Evaluation of theranostic nanocarriers for near-infrared imaging and photodynamic therapy on human prostate cancer cells

Accepted Manuscript Title: Evaluation of theranostic nanocarriers for near-infrared imaging and photodynamic therapy on human prostate cancer cells Authors: Fernanda Z. Leandro, J´ulia Martins, Aparecida M. Fontes, Antonio C. Tedesco PII: DOI: Reference:

S0927-7765(17)30160-1 http://dx.doi.org/doi:10.1016/j.colsurfb.2017.03.042 COLSUB 8452

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

31-8-2016 22-2-2017 18-3-2017

Please cite this article as: Fernanda Z.Leandro, J´ulia Martins, Aparecida M.Fontes, Antonio C.Tedesco, Evaluation of theranostic nanocarriers for near-infrared imaging and photodynamic therapy on human prostate cancer cells, Colloids and Surfaces B: Biointerfaceshttp://dx.doi.org/10.1016/j.colsurfb.2017.03.042 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Evaluation of theranostic nanocarriers for near-infrared imaging and photodynamic therapy on human prostate cancer cells

Fernanda Z. Leandro a, Júlia Martinsa, Aparecida M. Fontesb, Antonio C. Tedescoa,*

a

Departamento de Química, Laboratório de Fotobiologia e Fotomedicina, Faculdade de

Filosofia, Ciências e Letras de Ribeirão Preto (FFCLRP), Universidade de São Paulo, Ribeirão Preto, SP 14040-901, Brazil. b

Centro Regional de Hemoterapia, Faculdade de Medicina de Ribeirão Preto (FMRP),

Universidade de São Paulo, Ribeirão Preto, SP 14049-900, Brazil.

*Corresponding author: Antonio Claudio Tedesco, PhD, Laboratório de Fotobiologia e Fotomedicina/FFCLRP, Universidade de São Paulo, Av. dos Bandeirantes, 3900, 14040-901, Vila Monte Alegre, Ribeirão Preto SP, Brazil. Phone: +55 16 33153751. Fax: +55 16 33154838. Email: [email protected]

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Graphical Abstract

The in vitro theranostic effects of chloroaluminum phthalocyanine entrapped in nanocapsule and nanoemulsion on prostate cancer cells.

Highlights     

ClAl-phthalocyanine (ClAlPc) was entrapped in nanocapsule and nanoemulsion. Effects of ClAlPc-nanocarriers were evaluated on prostate cancer cells in vitro. Upon PDT, ClAlPc-nanocarriers efficiently killed prostate cancer cells. The cytolocalization of ClAlPc-nanocarriers was determined by confocal microscopy. ClAlPc-nanocapsule showed better theranostic behavior in prostate cancer cells.

Abstract This paper evaluates how effectively chloroaluminum phthalocyanine (ClAlPc) entrapped in colloidal nanocarriers, such as nanocapsule (NC) and nanoemulsion (NE), induces photodamage in human prostate cancer cells (LNCaP) during photodynamic therapy (PDT). The MTT cell viability assay showed that both ClAlPc-NC and ClAlPcNE induced phototoxicity and efficiently killed LNCaP cells at low ClAlPc-NC and ClAlPc-NE concentrations (0.3 µg mL-1) as well as under low light doses of 4 J cm-2 and 7 J cm-2, respectively, upon PDT with a 670-nm diode laser line.

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Confocal imaging studies indicated that ClAlPc-NC and ClAlPc-NE were preferentially localized in the perinuclear region of LNCaP cells both in the dark and upon irradiation with laser light. After PDT treatment, ClAlPc-NC-treated LNCaP cells exhibited a higher green fluorescence signal, possibly due to the larger shrinkage of the actin cytoskeleton, compared to ClAlPc-NE-treated LNCaP cells. Additionally, ClAlPc-NC or ClAlPc-NE and mitochondria showed a relatively high co-localization level. The cellular morphology did not change in the dark, but confocal micrographs recorded after PDT revealed that LNCaP cells treated with ClAlPc-NC or ClAlPc-NE underwent morphological alterations. Our preliminary in vitro studies reinforced the hypothesis that biocompatible theranostic ClAlPc-loaded nanocarriers could act as an attractive photosensitizer system in PDT and could serve as an interesting molecular probe for the early diagnosis of prostate cancer and other carcinomas.

Keywords:

photodynamic

therapy;

chloroaluminum

phthalocyanine;

colloidal

nanocarriers; photodiagnosis; theranostics; prostate cancer.

1. Introduction Prostate cancer (PCa) is one of the most common neoplastic malignancies and the second leading cause of death in men [1-3]. Conventional options to treat PCa include a radical prostatectomy, transperineal brachytherapy, radiotherapy, and androgen-deprivation therapy. However, most of these treatments have a number of adverse side effects and considerably impact the patient´s quality of life [4].

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Photodynamic therapy (PDT), a clinical therapeutic modality, can potentially become a useful theranostic approach to treat malignant and nonmalignant diseases noninvasively and to visualize cancer cells by fluorescence imaging [5-7]. The first clinical report of PDT for previously untreated PCa dates back to 1990 [8]. The PDT cancer treatment process involves the administration of a photosensitizer (PS) drug that reacts with oxygen upon exposure to visible light of an appropriate wavelength matched to the absorption maximum of the PS. This process generates reactive oxygen species (ROS) and subsequently destroys the tumor, preferably without harming the healthy tissue [9,10]. Moreover, the light-induced excitation of the PS can result in fluorescence emission, enabling photoactive drugs to act as both therapeutic and imaging agents [5-7]. In recent years, researchers have tested numerous first- and second-generation PS molecules both in vivo and in vitro to treat skin, esophageal, and liver cancer. Only a few porphyrin derivatives, such as Porfimer sodium, Talaporfin, Temoporfin, and Verteporfin, have received approval from the Food and Drug Administration (FDA) and other regulatory agencies [10-12] for cancer treatment in humans. Nevertheless, these PS drugs display low light absorption in the phototherapeutic window (600-900 nm). Intense infrared absorption is desirable in cancer imaging and PDT because NIR (near-infrared region) photons in the phototherapeutic window are the most penetrating and the least harmful to human tissues. Additionally, fluorescence imaging in the NIR optical window is promising because it leads to minimal tissue autofluorescence and light scattering [13,14]. In this context, phthalocyanines (Pcs) and their derivatives (known as second-generation PSs) have emerged as potential theranostic agents: they can absorb far-red and NIR light [7,13]. These PSs commonly bear large conjugated domains that result in strong energy

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absorption and high fluorescence quantum yield, which are required for effective PDT and fluorescence imaging. Pcs can form complexes with diamagnetic ions, such as Zn 2+, Al3+, and Ga3+, resulting in metallophthalocyanines (MPcs) with high triplet yields and long lifetimes [15,16]. MPcs carrying cationic substituents are advantageous over MPcs containing neutral and anionic substituents because they provide improved cellular uptake. In addition, studies have demonstrated that MPcs are selectively localized in the cell mitochondria, which induces apoptosis [17]. The photophysical properties of Pcs that favor their use in PDT depend on the core metal ion and on their molecular structure. Unfortunately, MPcs are hydrophobic and can aggregate in aqueous media. Aggregation significantly reduces the photosensitizer efficacy because only monomeric species are appreciably photoactive [18,19]. Interestingly, hydrophobic PSs have been shown to exhibit superior PDT performance compared to hydrophilic molecules, such as sulfonated aluminum phthalocyanine, which have received approval for use in clinical PDT in Russia [20]. Several drug delivery systems, such as liposomes, nanoemulsions, and nanocapsules, have helped to improve the solubility and bioavailability of hydrophobic Pcs [21-28]. Nanoemulsions (NEs) are fine oil-in-water (o/w) dispersions with droplet sizes ranging from 10 to 600 nm. They have been designed to transport hydrophobic materials that can be adsorbed at the oil-water interface. The organic phase is a homogeneous solution of oil, lipophilic surfactants, and water-miscible solvent. The aqueous phase consists of a hydrophilic surfactant and water [28]. Nanocapsules (NCs) are defined as submicronic vesicular systems composed of an oily core surrounded by a thin polymeric membrane. The hydrophobic drug can be entrapped in the central oily core and/or adsorbed onto the polymeric membrane of the nanocapsules [27].

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Our research group has used chloroaluminum phthalocyanine (ClAlPc, Figure 1) entrapped in different drug delivery system (liposomes, nanoemulsions, and nanocapsules) in various fields of nanomedicine application including the following: skin cicatrization, melanoma skin cancer, oral cancer, breast cancer, and glioblastoma [24-28]. Currently, we have extended the proposal to use this photosensitizer drug for the diagnosis and treatment of prostate cancer due to its favorable photophysical, photochemical and photobiological properties and behavior. Insert Figure 1 Chloroaluminum phtalocyanine has a long history of PDT and anti-neoplastic activity under visible light activation. As for Pc 4 in the past, the only phthalocyanine compound already approved by FDA to be used in clinical trials [29], we believe that ClAlPc, due to its long history of successful results, data and application, could be moved into clinical trials. This work examines and compares the effects of ClAlPc-NC and ClAlPc-NE on human prostate cancer cells (LNCaP, used as a starting model) both in the dark and upon irradiation with 670-nm light from a diode laser line. Confocal laser scanning microscopy (CLSM) helped to determine the cellular localization of the ClAlPc-loaded nanocarriers in LNCaP cells as well as the cellular morphology during both the dark assay and after PDT treatment. Our results also pave the way for a new imaging approach that uses ClAlPc to diagnose not only prostate cancer but also other carcinomas at the same time the patient is being treated. Theranostic properties allow the same compound to act both as a diagnostic and therapeutic agent, and this is a new useful feature expected of some drugs aimed at cancer therapy. To our knowledge, this is the first report of the evaluation of ClAlPc in human prostate cancer cells in vitro.

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2. Materials and Methods 2.1. Chemicals Chloroaluminum phthalocyanine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), paraformaldehyde, Poloxamer 188, poly(D,L-lactide-co-glycolide) (PLGA; 50:50), rhodamine-123 (Rh-123), and dimethylsulfoxide (DMSO) were purchased from the Sigma-Aldrich Co. (St. Louis, MO, USA). The natural soy phospholipids Epikuron 170® (7.5%) and Miglyol 812 N oil were obtained from Hulls Inc. (Puteaux, France). Alexa Fluor® 488 Phalloidin and ProLong Gold® antifade reagent containing 4’,6-diamidino-2-phenylindole (DAPI) were obtained from Invitrogen (São Paulo, SP, Brazil). The water used throughout the experiment was first bi-distilled and then deionized in a Milli-Q ultra-purification system (Millipore, Bedford, MA, USA). All of the other chemicals were of analytical grade and were used without further purification. 2.2. Preparation of the ClAlPc-loaded nanoemulsion The ClAlPc-loaded nanoemulsion (or simply ClAlPc-NE) was obtained by a spontaneous emulsification process as described by Primo et al., 2011 [28]. Initially, the surfactants were dissolved in 10 mL of acetone at 55 °C under magnetic stirring. Simultaneously, the ClAlPc was dissolved in Miglyol 812 N oil at 55 °C and was added to the phospholipid organic solution at a concentration of 1.0 mg mL -1 . The aqueous phase was obtained by dissolution of the polymer Poloxamer 188 in ultra-pure water. The NE was formed by slow injection of the organic phase into the aqueous phase under magnetic stirring (300 rpm for 30 min) at 55 °C. The solvent was evaporated under reduced pressure at approximately 75 °C, and the volume of the NE was concentrated to the initial volume of the aqueous phase. The amount of hydrophilic and lipophilic surfactants was fixed at 1% with a 1:1 weight ratio. 2.3. Preparation of ClAlPc-loaded nanocapsule

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ClAlPc-loaded nanocapsule (or simply ClAlPc-NC) was prepared by the interfacial deposition of a preformed polymer on the oil-in-water emulsion interface as described by Siqueira-Moura et al. [27]. Briefly, the organic phase (acetone) containing oil (2.5% v/v), PLGA (50:50, 0.75% w/v), lecithin (phosphatidylcholine from soybean), and ClAlPc (0.5 mg) was prepared at 40 °C. Subsequently, this organic solution was added to the aqueous phase containing Poloxamer 188 under magnetic stirring. The organic solvent was removed by evaporation under reduced pressure at 40 °C. Finally, the formulation was concentrated to a final volume of 10 mL. 2.4. Mean size, polydispersity index, and zeta potential analysis The mean diameters and polydispersity indices (PdI) of the colloidal nanocarriers were determined by photon correlation spectroscopy (PCS) at a scattering angle of 173° (Zetasizer® Nano ZS, Malvern PCS Instruments, UK). The  potentials of ClAlPc-NC and ClAlPc-NE were measured by electrophoretic mobility on a Zetasizer ® Nano ZS apparatus (Malvern PCS Instruments, UK). The analyses were conducted at 25 °C, and the samples were appropriately diluted (1/100) with ultra-purified water. Values are reported as the mean ± SEM of three different batches of each colloidal dispersion. 2.5 In vitro cytotoxicity assays 2.5.1. Cell culture The human prostate cancer cell line LNCaP clone FGC, obtained from the American Type Culture Collection (ATCC; Manassas, VA), was grown in 75-cm2 culture flasks and maintained in phenol red-positive RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 units mL-1 penicillin and 100 µg mL-1 streptomycin (Gibco, Grand Island, NY, USA). LNCaP cells were kept in a humidified incubator with 5% CO 2 at 37 °C. Cells in the logarithmic growth phase were used for further experiments. 2.5.2. Dark cytotoxicities of the ClAlPc-nanocarrier systems

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To evaluate the cytotoxic effects of ClAlPc-NC and ClAlPc-NE in the dark, LNCaP cells were incubated with a mixture of fresh medium and ClAlPc-NC or ClAlPc-NE at final concentrations of 0.3, 0.6, and 3.0 µg mL -1. The LNCaP cells were also incubated with unloaded NC or NE for a control. The incubated cells were kept in the dark in 5% CO 2 and at 37 °C for 3 h. After incubation, the medium was discarded, the cells were washed with phosphate-buffered saline (PBS) at a pH of 7.4, and fresh medium was added to the cells. The cell viability assay was performed 24 h after incubation with ClAlPc-NC and ClAlPc-NE as detailed in Section 2.5.4. 2.5.3. Phototoxicities of the ClAlPc-nanocarrier systems LNCaP cells were incubated with fresh medium containing a non-cytotoxic concentration of ClAlPc-NC or ClAlPc-NE (evaluated in Section 2.5.2) in 5% CO2 and at 37 °C for 3 h. After incubation, the medium was discarded. The LNCaP cells were washed with PBS (pH = 7.4) and sequentially re-incubated in fresh medium without phenol red. Finally, the LNCaP cells incubated with ClAlPc-NC or ClAlPc-NE were irradiated with a diode Eagle laser (Quantum Tech., São Carlos, SP, Brazil) operating at 670 nm in sterile conditions (average power = 650 mW; light irradiance = 92 mW cm-2); light doses ranged from 0.5 to 7 J cm-2. After PDT treatment, the colorless medium was discarded, and the LNCaP cells were re-incubated in fresh medium (5% CO2, 37 °C). The MTT cell viability assay was carried out 24 h later as described in Section 2.5.4. 2.5.4. MTT cell viability assay The MTT colorimetric assay helped to determine the percentage of viable cells after the dark cytotoxicity and phototoxicity assays. The MTT assay relies on the ability of the mitochondrial dehydrogenase of viable cells to cleave the tetrazolium rings of yellow MTT to form dark purple membrane-impermeable formazan crystals [30].

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Briefly, aliquots of 80 µL of MTT solution (5 µg mL -1) and 420 µL of fresh medium without phenol red were added to each well. Then, the LNCaP cells were kept in 5% CO2 and at 37 °C for 4 h. After incubation, the medium containing MTT solution was removed, and the formazan crystals were dissolved in 2-propanol. The optical density of the product was measured at 570 and 690 nm with the Safire2 microplate reader (TECAN Group Ltd., Grödig, Austria). Control cells were incubated with culture medium only without ClAlPcnanocarrier or light irradiation. The results are presented as the cell survival percentage, and the control was taken as 100%. The experiments were performed independently at least three times. Data were reported as a mean ± standard deviation (SD). Statistical analyses were conducted with one-way ANOVA followed by Tukey’s test to determine the statistical significance. All *p values < 0.05 were considered significant. 2.5.5. Confocal imaging of LNCaP prostate cancer cells Confocal laser scanning microscopy helped to evaluate the internalization, localization, and cell morphology of ClAlPc-NC and ClAlPc-NE in LNCaP cells both in the dark and after PDT treatment. LNCaP cells (untreated control LNCaP cells, ClAlPc-nanocarrier-treated LNCaP cells assayed in the dark, and ClAlPc-nanocarrier-treated LNCaP cells submitted to PDT) grown on 13-mm sterile glass coverslips were fixed in 2% paraformaldehyde at room temperature for 15 min, washed three times with PBS, and blocked with 0.1 mol L -1 glycine. The LNCaP cells were then permeabilized with 0.3% Triton X-100 for 5 min and blocked with 1% bovine serum albumin for 30 min at room temperature. Briefly, Alexa Fluor 488 phalloidin was added to the LNCaP cells for 30 min to label the F-actin cytoskeleton; the nuclei were stained with ProLong Gold antifade solution containing DAPI at room temperature. Mitochondria were stained by incubating the ClAlPcnanocarrier-treated LNCaP cells with Rh-123 in 5% CO2 and at 37 °C for 30 min prior to

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their fixation with paraformaldehyde. Finally, the LNCaP cells were visualized under a confocal laser scanning microscope (LSM 710; Carl Zeiss) equipped with an oil immersion 63x objective lens (numerical aperture = 1.4). A 590-nm argon laser was used to excite the ClAlPc, and the fluorescence emission was measured at 685 nm. The Alexa Fluor 488 phalloidin and Rh-123 fluorophores were excited at 488 nm, and the fluorescence emissions were measured at 525 nm. DAPI was excited at 405 nm, and the emitted fluorescence was measured at 461 nm. Visualization of light scattering for each of the excitation wavelengths was recorded in the multitracking mode by using separate detection channels. Images were analyzed with the ZEN 2008 software (Carl Zeiss). Identical settings were used to capture control images. Confocal images were processed by Fiji ImageJ software 1.51h [31] (U.S. National Institutes of Health, Bethesda, MD), an open-source platform for biological-image analysis, and assembled using Adobe Photoshop version 6.3. JaCoP [32], a plug-in of Fiji, was used to calculate the degree of colocalization between ClAlPc-nanocarriers and mitochondria using Pearson’s correlation R, where a R value of 0 corresponds to no colocalization and a R value of 1 is perfect colocalization, as well as Manders’ colocalization coefficient (MCC), i.e., percentage of the signal from ClAlPc-nanocarriers (red) overlapping with the (green) signal from the mitochondria. 3. Results and Discussion 3.1. Physicochemical characterization of the nanocarriers Table 1 lists the mean sizes, polydispersity indices, and zeta potentials of the colloidal nanocarriers. The results meet the criteria required for chemically stable drug delivery systems. The mean sizes of the colloidal nanocarriers were in the nanometer range (average diameter < 235 nm). The polydispersity indices showed a homogeneous distribution with

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a monodisperse profile for the particles in solution (< 0.3). Given the electrostatic equilibrium of superficial charges of colloidal systems, the electrokinetic potentials were negative with a modular value (> 30 mV). All of the parameters were monitored for 90 days. The nanocarrier samples stored at 4 °C displayed adequate physical-chemical stability profiles. Tedesco et al. [27, 28] had previously described the morphological characterization of the NE and NC employed herein by transmission electron microscopy (TEM). The NC and NE consisted of spherical and homogeneous nanomaterial systems with sizes of approximately 200 and 250 nm, respectively. Insert Table 1 3.2. In vitro dark and phototoxicity assays We assessed the dark cytotoxicity effects of different concentrations of ClAlPc-loaded nanocarriers (0.3, 0.6, and 3.0 µg mL-1) on LNCaP cells by the MTT colorimetric assay 24 h after incubating the prostate cancer cells with ClAlPc-NC or ClAlPc-NE. On the basis of Figure 2, neither ClAlPc-NC nor ClAlPc-NE was cytotoxic regardless of the tested ClAlPc concentration (as compared to the cytotoxicity of untreated control LNCaP cells). We also exposed LNCaP cells to unloaded NC and NE. The results showed that the LNCaP cells treated with the unloaded nanocarriers and the untreated LNCaP cells did not differ in terms of cell viability (data not shown). Insert Figure 2 Based on these data, we accomplished phototoxicity assays using the lowest ClAlPcnanocarrier concentration (0.3 µg mL -1) as a safe dose and tested the effect of increasing laser light doses ranging from 0.5 to 7.0 J cm-2. Figure 3 shows that the ClAlPc-NCtreated LNCaP cells submitted to PDT had a cell viability lower than 50% even at a low light dose of 0.5 J cm-2. This result indicated that ClAlPc-NC exhibited a higher degree

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of phototoxicity compared to ClAlPc-NE, a pattern that emerged for all of the light doses used in this phototherapeutic treatment (0.5-7 J cm-2). At a light dose of 4 J cm-2, 90% of the ClAlPc-NC-treated neoplastic cells died. Moreover, Figure 3 reveals that cell viability was affected in a light dose-dependent manner. Insert Figure 3 Both ClAlPc-loaded nanocarrier systems have Poloxamer 188 and Epikuron in their composition. However, the ClAlPc-loaded nanoemulsion is a drug delivery system that normally presents a faster release compared to ClAlPc-NC because ClAlPc-nanocapsule also displays a reservoir system consisting of a drug depot surrounded by the poly(D,Llactide-co-glycolide) barrier. This polymeric membrane (PLGA) of the nanocapsule must have culminated in better drug delivery to the LNCaP cells to afford higher photodamage during PDT treatment. Additionally, it has been reported that the burst release of drugs can be controlled in PLGA-based nanoparticles. This is significant as burst release can result in the majority of the drug being ineffective, with decreased cytotoxicity levels [3335]. As described by Tedesco et al. [27], fast release of the photosensitizer drug was detected between 4 and 7 hours, and a portion of ClAlPc was slowly released for up to 12 h. These data suggest the initial burst rate was reduced, allowing a stable release rate to be reached resulting in a prolonged and more controlled release profile. During the release process, the drug diffuses through the hydrated polymer membrane into the aqueous phase. In such a hydration process, the polymer chains relax improving the diffusion of drug molecules. Moreover, cell viability studies by MTT showed that ClAlPc-NC more efficiently triggered cell death through the generation of ROS upon irradiation with 680 nm light with a lower laser dose during PDT. Thus, these data suggest that cellular internalization

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and uptake of ClAlPc-loaded nanocapsule was more effective compared to the ClAlPcNE drug delivery system using LNCap cells as a prostate cancer model. 3.3. Confocal imaging of human prostate cancer cells 3.3.1. Cellular localization and cytotoxicity studies of ClAlPc-nanocarriers in the dark We evaluated the cellular localization, internalization, and cytotoxicity of ClAlPcnanocarriers in LNCaP cells by confocal scanning laser microscopy. Figures 4A and 4B depict the confocal images of LNCaP cells treated with ClAlPc-NC or ClAlPc-NE in the dark and stained with DAPI and Alexa Fluor 488 phalloidin. DAPI (blue fluorescence) and phalloidin (green fluorescence) bound to DNA and F-actin, respectively, and the ClAlPc red fluorescent signal appeared in the cytoplasm. The merged images (Figure 4A(d) and 4B(d)) indicated maximum ClAlPc fluorescence in the perinuclear region of the LNCaP cells, without obvious ClAlPc fluorescence in the nuclei. Moreover, the ClAlPc-NC-treated LNCaP cells (Figure 4A) exhibited a higher fluorescence signal than the ClAlPc-NE-treated LNCaP cells (Figure 4B), which suggested that ClAlPc-NC were internalized to a larger extent by the LNCaP cells. Figures 4A and 4B also indicated that actin filaments, as organized fibers, crossed the cytoplasm. These observations attested that there were no changes in the cellular morphology and suggested that the ClAlPc-nanocarrier concentration employed herein (0.3 µg mL-1) was not cytotoxic. These results agree well with the cell viability data depicted in Figure 2. Insert Figure 4 To assess the subcellular localization of ClAlPc-nanocarriers, we co-loaded ClAlPc-NCand ClAlPc-NE-treated LNCaP cells with rhodamine-123, a mitochondrion-specific dye. The mitochondria are important PS targets for the initiation of apoptotic response [36].

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Figures 5A and 5B illustrate the confocal micrographs of the ClAlPc-nanocarrier-treated LNCaP cells stained with DAPI and Rh-123. The merged images (Figures 5A(d) and 5B(d)) of the ClAlPc red fluorescence and the Rh-123 green fluorescence in the mitochondria attested to the high co-localization level. Comparison of the merged images revealed that the ClAlPc-NC-treated LNCaP cells exhibited a higher co-localization level with mitochondria (as indicated by the significant increase in the yellow staining), which suggested better ClAlPc-NC uptake by the LNCaP cells compared to the ClAlPc-NE uptake by these same cells. Insert Figure 5 To ensure these data, Fiji (ImageJ) software 1.51h was used to estimate the fluorescence intensity profile of the ClAlPc-loaded nanocarriers in prostate cancer cells from confocal imaging data. Moreover, the degrees of colocalization of the ClAlPc-NC and ClAlPc-NE with the mitochondria of the cells using Pearson’s Correlation Coefficient (R) and Manders’ colocalization coefficient (MCC) were calculated by using JACoP, a plugin of the Fiji Software. Figure 6A (LNCaP cells treated with the ClAlPc-nanocarriers) and Figure 6B (LNCaP cells treated with the ClAlPc-nanocarriers and also incubated with rhodamine-123) correspond to the fluorescence intensity profile extracted from the confocal imaging data. Within these results, it is possible to verify that prostate cancer cells incubated with ClAlPc-NC (Figure 6A(a) and Figure 6B(a)) exhibited a much higher fluorescence intensity than those LNCaP cells treated with ClAlPc-NE (Figure 6A(b) and Figure 6B(b)). Graphics were plotted on the same scale. Insert Figure 6 As demonstrated in confocal imaging (Figure 5A(d) and Figure 5B(d)), colocalization with cell mitochondria occurred in both ClAlPc-nanocarriers. In addition, prostate cancer

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cells incubated with ClAlPc-loaded NC presented a strong Pearson correlation coefficient (R = 0.69), and LNCaP cells treated with ClAlPc-NE showed a lower Pearson coefficient (R = 0.62). Moreover, Manders’ colocalization coefficient analyses revealed that the overlap of red and green channels is slightly higher in the prostate cancer cells treated with the ClAlPc-NC system (MCC = 0.887) than those cells treated with the ClAlPc-NE system (MCC = 0.811). Taken together, these results suggest that ClAlPc-NC was more efficiently internalized than ClAlPc-NE by the prostate cancer cells. These results pave the way for using ClAlPc-nanocarriers as a photodiagnostic agent in prostate cancer. The light-induced electronic excitation not only prompts a cytotoxic effect but also promotes fluorescence emission via relaxation of the PS excited state and its return to the ground state. 3.3.2. Phototoxic effects of ClAlPc-nanocarriers after PDT treatment Figures 7A and 7B contain confocal images of ClAlPc-nanocarrier-treated LNCaP cells after PDT treatment. The treated cells were incubated with DAPI (blue) and Alexa Fluor phalloidin 488 (green) for DNA and F-actin staining, respectively. Insert Figure 7 CLSM images of actin-stained (phalloidin) cells illustrate the changes in the actin cytoskeleton that occurred after photodynamic therapy (Figure 7A(d) and Figure 7B(d). According to Figure 7A(d), compared to ClAlPc-NE-treated LNCaP cells (Figure 7B(d)), ClAlPc-NC-treated LNCaP cells experienced much higher photodamage to the actin filament structure upon PDT treatment with a 4 J cm-2 light dose, as indicated by the significant change in cellular morphology. These results suggested that ClAlPc-NC induced LNCaP cell death to a larger extent than ClAlPc-NE upon PDT treatment, in agreement with the cell viability results displayed in

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Figure 3. Moreover, the ClAlPc-NE red fluorescence (Figure 7B(d)) appeared more in the LNCaP cells perinuclear region than in those ClAlPc-NC-treated cells (Figure 7A(d)). Fiji software was also used to estimate the fluorescence intensity profile of the actinstained (phalloidin) from confocal imaging data. The ClAlPc-NC-treated cells exhibited higher green fluorescence intensity of the actin cytoskeleton (Figure 7A(e)) compared to the ClAlPc-NE-treated LNCaP cells (Figure 7B(e)), possibly due to the larger shrinkage of the actin filaments in ClAlPc-NC-treated LNCaP cells. Silva et al. [37] evaluated the in vitro photodynamic activity of chloro(5,10,15,20tetraphenylporphyrinato)

indium

(III)-loaded

poly(lactide-co-glycolide)

(InTPP)

nanoparticles in LNCaP cells. Petri et al. [38] presented a comparative characterization of

the

cellular

uptake

and

photodynamic

efficiency

of

Foscan®

(meso-

tetra(hydroxyphenyl) chlorin, m-THPC) and its liposomal formulation Fospeg in a human prostate cancer cell line. Comparing our results with those of Silva et al. [37] (InTPP concentration: 2.9 µg mL-1; incubation time: 2 h; light dose: 30 to 45 J cm-2; and intracellular localization: perinuclear) and Petri et al. [38] (m-THPC concentration: 1.2 µg mL-1; Fospeg concentration: 0.15 µg mL-1 ; incubation time: 3 h; light dose: 180 mJ cm-2; and intracellular localization: perinuclear), we used a lower drug concentration (0.3 µg mL-1) than for InTPP and m-THPC as well as a lower light dose (4 J cm-2) for PDT treatment in relation to the report of Silva [37]. It is well known that the clinically phototherapeutic window for tumors should be between 600 and 900 nm. As for InTPP, Fospeg and m-THPC present low light absorption in the NIR window, and these molecules lose efficiency in PDT when compared to ClAlPc. Body tissue is easily penetrable in this spectral window, and thus NIR light can be used for the activation of PS drugs accumulated in deep-seated cancer tumors without causing phototoxicity to healthy tissue.

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Conclusion Our results showed that both ClAlPc-NC and ClAlPc-NE nanocarrier systems displayed imaging and phototherapeutic potential for human prostate cancer cells in vitro. However, ClAlPc-loaded nanocapsule exhibits better theranostic behavior than the ClAlPc-loaded nanoemulsion. These findings encourage further in vivo studies on animal models to explore the potential application of ClAlPc-loaded nanocapsule in the diagnosis and treatment of prostate cancer. Conflict of interest The authors declare that there is no conflict of interest regarding the work reported in this paper. Acknowledgments A.C.T. is thankful to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP Grants # 08/53719-4) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for financial support. F.Z.L. would like to thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the postdoctoral scholarship. J.M. is grateful to CNPq for the individual scholarship.

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Figure captions Figure 1. Chemical structure of chloroaluminum phthalocyanine (C32H16AlClN8). Figure 2. Dark toxicity of ClAlPc-loaded nanocarriers on LNCaP cells. Neoplastic cells were incubated with different concentrations of ClAlPc-nanocarriers in a 5% CO2 atmosphere and at 37 °C for 3 h. The MTT toxicity assay was carried out 24 h after incubation of the LNCaP cells with ClAlPc-nanocarriers. The data represent the mean ± SD of three independent experiments. Untreated LNCaP cells (control) were defined as 100% viable. ClAlPc: chloroaluminum phthalocyanine; LNCaP: prostate cancer cells; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; SD: standard deviation; Ctrl: control; NC: ClAlPc-loaded nanocapsule; and NE: ClAlPc-loaded nanoemulsion. Figure 3. Phototoxicity effects of ClAlPc-loaded nanocarriers on LNCaP cells. Cancer cells were incubated with ClAlPc-nanocarriers at 0.3 µg mL-1 in a 5% CO2 atmosphere and at 37 °C for 3 h, which was followed by irradiation with increasing light doses of a 670-nm diode laser (average power = 650 mW; light irradiance = 92 mW cm-2). The MTT toxicity assay was performed 24 h after PDT treatment. The data represent the mean ± SD of three independent experiments (*p < 0.05 compared to untreated cells). Untreated LNCaP cells (control) were defined as 100% viable. ClAlPc: chloroaluminum phthalocyanine; LNCaP: prostate cancer cells; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide; PDT: photodynamic therapy; SD: standard deviation; Ctrl: control; NC: ClAlPc-loaded nanocapsule; and NE: ClAlPc-loaded nanoemulsion. Figure 4. Localization of ClAlPc-loaded nanocarriers in LNCaP cells as determined by confocal laser scanning microscopy. Confocal images were processed by using Fiji (ImageJ) software 1.51h. The LNCaP cells were incubated with ClAlPc-nanocarriers at

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0.3 µg mL-1 for 3 h (37 °C, 5% CO2). Confocal analysis was carried out 24 h after incubation of the LNCaP cells with ClAlPc-nanocarriers. (A) ClAlPc-loaded nanocapsule. (B) ClAlPc-loaded nanoemulsion. (a) Nuclei were stained with DAPI (emission at 461 nm), (b) F-actin filaments were labeled with Alexa Fluor 488 phalloidin (emission at 525 nm), (c) red fluorescence of ClAlPc-nanocarriers, and (d) merged images of (a), (b), and (c). ClAlPc: chloroaluminum phthalocyanine; LNCaP: prostate cancer cells; and DAPI: 4’,6-diamidino-2-phenylindole. White arrows indicate the cellular localization of ClAlPc-nanocarriers that were found preferentially around the perinuclear region. Scale bars: 10 µm. Figure 5. Co-localization of ClAlPc-loaded nanocarriers with mitochondria in LNCaP cells as determined by confocal laser scanning microscopy and processed by using Fiji (ImageJ) software 1.51h. The LNCaP cells were incubated with ClAlPc-nanocarriers at 0.3 µg mL-1 for 3 h (37 °C, 5% CO2). After incubation, the ClAlPc-nanocarrier-treated LNCaP cells were co-loaded with Rh-123 for 30 min (37 °C, 5% CO2). Confocal analysis was conducted 24 h after incubation of the LNCaP cells with Rh-123. (A) ClAlPc-loaded nanocapsule. (B) ClAlPc-loaded nanoemulsion. (a) Nuclei were stained with DAPI (blue fluorescence), (b) mitochondria were labeled with Rh-123 (green fluorescence), (c) red fluorescence of ClAlPc-nanocarriers, and (d) merged images of (a), (b), and (c). ClAlPc: chloroaluminum phthalocyanine; LNCaP: prostate cancer cells; Rh-123: rhodamine-123; and DAPI: 4’,6-diamidino-2-phenylindole. Cellular localization of ClAlPc-nanocarriers was found preferentially in the perinuclear region. Scale bars: 10 µm. Figure 6. 3-D fluorescence intensity profile using Fiji (ImageJ) software 1.51h of confocal imaging from Figure 4A(c), Figure 4B(c), Figure 5A(c) and Figure 5B(c). (A) LNCaP cells treated with (a) ClAlPc-NC and (b) ClAlPc-NE. (B) LNCaP cells treated with ClAlPc-nanocarriers and also incubated with rhodamine-123: (a) ClAlPc-NC and

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(b) ClAlPc-NE. LNCaP: prostate cancer cells; ClAlPc: chloroaluminum phthalocyanine; NC: nanocapsule; and NE: nanoemulsion. Figure 7. Confocal imaging of LNCaP cells exposed to ClAlPc-loaded nanocarriers and submitted to PDT treatment. Confocal image data were processed by using Fiji (ImageJ) software 1.51h. The neoplastic cells were incubated with ClAlPc-loaded nanocarriers at 0.3 µg mL-1 for 3 h (37 °C, 5% CO2) and irradiated with a 670-nm diode laser (average power = 650 mW and light irradiance = 92 mW cm-2) at light dose of 4 J cm-2. (A) ClAlPcloaded nanocapsule. (B) ClAlPc-loaded nanoemulsion. (a) DAPI (blue fluorescence) bound to DNA for staining of the nucleus, (b) phalloidin (green fluorescence) bound to F-actin for staining of the cytoskeleton, (c) red fluorescence of ClAlPc-nanocarriers, (d) merged images of (a), (b), and (c), and (e) surface plot of the fluorescence intensity profile of phalloidin. LNCaP: prostate cancer cells; PDT: photodynamic therapy; ClAlPc: chloroaluminum phthalocyanine; and DAPI: 4’,6-diamidino-2-phenylindole. Scale bars: 10 µm.

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Figure 1

28

Figure 2

29

Figure 3

30

Figure 4

31

Figure 5

32

Figure 6 A

33

Figure 6 B

34

Figure 7

35

Table caption Table 1. Mean sizes, polydispersity indices (PdI), and zeta potentials of the colloidal nanocarriers.

Table 1. Colloidal Nanocarriers

Mean Size (nm)a

PdIb

Zeta Potential(mV)c

ClAlPc-NE

233.8 ± 7.8

0.274 ± 0.06

-43.4 ± 2.5

ClAlPc-NC

221.3 ± 2.6

0.258 ± 0.03

-40.9 ± 1.6

ClAlPc: chloroaluminum phthalocyanine. NE: nanoemulsion. NC: nanocapsule. a Mean size ± SD. b PdI ± SD. c  ± SD. SD: Standard deviation. Statistical number n=3.