. Photochem Photobiol Sci 2011;10:1773—82. [15] Ali-Seyed M, Bhuvaneswari R, Soo KC, Olivo M. PhotolonTM —– photosensitization induces apoptosis via ROS-mediated crosstalk between mitochondria and lysosomes. Int J Oncol 2011;39:821—31. [16] Rosenkranz AA, Jans DA, Sobolev AS. Targeted intracellular delivery of photosensitizers to enhance photodynamic efficiency. Immunol Cell Biol 2000;78:452—64. [17] Chen CY, Fancher RM, Ruan Q, Marathe P, Rodrigues AD, Yang Z. A liquid chromatography tandem mass spectrometry method for the quantification of indocyanine green in dog plasma and bile. J Pharm Biomed Anal 2008;47:351—9. [18] Lehmann BD, Bauer JA, Chen X, Sanders ME, Chakravarthy AB, Shyr Y, et al. Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Invest 2011;121:2750—67.
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Photodiagnosis and Photodynamic Therapy (2014) xxx, xxx—xxx
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/pdpdt
Indocyanine green loaded liposome nanocarriers for photodynamic therapy using human triple negative breast cancer cells Colby S. Shemesh a, Claire W. Hardy b, David S. Yu b, Brian Fernandez a, Hailing Zhang Ph.D. a,∗ a
Drug Delivery Laboratory, Department of Pharmaceutical Sciences, College of Pharmacy, Mercer University, 3001 Mercer University Drive, Atlanta, GA 30341, United States b Department of Radiation Oncology, Emory University School of Medicine, 1365 Clifton Road Northeast, Atlanta, GA 30322, United States
KEYWORDS Indocyanine green (ICG); Photodynamic therapy (PDT); Thermosensitive liposome; Triple negative breast cancer (TNBC); DNA damage
∗
Summary Background: The goal of the current research is to evaluate the potential of photodynamic therapy (PDT) in the treatment of triple negative breast cancer (TNBC) with the development of a theranostic thermosensitive liposome platform to deliver indocyanine green (ICG) as the near-infrared (NIR) photosensitizer excited by an 808 nm diode laser. Methods: In the PDT protocol, an optimized thermosensitive liposome formulation is investigated to formulate ICG as the photosensitizer, which is exited by laser light at the wavelength of 808 nm delivered by a fiber-coupled laser system. ICG in both free solution and thermosensitive liposomal formulation were evaluated as the NIR photosensitizer and compared in the PDT treatment on a panel of triple negative breast cancer cell lines along with the nontumorigenic mammary epithelial cell line MCF-10A. In addition to cytotoxicity, and clonogenic survival assessment, the role of DNA double strand break damage was evaluated. Results: Both MTT and clonogenic assays revealed that PDT using ICG inhibited the growth of several TNBC cell lines as well as the non-tumorigenic human breast epithelial cell line MCF-10A; and the liposomal formulation of ICG did not compromise the in vitro treatment potency, though free ICG performed slightly more effective in certain cell lines, but was not statistically significant. Cell viability was dose dependent in regards to ICG concentration and irradiation energy. Interestingly, PDT using the described protocol was more potent to inhibit the growth of MDA-MB-468 and HCC-1806 cells, coinciding with the observation that these cells are more sensitive toward DNA damaging agents. In comparison, cell lines HCC-70, BT-549, and
Corresponding author. Tel.: +1 678 547 6242; fax: +1 678 547 6423. E-mail address: zhang [email protected] (H. Zhang).
http://dx.doi.org/10.1016/j.pdpdt.2014.02.001 1572-1000/© 2014 Elsevier B.V. All rights reserved.
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C.S. Shemesh et al. MCF-10A were found to have less of an inhibitory effect. Furthermore, substantial DNA double strand breaks (DSBs) were observed 30 min after the PDT treatment via a ␥-H2AX staining assay. PDT induced DNA damage has the potential to lead to mutagenicity, which may have various responses depending on the repair capabilities of the cells. Conclusion: Our results suggest that PDT using indocyanine green loaded liposomes were effective in inhibiting tumor cell growth to varying extents with higher responses observed for MDA-MB-468 and HCC-1806 cells. © 2014 Elsevier B.V. All rights reserved.
Introduction In 2012 an estimated 226,000 new breast cancer cases were expected to develop in the United States with nearly 40,000 deaths indicating breast cancer as the leading cause of cancer mortality among women [1]. As a subclass of breast cancer, triple negative breast cancers (TNBCs) are characterized by neither expressing hormone receptors (estrogen or progesterone receptors) nor overexpressing human epidermal growth factor receptor 2 (HER2) [2]. About 15% of all diagnosed breast cancers belong to this subclass. Due to the characteristic genetic profiles, selective therapeutics that target hormonal or HER2 receptors are generally ineffective to treat TNBCs. Despite initial response to chemo/radio therapy treatment, relapse and recurrence occur more often in this subclass [3]. Poor prognosis and high mortality of patients with TNBCs have provoked a wide array of research aimed at seeking more effective treatments including recently developed poly(ADP-ribose) polymerase inhibitor and epidermal growth factor receptor targeted therapy [3], but the clinical outcomes of these new treatments remain controversial [3—5]. This manuscript encompasses our attempt at developing photodynamic therapy (PDT) as a potential treatment for TNBCs. During PDT, a photosensitizer is first administered, followed by excitation of the photosensitizer by exposing the tumor area to light of a specific wavelength. Then the excited photosensitizer may undergo a type I reaction to produce radicals and/or a type II reaction to generate reactive oxygen species (ROS) [6]. Moreover, PDT is based on the type II reaction that occurs to transfer energy of the activated photosensitizer to ground state oxygen forming singlet oxygen. The singlet oxygen and other ROS may then react with a large array of biomolecules in cells, such as proteins, nucleic acids, and lipids. Previous investigations have suggested that TNBC cell lines are more susceptible to H2 O2 through oxidative DNA damage due to mutations in the breast cancer susceptibility gene 1 (BRCA1) [7]. Therefore we hypothesized that TNBCs may be effectively treated by PDT, in which ROS and radicals are produced and cause DNA damage. We were particularly interested in using indocyanine green (ICG) as a photosensitizer. As a near infrared (NIR) dye used clinically to visualize the human vasculature, ICG is excited by light around 800 nm [8], which is of a longer wavelength compared to 630 nm that is used to excite photofrin (the photosensitizer used in clinical oncology) [6]. A light with a longer wavelength is presumably capable of penetrating deeper into tissue to treat more in-depth cancers owing to its lower absorption and scattering by endogenous biomolecules and intracellular organelles
[9]. Of course limitations in the feasibility of a localized photodynamic therapy exist due to very limited effects over 10 mm within tissue. However, ICG itself is not suitable for cancer treatment because of its very short circulation halflife [10] and low in vivo photo stability [11]. We attempted to address this issue by developing thermosensitive liposomes to formulate ICG as the photosensitizer in PDT. In our previous studies, liposomal ICG using a thermosensitive formulation was successfully and preferentially delivered to the tumor, as shown in the in vivo NIR imaging [12]. In addition to targeted delivery of ICG to tumor areas due to the enhanced permeability and retention effect (EPR), we expect the thermosensitive formulation will offer the potential for controlled release. Due to the EPR phenomena, in the translation to an in vivo procedure of PDT, tumor tissue may be selectively targeted and the PDT effect may be preferentially localized to tumor tissues while avoiding damage to normal tissue. The formulation is comprised of DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), soyPC (L-␣-phosphatidylcholine), cholesterol, and DSPC-PEG 2000 (N-(carbonyl-methoxypolyethyleneglycol 2000)-1,2distearoyl-sn-glycero-3-phosphoethanolamine) with a transition temperature around 42 ◦ C. We observed excellent stability at 37 ◦ C with this formulation, yet burst release was achieved at 42 ◦ C [12]. When ICG molecules encapsulated in thermosensitive liposomes are excited, we expect they will produce an increase in temperature within the liposomes [13]. The resulting hyperthermia may then increase the permeability of bilayers leading to the release of ICG, thereby reacting with O2 to produce ROS and radicals. Thermosensitive liposomes have the advantage of offering carrier stability at normal physiological temperatures with an increased permeation of the lipid bilayer above transition temperature. These phenomena may be exploited to better facilitate release of cargo in a more targeted approach by application of a very brief, limited, and localized hyperthermia with the intention to better minimize treatment resistance. Uniform heating of a tumorous tissue may be facilitated by utilization of a collimating lens to ensure a uniform distribution and focusing of the light. Liposome ICG is ultimately excited by NIR irradiation; it is hypothesized that ROS may diffuse freely through the liposomal bilayer due to their small size. In addition, ICG may also be released by an increase in temperature in liposomes due to the NIR excitation, which could react with the oxygen in the surrounding environment of cells or tissue to produce ROS. However, the direct investigation of releasing ICG from liposomes by laser irradiation is hampered by the high reactivity of excited ICG. Therefore, our initial focus was to evaluate the inhibition effectiveness of TNBC cell lines by
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Photodynamic therapy using human triple negative breast cancer cells photodynamic therapy when the aforementioned liposomal ICG was used as the photosensitizer. A panel of TNBC cells, including MDA-MB-468, MDA-MB-231, BT-549, HCC-1806, and HCC-70 were included in a MTT assay for cell viability evaluation post PDT treatment. Several TNBC cell lines including the non-tumorigenic epithelial cell line MCF10A were employed to evaluate the selectivity of PDT. As a subtype of breast cancer, the heterogeneity of TNBCs has been recognized due to the various genomic profiles associated with differences in TNBCs. The significance of including different TNBC cell lines is two-fold. Firstly, it helps us explore the selectivity of ICG-based PDT and further elucidate the possible mechanism. Secondly, translational and/or clinical relevance is more likely to be demonstrated by including different TNBC cell lines derived from humans. The sensitivity and selectivity of our treatment were further confirmed using a clonogenic assay to determine long-term and overall cell survival and proliferation ability after PDT treatment, which is presumably more reflective of the clinical outcome. Historically, the tumor-inhibiting effect of PDT is attributed to the damage of certain organelles, such as mitochondrial or endoplasmic reticular induced by ROS or radicals, as most photosensitizers target either the cell membrane or mitochondria [14,15]. Substantial PDT-induced DNA damage may be greatly enhanced by nuclear delivery of the photosensitizer [16]. Exploration of the cellular localization of photosensitizer may shed some light on the underlying mechanism of PDT to treat cancer. Our approach was to utilize a ␥-H2AX assay to evaluate whether part of ICG might be localized in the nucleus after internalization. This is particularly relevant for the treatment of TNBCs, because they are more susceptible to DNA damage. Due to the highly targeted selectivity of treatment, potential carcinogenesis and/or mutagenesis caused by the PDT-induced DNA damage may be minimized.
Materials and methods Materials 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and L-␣-phosphatidylcholine (soy-PC) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). N-(carbonyl-methoxypolyethyleneglycol 2000)-1,2distearoyl-sn-glycero-3-phosphoethanolamine (DSPE—PEG 2000) was purchased from NOF Corporation (Tokyo, Japan). Cholesterol was purchased from Alfa Aesar (Ward Hill, MA, USA). Indocyanine green, dimethylsulfoxide (DMSO >99.9% reagent grade), formic acid, cholera toxin, hydrocortisone, insulin, MTT, crystal violet, sodium and ammonium acetate, glutaraldehyde, paraformaldehyde, bovine serum albumin, poly-L-lycine and Sephadex G-75 were obtained from Sigma—Aldrich (St. Louis, MO, USA). Acetonitrile and fetal bovine serum were acquired from Fisher Scientific (Hanover Park, IL, USA). RPMI 1640, DMEM, DMEM-F12, phosphate buffered saline, horse serum, Alexa Fluor 488 and H2AX antibody were purchased from Life Technologies (Grand Island, NY, USA). Penicillin streptomycin (PS) was purchased from Atlanta Biologicals (Lawrenceville, GA, USA) and
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epidermal growth factor was obtained from Peprotech (Rocky Hill, NJ, USA).
Tissue culture Because TNBCs are a very heterogeneous subset of breast cancer a variety of cells were included in the evaluation panel. Cell types included MDA-MB-468, HCC-1806, and HCC-70, which are grouped into the basal type basal like subgroup, as well as cell lines MDA-MB-231 and BT-549 which are sub-grouped as basal like normal type. Normal mammary epithelial cell line MCF-10A was also used to assess the treatment selectivity. These cell lines have different genotypes and phenotypes and usually exhibit varying prognosis. These cells were selected to comprise the various subset of the TNBC group as a whole to allow for a more variable outcome. Human TNBC cell lines including MDA-MB-468, MDA-MB231, BT-549, HCC-1806, HCC-70 and mammary epithelial cell line MCF-10A were graciously donated from Dr. Ruth O’Regan’s research group of the Winship Cancer Institute at Emory University. Cell lines including MDA-MB-231, BT-549, HCC-1806, and HCC-70 were cultured in RPMI 1640 and supplemented with 100 units/mL of penicillin and 100 g/mL streptomycin in addition to 10% fetal bovine serum (FBS). Cell line MDA-MB-468 was cultured with DMEM with the same supplement. Cell line MCF-10A was cultured in DMEM-F12 and supplemented with 5% horse serum, 20 ng/mL of epidermal growth factor, 0.5 mg/mL of hydrocortisone, 100 ng/mL of cholera toxin, 10 g/mL of insulin, 100 units/mL of penicillin, and 100 g/mL streptomycin. Growth media was changed every three days and cell confluence and morphology were continuously monitored.
Preparation and characterization of liposomal ICG Liposomal ICG was formulated by a standard thin film/extrusion method, and the thermosensitive formulation was comprised of DPPC:SoyPC:Chol:DSPE-PEG 2000 at a molar ratio of 100:50:30:0.5, with a mass of total lipids weighing approximately 20 mg. Formulation components were weighed into a 25 mL round bottom flask and dissolved in ∼2 mL chloroform/methanol (2:1). Then the solvent was evaporated using rotary evaporator at 37 ◦ C in order to form a uniform lipid thin film, and an in-house vacuum was used overnight to remove the residual solvent. The resulted thin film was hydrated by 1.03 × 104 M ICG aqueous solution that was briefly pre-heated to 60 ◦ C. After spinning the flask at 110 rpm by a rotary evaporator at 60 ◦ C for 60 min, the resulting vesicle suspension was extruded 19 times through an Avanti mini-extruder equipped with a 100 nm polycarbonate membrane (Avanti Polar Lipids, Alabaster, AL, USA) and maintained at 65 ◦ C. Un-encapsulated ICG was isolated by standard size exclusion chromatography using Sephadex G75. The particle size distribution of liposome solution was estimated by using a Nano Zetasizer (Malvern Instruments, Malvern, United Kingdom), and liposomal ICG was quantified by a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI—MS—MS) assay. The assays were performed using an Agilent 1200 series binary pump liquid chromatographic system interfaced to an Agilent 6410B
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triple quadrupole mass spectrometer (Agilent, Santa Clara, CA, USA). As described previously, IR-820 was selected as a structural analog to ICG and used as a suitable internal standard [17]. Chromatographic separation was performed using an Agilent Zorbax Eclipse Plus (100 mm × 2.1 mm, 3.5 m) C18 column, and the column compartment was maintained at 25 ◦ C. The injection volume was 15 L. The mobile phase consisted of 10 mM ammonium acetate solution at pH 3.0 (adjusted by formic acid) and acetonitrile in 50:50 (v/v), and the flow rate was held at 0.45 mL/min. Samples were run using an isocratic elution. Ionization was achieved by electrospray operating in positive ion mode, and source conditions were optimized as follows: drying gas (N2 ) temperature 350 ◦ C and flow rate 12 L/min, nebulizer set at 40 psi, and capillary voltage set to 3500 V. Analysis was carried out in multiple reaction monitoring (MRM) mode with a dwell time of 100 ms and electron multiplier voltage of +400 V. For ICG, MRM transition of m/z 753→330 Da was monitored with a fragmentor voltage of 200 V and collision energy 37 V; IR-820 was monitored by MRM transition of m/z 827→330 Da with a fragmentor voltage of 185 V and collision energy of 40 V. The retention time for ICG and IR-820 was 1.5 and 3.6 min, respectively, and the total run time was 5.0 min. The limit of detection was 5.0 ng/mL and the limit of quantitation was 10.0 ng/mL. Samples were analyzed with a calibration curve between 50 and 2000 ng/mL. Internal standard was added to liposome samples whereby liposomes were lysed using a solution of 20% Triton X-100, vortexed briefly, and dried down. Samples were reconstituted and diluted in mobile phase followed by filtration using a 13 mm diameter and 0.2 m pore size Millex-LG polytetrafluoroethylene (PTFE) syringe driven filter unit (Millipore Corporation, Cork, Ireland). The procedure was validated in regards to precision and accuracy. Precision was calculated by the percent coefficient of variation (CV%) of the replicate procedure assayed at 100 and 1,000 ng/mL: the within-run CV% were 2.5% and 1.8%, respectively, while the betweenrun CV% were 4.9% and 5.2%. Accuracy was calculated as the absolute value of percent error at both 100 and 1000 ng/mL with values of 3.6% and 3.1% obtained.
Photodynamic therapy ICG solutions for cell culture were prepared by diluting ICG stock solution (in DMSO) with fresh cell culture growth media under sterile conditions after thawing the stock solution to room temperature. The concentration of DMSO was kept under 0.1%. In the photodynamic therapy care was taken to maintain cells under dark conditions when possible by covering plates with aluminum foil during brief transport and irradiating in a dark environment. Firstly, cells were incubated with ICG either in free solution or in liposomal formulation (ICG concentration ranging from 15 to 37.5 M) in the dark for 16 or 20 h, then cell media was replaced with 1× PBS to ensure uniform and maximum laser penetration. A fiber-coupled laser system with a wavelength of 808 nm (Edmunds Optics, Barrington, NJ, USA) was used to excite ICG. The laser system was calibrated in terms of power output ranging between 0 and 450 mW. Laser irradiation was delivered via a fiber (0.5 m in length and 100 m in diameter) coupled with a laser diode. A uniform
distribution of light was maintained in all experiments, which was capable of illuminating the entire cell containing wells of interest. The size of laser light remained the same for the same assay but varied in different assays in order to fully cover the cells. The diameter of the beam (spot size) varied slightly for certain procedures depending on the type of plate used (96 well or 6 well) but remained constant within batch. Temperature effects were monitored to be negligible and were very consistent. The different size of light was achieved by adjusting the distance of the fiber optic above the plate. The irradiation time was 3 min for all the assays. The light does were expressed by irradiation energy per unit area (J/cm2 ), which is calculated by multiplying the energy output per area (W/cm2 ) with irradiation time (180 s). The light dose ranging from 1 to 14 J/cm2 was evaluated. During the irradiation, the temperature of the cell media was periodically monitored. Upon the completion of the irradiation, PBS was removed and replaced with growth media, and then cells were returned to the dark in a humidified incubator for different time intervals for various assays.
MTT assay Cell viability after PDT treatment was investigated by a MTT assay. Briefly: 8,000 cells per well were seeded into 96 well plates in a humidified incubator overnight. The following day media was removed, and ICG, either in solution or liposomal formulation, at various concentrations was added to the appropriate wells; controls were processed in the same manner but with fresh media. The cell plates were immediately returned to the incubator for 16 or 20 h to facilitate cell internalization of the photosensitizer. The media was then replaced with 1× PBS. PDT or laser alone control groups were irradiated with laser at 808 nm at different light doses for 3 min, whereby 1× PBS was removed and replaced with 100 L of appropriate growth media. Cells were then returned to the incubator. After a 24-h incubation, 10 L of a 5 mg/mL solution of 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) dissolved in PBS was added and cells were returned to the incubator. After 3 h, media was removed from all wells, and 100 L of DMSO was added to solubilize the purple formazan crystal formed by actively metabolizing cells with maintained structural integrity, and the absorbance was measured at 570 nm using a Synergy HT plate reader (Bio-Tek Instruments, Winooski, VT, USA). Cell viability was determined by comparing each treatment group to the untreated control groups. All experiments were carried out in triplicate and also repeated three independent times, and data were plotted as mean ± SD.
Clonogenic assay A clonogenic assay was performed by plating 20,000 cells per well into 6 well plates and left to incubate in a humidified incubator overnight for cell adhesion. The following day media was removed whereby 37.5 of M indocyanine green in free solution was added and cells were further incubated overnight for 16 h. The media was replaced with PBS and cells were irradiated according to PDT protocol (light dose
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Photodynamic therapy using human triple negative breast cancer cells was 3.5 or 8 J/cm2 ). After PDT treatment, cells were immediately trypsinized, counted, diluted, and plated at 500 cells per well. Plates were returned to the incubator for 8—12 additional days to allow time for 5—10 cycles of cell divisions in order for colonies to contain 50 or more cells each. Cells were initially examined microscopically before staining to ensure colonies consisted of at least 50 cells. Then cell media was removed and cells were rinsed gently with 1× PBS, followed by the addition of 1 mL of a 6% aqueous glutaraldehyde solution containing 0.5% crystal violet. Cells were left to incubate with the adhesive and dye for 30 min, whereby plates were carefully submerged in water for stain removal. After air drying, plates were examined and colonies were scored with the percentage of colony formation calculated by comparing the number of colonies from each treatment group to that from the group without laser irradiation (untreated control). All experiments were carried out in triplicate and data were plotted as mean values ± SD.
Gamma-H2AX immunofluorescence assay Gamma-H2AX assay was performed by at first coating glass coverslips placed into 12 well plates with poly-L-lysine. MDA-MB-468 and BT-549 cells were seeded at a density of 80,000 cells per well and left to incubate overnight in a humidified incubator. The following day a 37.5 M ICG in free solution was added to the appropriate wells and left to incubate for 20 h, whereby ICG was then removed and replaced with 1× PBS. Cells were irradiated with a light dose of 3.5, 8, and 14 J/cm2 . After irradiation, PBS was replaced with fresh media and the culture plate was returned to a humidified incubator for 30 min. Media was then removed from the plate and cells were washed twice with 2 mL of ice-cold 1× PBS. Cells were fixed using 1 mL of 3%
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paraformaldehyde for 10 min, whereby cells were rinsed with ice-cold 1× PBS twice and permeabilized using 1 mL of 0.5% Triton-X-100 in PBS and left to incubate for 10 min. After rinsing twice with ice-cold 1× PBS, cells were blocked using 1 mL of 5.0% bovine serum albumin (BSA) in PBS and left to incubate for 15 min at room temperature. Cells were incubated with the primary antibody solution which contained ␥-H2AX and 1% BSA in PBS at a 1:4500 ratio for 1 h at 37 ◦ C (Abcam, Cambridge, MA, USA). After thoroughly washing cells of primary antibody, cells were incubated for an additional 30 min in a secondary antibody with a concentration of 1:500, using Alexa Fluor 488 in 1× PBS. To control for background staining, secondary antibody control samples were incubated with 1% BSA and incubated under the same conditions as the primary control. After incubation samples were rinsed with PBS three times and mounted to glass slides using mounting media with 4 ,6-diamidino-2-phenylindole (DAPI). Slides were dried and stored at 4 ◦ C. Results were analyzed under oil immersion 63× magnification, and foci were counted using an Observer Z1 fluorescent microscope with AxioVision software (Carl Zeiss, Thornwood, NY, USA). Cells exhibiting more than 6 distinct foci were scored as positive, and percentage of foci positive cells was determined by analyzing 100 healthy cells and then compared to the untreated control group. All experiments were carried out in triplicate and data were plotted as mean values ± SD.
Statistical analysis All experiments were performed in triplicate and the data were expressed as mean plus and minus the standard error of the mean. Analysis of variance (ANOVA) and Student’s t-test were used to determine the statistically significant difference among different groups when appropriate.
Figure 1 Cell viability of HCC-1806 cells assessed by a MTT assay after PDT treatment with 37.5 M ICG either in free solution or encapsulated in the thermosensitive liposomes. Cells were incubated with ICG for 16 h. Solid bars represent ICG in free solution, while cross-hatched bars represent liposomal ICG. Data were presented as the mean cell viability with standard deviations after triplicate analysis.
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C.S. Shemesh et al. after measurements taken from three independent batches. The concentration of encapsulated ICG ranged from 446.5 to 548.4 M, determined by the optimized LC-ESI-MS-MS assay. Based on our previous research [12], liposomal ICG demonstrated excellent short term stability at 4 ◦ C for up to 4 days in regards to particle size and ICG concentration. Formulations used in this study were prepared fresh or up to 24 h prior.
Cell viability assessment after PDT treatment Figure 2 Cell viability of HCC-1806 cells assessed by a MTT assay after PDT treatment using 15, 25, and 37.5 M ICG, either in free solution or encapsulated in the thermosensitive liposomes. Cells were incubated with ICG for 16 h. Solid bars represent ICG in free solution, while cross-hatched bars represent liposomal ICG. Data were presented as the mean cell viability with standard deviations after triplicate analysis.
Results Liposomal ICG preparation and characterizations Thermosensitive liposomes that encapsulated ICG ranged from 51 to 85 nm in diameter with an average of 71 ± 10 nm
Optimization of PDT treatment protocol was carried out with HCC-1806 cells. During the brief irradiation the temperature of the PBS solution remained very close to room temperature at 22.7 ◦ C and did not rise. Initially, three doses of ICG: 15, 25, and 37.5 M, respectively, either in free solution or formulated with thermosensitive liposomes, were evaluated as the photosensitizer. Compared to the untreated control, the group that was treated with laser alone at a dose of 14 J/cm2 (the highest tested) showed 100% cell viability while 37.5 M of ICG alone, either in free solution or liposomal formulation, showed slightly decreased cell viability (Fig. 1). After the treatment, cell viability was decreased significantly but similar between groups using liposomal ICG or free ICG solution as the photosensitizer. The potency of the PDT treatment was closely correlated with ICG
Figure 3 Cell viability of MDA-MB-468 (A), HCC-1806 (B), MDA-MB-231 (C), HCC-70 (D), BT-549 (E), and MCF-10A (F) evaluated by a MTT assay after PDT treatment with 37.5 M ICG either in free solution or encapsulated in the thermosensitive liposomes. Cells were incubated with ICG for 20 h. Solid bars represent ICG in free solution, while cross-hatched bars represent liposomal ICG. Data were presented as the mean cell viability with standard deviation after triplicate analysis.
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DNA damage evaluation by Gamma-H2AX (␥-H2AX) assay
Figure 4 Comparison of cell viability of MDA-MB-468 (dashed lines) and HCC-1806 (solid lines) with MDA-MB-231, HCC-70, BT549, and MCF-10A after PDT treatment with 37.5 M liposomal ICG and 14 J/cm2 laser irradiation. Cell viability was evaluated using a MTT assay and data were presented as the mean cell viability with standard deviation after triplicate analysis. Student t-test was performed to compare cell viability of MDA-MB-468 and HCC-1806 to that of MDA-MB-231, HCC-70, BT-549, and MCF10A; P value < 0.001 are indicated by three asterisks (***) and P value < 0.01 are indicated by two asterisks (**).
concentrations, where 37.5 M of ICG was the most effective in inhibiting the growth of HCC-1806 cells (Fig. 2). Therefore, 37.5 M of ICG was used for the subsequent experiments described in this article. The data in Figs. 1 and 2 also indicate that the efficacy of PDT depends on the irradiation energy of the laser used to excite ICG in the treatment. Upon expansion of the cytotoxicity studies to four other TNBC cell lines, a similar light dose response was observed (Fig. 3). Once again, liposomal ICG showed similar inhibitory potency compared with ICG in free solution. The results in Fig. 3 also suggested that PDT treatment inhibited the growth of different TNBC cell lines to different extents. The variable responses of treatment may be related to differing phenotypes, gene expression, molecular behavior, and DNA repair processes of these cells. After PDT treatment with a 14 J/cm2 laser dose, cell viability of MDA-MB-468 and HCC-1806 is significantly lower than that of MDA-MB-231, BT-549, and HCC-70 cell lines (P < 0.001). Furthermore, nontumorigenic mammary epithelial cell line MCF-10A is shown to be more resistant to the treatment when compared to MDA-MB-468 and HCC-1806 cell lines (P < 0.01) (Fig. 4).
Clonogenic assay assessment In our experiment, 37.5 M of ICG in free solution was used as the photosensitizer, as the MTT assay revealed that the liposomal formulation of ICG has negligible impact on the in vitro PDT treatment. Light doses of 3.5 J/cm2 and 8 J/cm2 were selected in PDT in order to observe colony formation with certain cell lines. Among the five cell lines evaluated, HCC-1806 and MDA-MB-468 showed higher sensitivity toward the treatment therefore having fewer colonies formed (Figs. 5 and 6), confirming the findings in the MTT assay experiment.
In an effort to reveal the possible role of DNA damage in PDT treatment, a ␥-H2AX assay based on immunofluorescence microscopy was conducted to examine if the treatment could induce DSBs. We specifically examined MDA-MB-468 and BT-549 cell lines due to their significantly different response to the treatment. Indeed, ␥-H2AX foci were observed 30 min after the treatment even with a low laser dose of 3.5 J/cm2 , indicating some level of DSBs induced by PDT (Fig. 7). The degree of DSBs seemed positively correlated to the dose of laser, as more ␥-H2AX foci were found with higher doses of laser. However, a similar amount of DSBs were seen for MDA-MB-468 and BT-549 (pictures of BT-549 cells not shown). This is further confirmed by the quantitative comparisons of these two cell lines as shown in Fig. 8.
Discussion Presented in this article was the exploration of liposomal ICG as the photosensitizer in PDT to treat TNBCs. ICG concentration, liposomal formulation, and laser dose were investigated in PDT on a panel of five TNBC cell lines. The results from both the MTT assay and clonogenic assay indicate PDT treatment using ICG is effective in inhibiting the growth of TNBC cell lines in vitro, and liposomal formulation of ICG did not compromise the phototoxicity, suggesting that thermosensitive liposomes could serve as the delivery platform for ICG in a PDT treatment. While a slight loss of cell viability was observed for HCC-1806 cells after a 16 h incubation with ICG was observed, this was found to be unique for this cell line as these cells were much more sensitive compared to the remaining panel. This effect may be minimized by incubation for shorter time intervals at a lower concentration while exposing to longer irradiation times. Interestingly, MDA-MB-468 and HCC-1806 cell lines are particularly sensitive to the treatment. If further classification is applied according to the gene expression profile, MDA-MB-468 and HCC-1806 belong to basal-like subtype of TNBC cell lines. This subtype of TNBCs is significantly more sensitive to DNA damage agents such as cisplatin due to the elevated cell-cycle and DNA damage response genes [18]. Similarly, PDT is more effective in inhibiting these two basallike TNBC cell lines, and this observation suggests that DNA damage might play a significant role in the PDT treatment. PDT induced DNA damage may be unique to the type of photosensitizer, based on different physiochemical properties, which may be highly dependent on the ultimate localization inside of a cell. Nuclear delivery of photosensitizer may be facilitated using high-cost peptides, nuclear localization signals, or other sophisticated delivery systems. However, most photosensitizers target either cell membranes or mitochondria. To the best of our knowledge, PDT induced DSB DNA damage by PS itself was firstly demonstrated in our work. Due to the extreme short life time and diffusion distance of ROS the intracellular damage by PDT may be limited to the subcellular location of the photosensitizer. Therefore, nuclear targeting of the photosensitizer is necessary for achieving significant DNA damage. Although we were not
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Figure 5 A clonogenic assay was carried out with MDA-MB-468, HCC-1806, MDA-MB-231, HCC-70, BT-549, and MCF-10A cell lines after PDT treatment with 37.5 M ICG solution as the photosensitizer and laser doses was 3.5 or 8 J/cm2 . Images are representative colonies for 500 seeded cells after 8—12 days of incubation.
able to study the subcellular location of ICG, the results from the ␥-H2AX revealed obvious DSB DNA damage after the treatment, which may indicate the partial nuclear delivery of ICG.
Earlier investigations suggested that PDT mainly targeted mitochondria and endoplasmic reticulum to induce apoptosis in tumor cells [14]. However, our preliminary studies suggested that the PDT may kill cancer cells through a
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Figure 6 Quantitative comparison of percentage of colony formation among MDA-MB-468, HCC-1806, MDA-MB-231, HCC70, BT-549, and MCF-10A cell lines after PDT treatment with 37.5 M ICG solution as the photosensitizer and laser dose was 3.5 or 8 J/cm2 . Data were presented as the mean colony formation percentage with standard deviation after triplicate analysis.
different mechanism. As observed by the MTT and clonogenic assays, basal-like cell lines HCC-1806 and MDA-MB-468 are more sensitive to the treatment especially compared to mesenchymal-like cell lines BT-549 and MDA-MB-231. A similar trend was reported for DNA damage agents such as cisplatin by other investigators [18]. Hence, we speculated that PDT may kill cancer cells through DNA damage by ROS or radicals, which have shown to cause direct or indirect double-strand breaks (DSBs) among DNA [19]. DSBs are generally considered to be the most toxic DNA lesion, and a single DSB can ultimately lead to cell death if the cell is left without efficient DNA repair [20]. Followed by DSBs, histone H2AX is rapidly phosphorylated to produce ␥-H2AX, and measurement of ␥-H2AX foci with an immunocytochemical assay was utilized as a sensitive method to detect and quantify DSBs [21,22]. The results from the ␥-H2AX assay showed an increased level of ␥-H2AX foci after the PDT, suggesting that DNA damage, particularly DSBs, may be one of mechanisms that underlie the PDT’s inhibition on cancer cell growth. MDA-MB-468 and BT-549 cell lines showed similar level of ␥-H2AX foci after the treatment. The difference in cell viability after treatment found with these two cell lines may result from their different efficiency to repair DSBs in response to DNA damage induced by the PDT treatment. PDT may lead to more extensive cell death via DNA damage among basal-like TNBC cells such as MDA-MB468 and HCC-1806, due to their elevated DNA repair gene mutation. In summary, we have demonstrated that PDT using ICG as the photosensitizer can effectively inhibit the growth of several TNBC cell lines in vitro, particularly the basal-like cell lines MDA-MB-468 and HCC-1806. In addition, liposomal ICG performed similarly to free ICG solution in the in vitro PDT treatment, suggesting the potential application of the thermosensitive liposome formulation for the delivery of ICG in a PDT treatment. Although a complete understanding of this liposomal ICG formulation in the PDT treatment begs for more detailed investigations on the cellular uptake, the stability of liposomal ICG, the release characteristics of ICG,
Figure 7 Induction of ␥-H2AX foci in MDA-MB-468 cells 30 min after PDT treatment with 37.5 M ICG solution as the photosensitizer. Increased levels ␥-H2AX foci were observed with higher laser doses. A, B, C, D, and E are corresponding to laser control, ICG control, PDT treatment with 3.5, 8, and 14 J/cm2 , respectively. Very similar levels of ␥-H2AX foci were observed with the BT-549 cell line (pictures not included). DAPI, 4 ,6-diamidino-2phenylindole (fluorescent stain). Only cells exhibiting more than 6 foci were scored which were found in PDT treatment groups.
etc., a higher in vivo efficacy is predicted based on the targeted delivery of ICG to the tumor area from our previous in vivo imaging studies [12]. We also investigated DSBs on DNA after treatment by a ␥-H2AX assay, which revealed a certain degree of DNA damage 30 min after the treatment, even with a relatively low light dose. This is indicative of ROS and radicals produced during PDT, which may cause DNA damage and lead to cell death, but further investigations are necessary to validate this statement. The primary aim of this study was to investigate the effectiveness of liposomal ICG using a thermosensitive formulation as the photosensitizer in photodynamic therapy to treat TNBCs. Both MTT and clonogenic assays revealed PDT is effective at inhibiting the growth of TNBCs to different extents, which might due to the reason that TNBC is a very heterogeneous
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References
Figure 8 Quantitative comparison of percentage of ␥-H2AX foci positive cells between MDA-MB-468 and BT-549 cell lines after PDT treatment with 37.5 M ICG solution as the photosensitizer. Data were presented as the mean foci positive cells percentage with standard deviation after triplicate analysis.
type of cancer with a wide variability in receptor expression. Moreover, the thermosensitive liposome formulation does not compromise potency of the in vitro PDT treatment. DNA damage by PDT was investigated by a ␥-H2AX assay, suggesting that DNA damage could be the main event leading to the extensive cancer cell death. Thermosensitive ICG loaded liposomes would ideally be administered to a patient as a sterile parenteral formulation reconstituted in saline for intravenous administration. After i.v. administration, ICG is confined to the circulation due to its high binding affinity to plasma proteins; in addition, the half-life of free ICG in humans is about 3—5 min and it is also prone to chemical and photo-degradation. Hence the liposome vesicles are essential to protect ICG, prolong the circulation time, and eventually deliver ICG to the tumor site for subsequent illumination. This therapy has the advantage to be conducted multiple times by repeat administration and illumination in a targeted approach. We would anticipate this therapy to hold promise to be exploited as a form of personalized medicine tailored to the patient. In an ideal setting a physician may adjust the photosensitizer dosage and light energy for the patient to obtain the optimal therapeutic response. However, due to the aggressive nature of TNBC and the limitations of a localized PDT treatment, this therapy may offer potential for early-treatment of TNBC either alone or in a combination therapy.
Conflict of interest The authors report that there are no conflicts of interest regarding any aspects of this manuscript.
Acknowledgements We thank Dr. LaTonia Taliaferro-Smith and Tongrui Liu of the Winship Cancer Institute for their valuable assistance in obtaining the various TNBC cells. This research was carried out using the facilities of the Department of Pharmaceutical Sciences at Mercer University College of Pharmacy in addition to the Winship Cancer Institute located at Emory University. This work was funded in part by the Georgia Cancer Coalition’s Cancer Research Award.
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