Semi-synthesis and PDT activities of a new amphiphilic chlorin derivative

Accepted Manuscript Title: Semi-synthesis and PDT activities of a new amphiphilic chlorin derivative Author: Milene N.O. Moritz Joyce L.S. Gonc¸alves Irwin A.P. Linares Janice R. Perussi Kleber T. de Oliveira PII: DOI: Reference:

S1572-1000(16)30189-2 http://dx.doi.org/doi:10.1016/j.pdpdt.2016.10.005 PDPDT 841

To appear in:

Photodiagnosis and Photodynamic Therapy

Received date: Revised date: Accepted date:

13-9-2016 13-10-2016 17-10-2016

Please cite this article as: {http://dx.doi.org/ 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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Semi-synthesis and PDT Activities of a new Amphiphilic Chlorin Derivative

Milene N. O. Moritz,a Joyce L. S. Gonçalves,b Irwin A. P. Linares,b Janice R. Perussi a,b* and Kleber T. de Oliveira c*

a

Programa de Pós-Graduação Interunidades Bioengenharia EESC/FMRP/IQSC, Universidade de São Paulo,

São Carlos, SP, Brazil b

Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil.

c

Departamento de Química, Universidade Federal de São Carlos, São Carlos, SP, Brazil.

Corresponding author address: Departamento de Química, Universidade Federal de São Carlos, Rodovia Washington Luís, km 235 - SP-310 São Carlos - São Paulo - Brazil CODE: 13565-905

*E-mail address: [email protected]/ [email protected]

Graphical abstract

2

Highlights -Semi-synthesis of a new amphiphilic chlorin derived from chlorophyll a is described -Efficient singlet oxygen generation was obtained compared to control photosensitizers -The phototoxicity presented by the new chlorin is ten times higher than the controls

3

Abstract An amphiphilic chlorin derivative (CHL-T) was prepared from methylpheophorbide a (CHL) and

2-Amino-2-(hydroxymethyl)-1,3-propanediol

(TRISMA®).

The

new

chlorin

was

compared to other dyes (CHL and Hypericin) in relation to photophysical and photobiological activities in tumor and non-tumor cell lines. Cytotoxicity and cell death target were determined to evaluate the CHL-T efficiency, comparing to the precursor CHL and to the well-known dye hypericin (HY). All of the studied compounds exhibited absorption bands in the therapeutic window and presented a small fluorescence quantum yield compared to the reference dye (rhodamine B). CHL-T was about three times more efficient on singlet oxygen generation than the others photosensitizers. The lipophilicity order of the photosensitizers was CHL>HY>CHL-T. The tumoral HeLa cells presented improved accumulation for CHL and CHL-T compared to HY. The phototoxicity presented by the CHL-T was about ten times higher than by CHL, as demonstrated by the MTT assay. CHLT showed more cytotoxicity to tumoral cell, comparing to non-tumoral cell in short incubation time. The cell death rises proportionally with increasing PSs concentrations, mainly by necrosis. These findings suggest that CHL-T is a potential new photosensitizer for PDT.

Keywords: amphiphilic photosensitizer; chlorins; photodynamic therapy; tumor cells; phototoxicity; necrosis

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Introduction

Photosensitizers (PSs) are compounds that in the presence of light absorb the light and transfer adequate radiations to molecular oxygen or biological molecules, inducing the formation of Reactive Oxygen Species (ROS) which cause biological tissue destruction. These properties have been explored in Photodynamic Therapy (PDT) treatments for the eradication of tumors and other diseases such as psoriasis, mycoses, rheumatoid arthritis, age-related macular degeneration, bacterial and viral infections. PSs may be administered systemically or topically to the patient depending on the stage of disease and its location (1, 2). It is suggested that there are three mechanisms responsible for tumor eradication in PDT treatments: (i) directly by the generation of ROS, causing cellular necrosis or apoptosis, (ii) indirectly by damaging the vasculature of the tumor region, resulting in oxygen and nutrient limitation to the tissue, and (iii) by activation of the immune response against the tumor (3, 4). New photosensitizers have been designed in order to obtain better performances in photodynamic processes (5, 6). The porphyrins are considered the first generation of PSs, and the hematoporphyrin (Hp) derivatives represent the most important porphyrinoids in use (7). Photofrin® was the first commercially approved porphyrinoid for use in PDT treatments to the lung, cervix, esophagus and bladder cancers (8, 9). Despite the success of the first generation PSs, they have a slow elimination from the organism and the absorption bands of these compounds are out of the best therapeutic window wavelength. Aiming to overcome these disadvantages, a number of second generation PSs were developed,

such

as

chlorins,

bacteriochlorins,

benzoporphyrins,

phthalocyanines,

naphthalocyanines, and others (10, 11). Some second generation PSs, however, present low solubility in aqueous media, thus limiting some uses in PDT treatments. One strategy to overcome this limitation is the use of vehicles that transport and deliver PSs to the target tissue with selectivity and specificity (third generation PSs) (12-14), but formulations always require massive studies to attest their efficient use. Other strategies have also been provided to increase the efficiency of PDT, such as the simultaneous use of two PSs in therapy combinations (15-17). It is well known that porphyrinoid systems comprise the most successful photosensitizers in use for PDT treatments; however, hypericin (HY) is also an efficient photoactive natural pigment extracted from Hypericum perforatum, commonly known as St. John’s plant, which has anti-inflammatory, antiseptic, anti-infectious and antiviral activities

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(10), and mainly PDT activity. Studies carried out in our laboratory with hypericin have demonstrated its photoactivity in cells and in microorganisms, as well as a good intracellular accumulation rate (18, 19), thus making this compound very useful for PDT. Considering all of the described and well-known limitations of current commercial photosensitizers, we have proposed a new semi-synthetic compound derived from methylpheophorbide a and TRISMA® in order to obtain an amphiphilic molecule with highlighted photosensitizing properties. In this study, we have investigated physical-chemical properties of this new chlorin derivative as well as its photodynamic efficiency against the tumoral HeLa cells. The CHL-T accumulation, cytotoxicity and type of cell death (apoptosis or necrosis) were evaluated in comparison to the precursor chlorin and to hypericin. Materials and methods

Chemicals

Methylpheophorbide a (CHL) was semi-synthesized from chlorophyll a as described in the literature (20) and hypericin (HY) was prepared from natural emodin according to the literature as well (21-23). The CHL-T was prepared from CHL after reaction with excess of TRISMA® in tetrahydrofuran at 120oC (48h) in a glass pressure tube (Scheme 1). The compound CHL-T was purified on silica column chromatography as detailed below. The structure of the compound CHL-T was completely assigned by 1H and

13

C-NMR spectroscopy (1D and 2D)

and HRMS-TOF. The site-selectivity of this reaction was determined by the HMBC analysis (2D–NMR) where the correlation between H-132 and C-133 and also H-134 (NH) and C-133 completely defined the position of the transamidation (C-133). Probably, an internal acid catalysis performed by a keto-enol form makes the ester portion at the position C-133 more reactive than at the position C-173 (Scheme 1).

6

Scheme 1. Synthesis and site-selectivity proposal for the CHL-T preparation.

Synthesis of CHL-T: 152.0 mg of CHL (0.250 mmol) and 151.0 mg of TRISMA® (1.25 mmol) were dissolved in 5 mL of dry and deoxygenated tetrahydrofuran in a glass pressure tube. The reaction was protected from light and heated at 120 oC for 48 h. After that, 50 mL of ethyl acetate was added and the reaction mixture extracted with water (3 x 50 mL). The organic layer was dried with Na2SO4 and purified over silica gel column chromatography using a 9:1 CH2Cl2:CH3OH mixture as eluent (Rf = 0.2) yielding compound CHL-T in 63% yield (119.5 mg, 0.16 mmol). 1H-NMR, CDCl3, δ (ppm), 400.13 MHz: 9.37 (s, 1H), 9.30 (s, 1H) and 8.52 (s, 1H) (H-5, H-10 and H-20), 7.94 (dd, 1H, J = 17.7 Hz and J = 11.7 Hz, H31), 7.94 (br.s., 1H, H-134, NH), 6.26 (dd, J = 17.7 Hz and J = 1,4 Hz, 1H, H-32α), 6.22 (s, 1H, H-132), 6.16 (dd, J =11.7 Hz and J = 1.4 Hz, 1H, H-32β) 4.22-4.43 (m, 5H, H-17, H-18, 3 x OH), 3.86-3.98 (m, 6H, H-136), 3.66 (s, 3H, H-121), 3.56-3.64 (m, 5H, H-174 and H-81), 3.37 (s, 3H, H-21), 3.18 (s, 3H, H-71), 2.86 – 2.96 (m, 1H), 2.51-2.60 (m, 1H), 2.39-2.49 (m, 1H), 1.86-1.95 (m, 1H) (H-171, H-172), 1.83 (d, J = 7.2 Hz, 3H, H-81), 1.65 (t, J = 7.6 Hz, 3H, H-82), 0.50 and -1.68 (br.s., 2H, H-23 and H-21). 1

13

C-NMR (CDCl3, 100.04 MHz) δ

3

(ppm): 192.0 (C-13 ), 174.6 (C-17 ), 172.4 (C-19), 169.4 (C-133), 162.9 (C-16), 155.9 (C-6), 151.0 (C-9), 150.0 (C-14, C-15), 145.2 (C-8), 142.3 (C-1), 137.9 (C-11), 136.7 (C-4), 136.3 (C-7), 136.2 (C-3), 131.9 (C-2), 129.0 (C31), 128.9 (C-12), 122.8 (C-32), 104.5 (C-13), 104.4 (C-10), 97.5 (C-5), 93.3 (C-20), 65.9 (C-132), 65.0 (C-136), 62.2 (C-135), 51.8 (C-174), 51.1 (C-17), 50.2 (C-18), 30.7 (C-172), 30.1 (C-171), 23.1 (C-181), 19.4 (C-81), 17.4 (C-82), 12.1 (C-121, C-21), 11.2 (C-71). MS (ESI): (m/z) calculated for [M-H]- 694.3, C39H44N5O7, found

7

694.0. HRMS-TOF (m/z) calculated for [M+H]+ 696.3392, C39H46N5O7, found 696.3409 (δ = 2.4 ppm). CHL: UV/Vis (DMSO): max (log) = 411 (4.77), 506 (3.84), 538 (3.72), 607 (3.75), 667 (4.39) nm and CHL-T: UV/Vis (DMSO)max (log)= 411 (5.24), 506 (4.28), 538 (4.25), 607 (4.19), 667 (4.85) nm. Only freshly distilled water and ultra-pure water (Milli-Q, Millipore, USA) were used. All other chemical reagents were commercially obtained as reagent-grade products. DMSO stock solutions of 1.65 x 10-3 mol dm-3 (CHL); 1.44 x 10-3 mol dm-3 (CHL-T) and 1.98 x 10-3 mol dm-3 (HY) were prepared, and stored at 4oC in the absence of light. The working solutions to treat the cells contained less than 1% of DMSO (v/v), which is a concentration that is not cytotoxic to mammalian cells (24).

Figure 1. Structures of the studied photosensitizers.

Aggregation studies

UV-Vis analysis was used to evaluate the aggregation degree as function of the PS concentration. Spectra of the PS solutions ranging from 1 x 10-6 to 1 x 10-5 mol L-1 using DMSO as solvent were obtained and the coefficient of molar absorptivity was determinated at 598 and 667 nm for HY and chlorins, respectively.

Fluorescence measurements

8

Fluorescence quantum yields (ΦF) were determined by a relative method (25) using rhodamine B in ethanol as a reference (0.65) (26). Solutions in the same solvent were excited at 515 nm and the spectra were obtained from 525 to 800 nm for calculation using Equation 1: Equation 1

where F is the integrated area under the fluorescence emission spectrum, A is the absorbance of the solutions at the excitation wavelength (lower than 0.05 to avoid the filter effect) and the subscripts R and S refer to the reference and the sample, respectively. Fluorescence measurements were performed in a spectrofluorimeter (HITACHI F-4500, Japan).

Determination of photodynamic potential using uric acid as a chemical dosimeter

In order to evaluate the photodynamic activity of the photosensitizers, uric acid (UA) was used as a singlet oxygen scavenger using an adaptation of Fischer´s method (27). Twentyfive milliliters of 7.2 x 10-7 mol L-1 of photosensitizers and UA 8.5 x 10-5 mol L-1 in phosphate buffer with 2% SDS, pH 7.0, were magnetically stirred in the dark. The solutions were irradiated for 0, 300, 600 and 1200s using a Biotable consisting of an illumination table containing a matrix of 5 x 10 LEDs centered at 630 ± 10 nm (I= 18 mW cm-2, CEPOF, IFSCUSP). The pseudo-first order kinetics of UA oxidation was determined as a function of the irradiation time by the decrease in UA absorption in the solution at 293 nm (ΔAUA): Equation 2

where

,

,

and

are the photodynamic activity (m2 J-1), the irradiance (mW cm-2),

the irradiation time and the absorbance of the photosensitizer in solution at the irradiation wavelength,

respectively.

Absorbance

spectrophotometer (HITACHI U-2800, Japan).

measurements

were

performed

in

a

9

Determination of organic/aqueous phase partition of the photosensitizers

The lipophilicity of the photosensitizers was evaluated by the partition coefficient (P) in 1octanol and phosphate buffer 20 mM at pH 7.0, each one pre-saturated with the other using the shake-flash method (28). Equal amounts of buffer and 1-octanol with photosensitizer at 1.4 x 105 mol L-1 were mixed for 3 h on a magnetic stirrer in the dark. The phase separation was followed by 10 min centrifugation at 1000 rpm (Excelsa® II 206 BL, Fanem-Brazil). The absorbance of photosensitizers (667 nm for CHL and CHL-T and 590 nm for HY) were measured before and after the partition to determine the log P, referred in Equation 3: (

)

(

where

and

)

Equation 3

are the absorption of the photosensitizer before and after partitioning in

the organic phase, respectively.

Cell culture

The HeLa cell line (CCL-2TM, ATCC, USA) was used to evaluate the phototoxicity, intracellular accumulation and cell death induced by the PSs after photodynamic treatment. These human cervical carcinoma cells were grown in Iscove’s Modified Dulbecco’s media (Sigma–Aldrich, Brazil) supplemented with 10% FBS (Fetal Bovine Serum, Cultilab, Brazil) and 0.01% of antibiotics (penicillin and streptomycin) in 25 cm2 cell culture flasks and incubated at 37oC and 5% CO2 (Forma Scientific Incubator). We also carried out cytotoxic assays with the non-tumoral epithelial kidney cells Vero (CCL81™, ATCC, USA) grown at the same conditions as HeLa cells. Before the experiments, the cell viability was measured using the trypan blue exclusion method.

Intracellular accumulation of photosensitizers Cells were seeded (1 x 105 cell mL-1) in 60 mm diameter Petri dishes. After 24 h, the culture media was removed and replaced with the same amount of fresh culture media containing

10

the PSs (CHL, CHL-T or HY) at 1.4 x 10-6 M. Following incubation times of 1, 2, 4, 8, 16, and 24 h, the media was removed and the cells were washed with phosphate buffered saline (PBS). The cells were then lysed with ethanol, centrifuged and the supernatant was quantified by fluorescence to determine the PS concentration incorporated by the cells over time. Calibration curves were previously prepared from the fluorescence intensity of each PS in ethanol. The fluorescence of each sample was measured in a spectrofluorimeter and normalized by the protein concentration according to the Lowry method (29).

Cell photosensitization

For photodynamic assays, cells were incubated with the PSs in absence of light with different concentrations of CHL, CHL-T or HY for a period of 2 or 16 h (previously determined by the accumulation assays). In these experiments, 1 x 105 cells mL-1 for MTT assay and 1x106 cells mL-1 for apoptosis/necrosis assays were seeded in Iscove’s Modified Dulbecco’s medium supplemented with 10% FBS in 96-well plates and grown at 37°C and 5% CO2. Following the incubation period, the media with PS was removed, the cells were washed with PBS and a fresh medium was added. Then, the cells were irradiated at 630 ± 10 nm LED with a red light of 6.0 J cm-2 and irradiance of 18 mW cm-2. Three independent assays with triplicates (accumulation and cell death assays) or sextuplicates (MTT assay) were performed.

Phototoxicity assay The cells were seeded (1 x 105 cells mL-1) in 96-well plates and submitted to photodynamic treatments with CHL, CHL-T and HY in order to evaluate cell viability using the MTT assay (30). This method is based on the reduction of thiazolyl blue tetrazolium into a majority of purple formazan by active mitochondrial dehydrogenases, which are found only in metabolically active cells (31). With 24 hours of post-irradiation, the medium was removed and the cells were incubated for 4h with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution at 1 mg mL-1 in culture media. Following the incubation period, the media containing MTT was removed and the formazan crystals formed were solubilized in 50 µL of absolute ethanol and 150 µl of isopropyl alcohol. The absorbance was read at 570 nm by a spectrophotometer (Benchmark BIO-RAD). The cell survival rate (%) was assessed dependent on the photosensitizer concentration. The average inhibitory

11

concentrations (IC50) of the PSs were determined by the Calcusyn® program. The analysis of controls with no treatment, treated only with PSs (not irradiated), and treated only with irradiation (without PSs) were also carried out in parallel.

Detection of cell death by fluorescence microscopy

It is known that cells that undergo necrosis are red, marked with ethidium bromide, as this compound only penetrates those cells that have lost their membrane integrity. Cells undergoing apoptosis appear green, with apoptotic bodies marked by acridine orange, or appear orange when marked by the two fluorochromes. Living cells are stained by acridine orange and appear green with arranged structures that can be observed by fluorescence microscopy (32). HeLa cells were seeded (1 x 106 cells mL-1) in 6-well plates with culture medium. After 24 h the cells were incubated with different concentrations of PSs for 2 or 16 h. Then the cells were washed with PBS and maintained in culture medium and irradiated with a red light of 6 J cm-2. After 24 h, the cells were stained with 1 µL of ethidium bromide and acridine orange (100 µg mL-1). The labeled cells were placed in glass slides and overlaid with coverslips for the visualization under fluorescence microscope (Olympus BX41) at 20x magnification, excitation filter of 460/90 nm, dichromatic mirror of 50 nm and barrier filter of 520 nm. Images of five fields of each slide (sample) to count a minimum of 200 cells were captured (Olympus DP72 camera).

Statistical analysis

The results were expressed as the mean ± S. E. (average of three independent assays). For chemical analysis, Q-Dixon and Student-t tests were used. In biological analysis (cell death by fluorescence microscopy), ANOVA followed by Tukey or Student t-test were used. Both analyses were accessed with a confidence interval of 95%.

Results and Discussion

12

The absorption spectra of CHL and CHL-T solutions in DMSO and phosphate buffer 2.0 × 10-2 mol L-1, pH 7.0 are shown in Figure 2. Both chlorins presented a typical UV-Vis spectrum of reduced tetrapirrol compounds: a Soret band centered at 413 nm and four Q bands centered at 506, 538, 607 and 667 nm due to the * and n-* transitions, respectively. The ratio between Soret/Q band at 667 nm was about 2.4.

200

(a) DMSO

CHL CHL-T

150

50

-1

-1

 (x10 L mol cm )

100

0

3

(b) Phosphate buffer pH 7.0 150

100

50

0 300

400

500

600

700

Wavelength (nm)

Figure 2. Absorption spectra of CHL and CHL-T in (a) DMSO and (b) phosphate buffer 2.0 × 10-2 mol L-1 pH 7.0. As can be observed in the absorption spectra, the insertion of the TRISMA® fragment did not modify the chromophore group; consequently, the spectroscopic properties of the chlorin are preserved. However, these properties can be highly influenced by their environment. In phosphate buffer, the absorption spectra of the PS exhibited the same

13

bands, but with a slight red-shift at the Q band and a blue-shift at the Soret band. These changes can be explained by the low solubility of these compounds in aqueous medium. It has been described that HY and some of its derivatives show low solubility in aqueous medium (33). As previously described, the absorption spectra of HY in organic solvents show two main bands centered at 556 and at 598 nm assigned to dimeric and monomeric forms, respectively. Similar to chlorins in aqueous medium, HY shows not only the same bands but also a change in the band intensity relative to the monomeric and dimeric forms. All of the studied compounds exhibited absorption bands in the therapeutic window (around 600 nm, in which the light has a higher skin penetration capacity), so their photodynamic potential as photosensitizers was evaluated considering their amphiphilic behavior, the singlet oxygen generation and tumor cell death. The Beer-Lambert law behavior of the PS was investigated in DMSO in concentrations ranging from 1 x 10-6 to 1 x 10-5 mol L-1. The data showed that the intensity of absorption bands increased linearly with the concentration of the PS and no new bands were observed. This means that all PSs followed the Beer–Lambert law in the studied concentration range (Table 1), suggesting that there is no aggregation in this concentration range. It is important to mention that in the cytotoxicity experiments, the concentrations used are lower than those tested by Beer-Lambert law. Table 1. Physical-chemical parameters obtained for the studied photosensitizers 2

-1

i

PS

log ε (DMSO)

ΦF

PA (m J )

log P

CHL

4.36

0.0118 ± 0.0006

35±7

∞*

CHL-T

4.86

0.0112 ± 0.0002

92±7

-2.02±0.61

HY

4.37

0.0520 ± 0.0080

26±5 (34)

0.48±0.14

i

PS is the photosensitizer; ε is the coefficient of molar absorptivity at 667 nm for CHL and CHL-T in DMSO and 598 nm for HY; ΦF is the fluorescence quantum yield in ethanol; PA is photodynamic activity and log P is the partition coefficient.

Fluorescence measurements

The fluorescence quantum yield (ΦF) values for the photosensitizers using rhodamine B in ethanol as a reference were also determined (Table 1). There were no significant

14

differences in ΦF between CHL and CHL-T (Student t test, 95% confidence). These results are consistent with the spectroscopic studies since both chlorins have the same chromophore group and no changes were caused in fluorescence emission spectrum by addition of the hydrophilic group TRISMA® in CHL. The ΦF value of HY has been previously determined in our group as approximately five times higher than the chlorins. On the other hand, all photosensitizers have a small ΦF compared with the reference dye (rhodamine B, 0.65 (35)).

Photodynamic activity

It has been described that UA absorbs at the UV range of the spectrum between 220 and 293 nm (27). As demonstrated before, CHL and CHL-T can absorb in the visible region of the spectrum and no significant absorbance in UV wavelengths was identified (Figure 1). When UA solutions containing PSs were irradiated with red light, there was a decrease in the intensity of the bands centered at 220 and 293 nm, caused by oxidation of this singlet oxygen scavenger (figure 3). In the absence of PS, there were no changes in absorbance of the solutions, even through irradiation (data not shown). This UA oxidation occurs by the capture of singlet oxygen formed by photosensitizers in the triplet state and molecular oxygen, which leads to the formation of triuret, sodium oxalate, allantoxaidin and CO2 (36). Photodynamic activity of photosensitizers (Table 1) was determined after 300s of irradiation, showing a linear correlation between

as function of the irradiation time.

15

1.4

Time (s) 1.2

0 300 600 900 1200

ln (Absorbance)

Absorbance

1.0 0.8 0.6 0

200

400

600

800

1000 1200

Wavelength (nm)

0.4 0.2 0.0 300

400

500

600

700

Wavelength (nm)

Figure 3. Absorption spectra of the mixture of CHL-T (7.2 x 10-7 mol L-1) and uric acid (8.5 x 10-5 mol L-1) in phosphate buffer with 2% SDS (pH 7.0), irradiated with red light (= 630 ± 10 nm and I= 18 mW cm-2). The inset graphic shows the linearity between the natural logarithm of the absorbance values as a function of the wavelength.

All of the photosensitizers studied, when excited by red light, proved to be able to interact with molecular oxygen, thus generating singlet oxygen. There were no significant differences between the photodynamic activity values of CHL and HY (Student t test, 95% confidence), but CHL-T was about three times more efficient for singlet oxygen generation than the other photosensitizers. As these photosensitizers are not soluble in aqueous media, we adapted the UA method by adding 2% SDS in phosphate buffer to ensure that there is no difference in the level of aggregation, i.e., both PSs are completely monomerized in the experimental system. The aggregate formation was already demonstrated as a suppressor of singlet oxygen evaluated not only by the UA method (23) but also by other chemical dosimeters such as 1,3-Diphenylisobenzofuran (DPBF) (37) and p-nitrosodimethylaniline (RNO) (38). It was considered that cell culture medium can

16

solubilize the photosensitizers; thus, the studies in similar conditions for the photodynamic activity were very helpful. Log P

Log P values are directly proportional to the lipophilicity of the substance (39). Considering Equation 3, a higher Log P value indicates a more lipophilic substance. Thus, positive values of Log P characterize lipophilic compounds, while negative values indicate hydrophilic compounds. The data presented show that the lipophilicity order of the photosensitizers is CHL>HY>CHL-T (Table 1). Log P for CHL was considered as high because its absorption spectrum was the same before and after partition. Such behavior characterizes the strong affinity of CHL with the organic phase and, consequently, ratifies that it is not possible to solubilize this compound in aqueous phase. However, the negative value of CHL-T confirms that the hydrophilic TRISMA® group provided a greater wateraffinity to the CHL compound. Log P values above zero may indicate substances with intermediate behavior, such as amphiphilic compounds. It has been demonstrated that lipophilic photosensitizers can easily cross biological membranes. On the other hand, as photosensitizers usually are administrated systemically in the bloodstream, they are expected to present hydrophilic character as well. Therefore, the hydro/lipophilic balance is considered an important parameter in the photosensitizer biodistribution.

Photosensitizers accumulation assay

First, a standard absorbance curve for BSA at 750 nm was obtained, and the values were used to estimate the protein concentration of each sample and also to normalize the obtained fluorescence values. Each PS analytical curve was previously obtained by fluorescence of the PS in ethanol (data not shown). The curves of intracellular PS accumulation are shown in Figure 4.

17

Figure 4. Intracellular PS accumulation (mg PS/mg protein) after incubation with CHL, CHL-T or HY at 1.4 x 10-6 M in HeLa cells. Values are the means ± S.D. (average of three independent assays).

It can be observed that the cell PS uptake increases with the incubation time for the three PSs and the chlorin accumulation being higher compared to hypericin. The accumulation assay data were consistent with the Log P results, as they show a lipophilic character of CHL with a high accumulation rate.

Phototoxicity

Figure 5 shows that the cell survival rates after irradiation for the HeLa cell line decrease as a function of photosensitizer concentration.

18

A)

B)

Figure 5. Cell survival rate of HeLa cell line at different PSs concentrations after irradiation with red light (6 J cm-2, I= 18 mW cm-2), 24 h after irradiation. A: Incubation time of 2 h. B: Incubation time of 16 h. Average of three independent assays.

The IC50 values are shown in Table 2. It can be observed that the chlorin structural modification improved the phototoxicity by about ten times for the two incubation times studied. Other modified PSs have been studied for use in PDT, such as metallochlorins, which presented high efficiency in 1O2 generation and phototoxicity around 0.5 µM in Hela cells (40). The CHL-T, however, seems to induce a highly superior cytotoxic response compared to the metallochlorins. Studies with other hydrophilic chlorins have shown efficacy in vivo and in patients too (41, 42). Table 2. Median inhibitory concentration (IC50) for the PSs incubated for 2 and 16 h and irradiated with red -2 -2 light (6 J cm , I= 18 mW cm ). The values are the mean ± S.D. (average of three independent assays).

Incubation time

IC50 (HeLa)

IC50 (Vero)

[µM]

[µM]

2h

16 h

2h

16h

CHL

0.43 ± 0.04

0.54 ± 0.05

0.31 ± 0.05

0.10 ± 0.02

CHL-T

0.04 ± 0.01

0.05 ± 0.01

0.68 ± 0.10

0.06 ± 0.04

HY

0.56 ± 0.03

0.04 ± 0.02

0.88 ± 0.10

0.40 ± 0.08

19

Both CHL and CHL-T practically do not change their IC50 values with the increase of incubation time for HeLa cells. On the other hand, the HY IC50 value decreases with an increase of incubation time from 2 to 16 h. These results suggest that only 2 h of incubation is enough to achieve a good chlorin IC50 value while hypericin needs more time of incubation to accumulate in the cells and reach a low IC50 value. This result concerning HY has already been reported elsewhere by our group (19, 34). Besides, it is clear from table 2 that CHL-T is already very phototoxic just after 2 h of incubation and becomes 25% more phototoxic if the incubation goes from 2 to 16 h, becoming as phototoxic as HY, a wellknown highly photoactive compound. These data suggest the elevated potential of this new chlorin for PDT. Comparing tumoral cell to non-tumoral cells at 2h of incubation time, either CHL-T or HY were more cytotoxic to the tumoral cell. However CHL presented similar IC50 for both cell lines. These results indicated that for short incubation times the modification in CHL structure improved its cytotoxicity, avoiding non-tumoral cell damage. At 16 h of incubation time, CHL-T has similar phototoxicity for tumor and non-tumor cell lines. This result shows that long periods of incubation should be avoided, because non-tumoral cells can be damaged too. For the HeLa cell line, comparing CHL and CHL-T accumulation rates, CHL was expected to be more cytotoxic because of its higher accumulation rate. However, CHL-T showed a ten times greater phototoxicity rate than CHL. These discrepant data between uptake and phototoxicity have also been found in other studies (43). Despite being uncommon, it suggests that additional factors determine the cellular damage. Our results indicate that little amounts of the new PS inside the cell is able to promote a highly cytotoxic effect on tumor cells. To confirm that the cytotoxic effect of the studied PSs is due to irradiation, cytotoxic assay was performed with the same PSs concentrations, but without irradiation. This control showed that the PSs concentrations used were non-toxic without light. On the other hand, only the irradiation up to 6 J cm-2 presented around 100 % of viability, suggesting that the light dose alone is not cytotoxic to these cells.

20 Table 3. Medium inhibitory concentration (IC50) for the PSs incubated for 2 and 16 h without irradiation. The values are the mean ± S.D. (average of three independent assays). IC50 (Vero) [µM] Incubation time

2h

16 h

CHL

1.29 ± 0.40

5.86 ± 1.33

CHL-T

8.68 ± 0.70

2.74 ± 0.40

HY

4.85 ± 0.58

3.28 ± 0.56

In Table 3, it can be noticed that without irradiation the PSs have high IC50 values in nontumoral cells, showing that the PSs have no intrinsic cytotoxicity in the concentration range used.

Detection of cell death by fluorescence microscopy

The quantification of total cell death rate as well as the proportion of apoptosis and necrosis processes is presented in Tables 4 and 5. Controls of cells only with PS (not irradiated), and cells treated only with light (without PS) were also carried out. Such control samples showed viability above 80%.

21 Table 4. Cell death type (apoptosis, necrosis and total) obtained by fluorescence microscopy of HeLa cells stained with ethidium bromide and acridine orange after photodynamic treatment with the three PSs at -2 -2 different concentrations and incubation time of 2 h, red light of 6 J cm and irradiance of 18 mW cm . * Significant values, ANOVA followed by Tukey, C.I: 95%.

PS

[PS] (µM)

CHL

0.52 (IC50)

CHL

HeLa Apoptosis (%)

Necrosis (%)

Total (%)

3.6

10.6

14.2

1.04 (2 x IC50)

1.6

5.6

7.3

CHL

2.08 (4 x IC50)

8.7*

28.4

37.1

CHL

8.49 (IC100)

3.8

95.8*

99.6

CHL-T

0.06 (IC50)

4.4

41.6*

46.0

CHL-T

0.12 (2 x IC50)

3.9

86.8*

90.5

CHL-T

0.24 (4 x IC50)

2.1

97.1*

99.2

CHL-T

0.92 (IC100)

4.4

95.6*

100

HY

0.72 (IC50)

10.2*

26.9*

37.1

HY

1.44 (2 x IC50)

2.9

62.4*

65.4

HY

2.88 (4 x IC50)

2.4

96.7*

99.1

HY

12.70 (IC100)

2.3

97.7*

100

The increasing PS concentration leads to an increased percentage of cell death, as expected. It is noteworthy that the concentrations used for CHL-T were about ten times lower than the concentrations used for the other two PSs corroborating with the previous experiments described here. Therefore, it can be observed that the new proposed PS has great potential as a photosensitizer for PDT. Some studies in the literature have shown that concentrations above 1 µM of HY and a light dose near 6 J cm-2 induce more necrosis than apoptosis in photodynamic assays (44), which is in agreement with our results.

22

A

B

C

E

D

Figure 6. Fluorescence microscopy of HeLa cells with 2h incubation with CHL-T. A: dark = negative control without PS; B: Dark control with 0.06 µM of CHL-T; C: Dark control with 0.92 µM of CHL-T; D: 0.06 µM of CHL-T and red light (6 J cm-2, I= 18 mW cm-2); E: 0.92 µM of CHL-T and red light (6 J cm-2, I= 18 mW cm-2).

The images in figure 6 show that only when the cells were treated with 0.92 µM of CHL-T and light (E) was a higher amount of cell death (necrosis) reached, comparing to the controls. Cell death (apoptosis and necrosis) after 16 h of incubation was also determined with PS concentration equivalent to incubation 2 x IC50.These results are presented in Table 5. Table 5. Cell death type (apoptosis, necrosis and total) obtained by fluorescence microscopy of HeLa cells stained with ethidium bromide and acridine orange after photodynamic treatment with the three PSs at -2 different concentrations and incubation time of 16 h, irradiation with red light of 6 J cm and irradiance of 18 -2 mW cm . * Significant values, ANOVA followed by Tukey, C.I: 95%.

HeLa PS

[PS] (µM)

Apoptosis (%)

Necrosis (%)

Total (%)

CHL

1.04

18.9

57.5*

76.4

CHL-T

0.12

20.4

78.8*

99.3

HY

1.44

40.2*

53.1*

93.2

23

The results presented in Table 5 show that necrosis was prevalent to apoptosis in the presented conditions as observed after 2 h of incubation (Table 4). Figure 7 shows HeLa cell line treatments after 16 h of incubation. It can be seen that with 0.12 µM CHL-T in the absence of light (B) a few dead cells appear, while at the same concentration and light dose of 6 J cm-2 (C), almost all cells undergo necrosis.

A

B

C

Figure 7. Fluorescence microscopy of HeLa cells after incubation for 16 h with CHL-T 0.12 µM with and without light irradiation. A: dark = negative control without PS; B: Dark control with 0.12 µM of CHL-T; C: 0.12 µM of CHL-T and irradiation with red light (6 J cm-2, I= 18 mW cm-2).

Quantification of cell death is crucial for determining the potential of a PS in inducing cell death after irradiation. Activation of necrosis may be desirable when resistance to apoptosis occurs (45). Some studies show apoptosis resistance in HeLa cells treated with PDT (4648). Therefore, the higher percentage of necrosis observed in HeLa cells treated with the new chlorin represents a good response to eradicate tumor cells.

Conclusions

In this study, three PSs were evaluated: CHL, HY and CHL-T. The modified chlorin CHL-T showed an enhanced rate for singlet oxygen generation (about three times more efficient than the other photosensitizers), presenting high potential as a photosensitizer. Comparing the PS accumulation in cells, it was observed that both chlorins accumulate more than hypericin. Despite the results showing higher lipophilicity and accumulation rates for CHL, the new PS proved to be more efficient, where better cytotoxicity effects could be achieved using lower concentrations. The intracellular accumulation of PS did not necessarily result

24

in greater efficacy in inducing cell death, since other aspects, such as cell type, may influence the cytotoxicity of these compounds. The phototoxicity presented by CHL-T was about ten times higher than that of CHL, given that the IC50 values obtained were ten times smaller. Using HeLa and Vero cells, It can be concluded that lower incubation times are preferred in order to have a cell selectivity. Fluorescence microscopy showed that the percentage of cell death increases with increasing concentrations of PS. Necrosis was the predominant type of cell death caused by CHL-T after PDT treatments in the HeLa cell line. Resistance to apoptosis for this cell line is known; thus, the result of inducing necrosis was favorable. Therefore, this study shows that CHL-T can be employed as an amphiphilic photosensitizer, resulting in higher bioavailability and cytotoxicity to the tumor cells.

Acknowledgements

The authors thank Prof. Ilce Mara de Syllos Cólus for kindly proving the HeLa cell line and to CEPOF for the Biotable. This study was sponsored by Brazilian funding agencies (FAPESP 2013/06532-4, 2013/07276-1, 2015/21110-4, CNPq and CAPES). Thanks are also due to Prof. Dr. Timothy J. Brocksom by the English editing and proofreading.

References 1.

Robertson C, Evans D, Abraharnse H. Photodynamic therapy (PDT): A short review

on cellular mechanisms and cancer research applications for PDT. Journal of Photochemistry and Photobiology B-Biology. 2009;96(1):1-8. 2.

Sibata C, Colussi V, Oleinick N, Kinsella T. Photodynamic therapy: a new concept in

medical treatment. Brazilian Journal of Medical and Biological Research. 2000;33(8):86980. 3.

Castano A, Demidova T, Hamblin M. Mechanisms in photodynamic therapy: part

one-photosensitizers, photochemistry and cellular localization. Photodiagnosis and Photodynamic Therapy. 2004;1(4):279-93.

25

4.

Castano A, Demidova T, Hamblin M. Mechanisms in photodynamic therapy: part

two-cellular signaling, cell metabolism and modes of cell death. Photodiagnosis and Photodynamic Therapy. 2005;2(1):1-23. 5.

de Assis F, de Souza J, Assis B, Brocksom T, de Oliveira K. Synthesis and

photophysical studies of a chlorin sterically designed to prevent self-aggregation. Dyes and Pigments. 2013;98(1):153-9. 6.

dos Santos F, Uchoa A, Baptista M, Iamamoto Y, Serra O, Brocksom T, et al.

Synthesis of functionalized chlorins sterically-prevented from self-aggregation. Dyes and Pigments. 2013;99(2):402-11. 7.

Nyman

E,

Hynninen

P.

Research advances in

the

use of

tetrapyrrolic

photosensitizers for photodynamic therapy. Journal of Photochemistry and Photobiology BBiology. 2004;73(1-2):1-28. 8.

Dougherty T. An update on photodynamic therapy applications. Journal of Clinical

Laser Medicine & Surgery. 2002;20(1):3-7. 9.

Yano S, Hirohara S, Obata M, Hagiya Y, Ogura S, Ikeda A, et al. Current states and

future views in photodynamic therapy. Journal of Photochemistry and Photobiology CPhotochemistry Reviews. 2011;12(1):46-67. 10.

Karioti A, Bilia A. Hypericins as Potential Leads for New Therapeutics. International

Journal of Molecular Sciences. 2010;11(2):562-94. 11.

O'Connor A, Gallagher W, Byrne A. Porphyrin and Nonporphyrin Photosensitizers in

Oncology: Preclinical and Clinical Advances in Photodynamic Therapy. Photochemistry and Photobiology. 2009;85(5):1053-74. 12.

Konan Y, Gurny R, Allemann E. State of the art in the delivery of photosensitizers for

photodynamic

therapy.

Journal

of

Photochemistry

and

Photobiology

B-Biology.

2002;66(2):89-106. 13.

Sun Y, Chen Z, Yang X, Huang P, Zhou X, Du X. Magnetic chitosan nanoparticles as

a drug delivery system for targeting photodynamic therapy. Nanotechnology. 2009;20(13). 14.

Garcia-Diaz M, Kawakubo M, Mroz P, Sagrista M, Mora M, Nonell S, et al. Cellular

and vascular effects of the photodynamic agent temocene are modulated by the delivery vehicle. Journal of Controlled Release. 2012;162(2):355-63. 15.

Cohen E, Ding H, Kessinger C, Khemtong C, Gao J, Sumer B. Polymeric micelle

nanoparticles for photodynamic treatment of head and neck cancer cells. OtolaryngologyHead and Neck Surgery. 2010;143(1):109-15.

26

16.

Schneider-Yin X, Kurmanaviciene A, Roth M, Roos M, Fedier A, Minder E, et al.

Hypericin

and

5-aminolevulinic

acid-induced

protoporphyrin

IX

induce

enhanced

phototoxicity in human endometrial cancer cells with non-coherent white light. Photodiagnosis and Photodynamic Therapy. 2009;6(1):12-8. 17.

Verma S, Watt G, Mal Z, Hasan T. Strategies for enhanced photodynamic therapy

effects. Photochemistry and Photobiology. 2007;83(5):996-1005. 18.

Bernal C, Rodrigues J, Guimaraes A, Ribeiro A, de Oliveira K, Imasato H, et al.

Selective photoinactivation of C. albicans and C. dubliniensis with hypericin. Laser Physics. 2011;21(1):245-9. 19.

Lima A, Dal Pizzol C, Monteiro F, Creczynski-Pasa T, Andrade G, Ribeiro A, et al.

Hypericin encapsulated in solid lipid nanoparticles: Phototoxicity and photodynamic efficiency. Journal of Photochemistry and Photobiology B-Biology. 2013;125:146-54. 20.

Uliana M, Pires L, Pratavieira S, Brocksom T, de Oliveira K, Bagnato V, et al.

Photobiological characteristics of chlorophyll a derivatives as microbial PDT agents. Photochemical & Photobiological Sciences. 2014;13(8):1137-45. 21.

Falk H, Meyer J, Oberreiter M. A convenient semisynthetic route to hypericin.

Monatshefte Fur Chemie. 1993;124(3):339-41. 22.

Huygens A, Kamuhabwa A, de Witte P. Stability of different formulations and ion

pairs

of

hypericin.

European

Journal

of

Pharmaceutics

and

Biopharmaceutics.

2005;59(3):461-8. 23.

Lima AM, Pizzol CD, Monteiro FB, Creczynski-Pasa TB, Andrade GP, Ribeiro AO, et

al. Hypericin encapsulated in solid lipid nanoparticles: phototoxicity and photodynamic efficiency. J Photochem Photobiol B. 2013;125:146-54. 24.

Da Violante G, Zerrouk N, Richard I, Provot G, Chaumeil J, Arnaud P. Evaluation of

the cytotoxicity effect of dimethyl sulfoxide (DMSO) on Caco(2)/TC7 colon tumor cell cultures. Biological & Pharmaceutical Bulletin. 2002;25(12):1600-3. 25.

Fery-Forgues S, Lavabre D. Are fluorescence quantum yields so tricky to measure?

A demonstration using familiar stationery products. Journal of Chemical Education. 1999;76(9):1260-4. 26.

Murov SL, Carmichael I, Hug GL. Handbook of photochemistry. 2 ed. New York:

Marcel Dekker; 1993. 420 p. 27.

Fischer F, Graschew G, Sinn HJ, Maier-Borst W, Lorenz WJ, Schlag PM. A chemical

dosimeter for the determination of the photodynamic activity of photosensitizers. Clinica chimica acta; international journal of clinical chemistry. 1998;274(1):89-104.

27

28.

Pooler JP, Valenzeno DP. Physicochemical determinants of the sensitizing

effectiveness for photooxidation of nerve membranes by fluorescein derivatives. Photochemistry and photobiology. 1979;30(4):491-8. 29.

Lowry O, Rosebrough N, Farr A, Randall R. Protein measurement with the folin

phenol reagent. Journal of Biological Chemistry. 1951;193(1):265-75. 30.

Denizot F, Lang R. Rapid colorimetric assay for cell-growth and survival -

modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. Journal of Immunological Methods. 1986;89(2):271-7. 31.

Carmichael J, Degraff W, Gazdar A, Minna J, Mitchell J. Evaluation of a tetrazolium-

based semiautomated colorimetric assay - assessment of chemosensitivity testing. Cancer Research. 1987;47(4):936-42. 32.

Ribble D, Goldstein N, Norris D, Shellman Y. A simple technique for quantifying

apoptosis in 96-well plates. Bmc Biotechnology. 2005;5. 33.

Altmann R, Falk H. The deprotonation and protonation equilibria of a hypericin

derivative in aqueous solution. Monatsh Chem. 1997;128(6-7):571-83. 34.

Bernal C, Ribeiro AO, Perrusi JR. Photodynamic efficiency of hypericin compared

with a chlorine and a porphyrin derivative in HEp-2 and Vero epithelial cell lines. 35.

Murov SLC, I.; Hug, G. L. Handbook of photochemistry. 2 ed. New York: Marcel

Dekker; 1993. 420 p. 36.

Matsuura T, Saito I. Photoinduced Reactions .21. Photosensitized Oxygenation of N-

Unsubstituted Hydroxypurines. Tetrahedron. 1968;24(22):6609-&. 37.

Spiller W, Kliesch H, Wohrle D, Hackbarth S, Roder B, Schnurpfeil G. Singlet oxygen

quantum yields of different photosensitizers in polar solvents and micellar solutions. J Porphyr Phthalocya. 1998;2(2):145-58. 38.

Kraljic I. Detection of singlet oxygen and its role in dye-sensitized photooxidation in

aqueous and micellar solutions. Biochimie. 1986;68(6):807-11. 39.

Smith DW, H.; Walker, D.; Mannhold, R.; Kubinyi, H. e Timmerman, H.

Pharmacokinetics and Metabolism in Drug Design. 3 ed: Wiley Verlag; 2012. 268 p. 40.

Obata M, Hirohara S, Tanaka R, Kinoshita I, Ohkubo K, Fukuzumi S, et al. In Vitro

Heavy-Atom Effect of Palladium(II) and Platinum(II) Complexes of Pyrrolidine-Fused Chlorin in Photodynamic Therapy. Journal of Medicinal Chemistry. 2009;52(9):2747-53. 41.

Mori H, Sakata I, Hirano T, Obana A, Nakajima S, Hikida M, et al. Photodynamic

therapy for experimental tumors using ATX-S10(Na), a hydrophilic chlorin photosensitizer, and diode laser. Japanese Journal of Cancer Research. 2000;91(7):753-9.

28

42.

Kato H, Furukawa K, Sato M, Okunaka T, Kusunoki Y, Kawahara M, et al. Phase II

clinical study of photodynamic therapy using mono-L-aspartyl chlorin e6 and diode laser for early superficial squamous cell carcinoma of the lung. Lung Cancer. 2003;42(1):103-11. 43.

Kiesslich T, Neureiter D, Alinger B, Jansky G, Berlanda J, Mkrtchyan V, et al. Uptake

and phototoxicity of meso-tetrahydroxyphenyl chlorine are highly variable in human biliary tract cancer cell lines and correlate with markers of differentiation and proliferation. Photochemical & Photobiological Sciences. 2010;9(5):734-43. 44.

Kubin A, Wierrani F, Burner U, Alth G, Grunberger W. Hypericin - The facts about a

controversial agent. Current Pharmaceutical Design. 2005;11(2):233-53. 45.

Kepp O, Galluzzi L, Lipinski M, Yuan J, Kroemer G. Cell death assays for drug

discovery. Nature Reviews Drug Discovery. 2011;10(3):221-37. 46.

Assefa Z, Vantieghem A, Declercq W, Vandenabeele P, Vandenheede J, Merlevede

W, et al. The activation of the c-Jun N-terminal kinase and p38 mitogen-activated protein kinase signaling pathways protects HeLa cells from apoptosis following photodynamic therapy with hypericin. Journal of Biological Chemistry. 1999;274(13):8788-96. 47.

Kocanova S, Buytaert E, Matroule J, Piette J, Golab J, de Witte P, et al. Induction of

heme-oxygenase 1 requires the p38(MAPK) and PI3K pathways and suppresses apoptotic cell death following hypericin-mediated photodynamic therapy. Apoptosis. 2007;12(4):73141. 48.

Hendrickx N, Volanti C, Moens U, Seternes O, de Witte P, Vandenheede J, et al. Up-

regulation of cyclooxygenase-2 and apoptosis resistance by p38 MAPK in hypericinmediated photodynamic therapy of human cancer cells. Journal of Biological Chemistry. 2003;278(52):52231-9.