Chitosan System for Photodynamic

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Characterization of a Squaraine/Chitosan System for Photodynamic Therapy of Cancer Diana P. Ferreira, David S. Conceição, Fabio Fernandes, Tânia Patrícia Marques de Sousa, Ricardo C. Calhelha, Isabel C.F.R. Ferreira, Paulo Fernando Da Conceição Santos, and Luis Filipe Vieira Ferreira J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b11604 • Publication Date (Web): 01 Feb 2016 Downloaded from http://pubs.acs.org on February 5, 2016

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The Journal of Physical Chemistry

Characterization of a Squaraine/Chitosan System for Photodynamic Therapy of Cancer Diana P. Ferreiraa, David S. Conceiçãoa, F. Fernandesa, T. Sousaa, Ricardo C. Calhelhab, Isabel C.F.R. Ferreirab, Paulo F. Santosc, L.F.Vieira Ferreiraa,*

a

Centro de Química-Física Molecular and IN-Institute of Nanoscience and Nanotechnology,

Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa.

b

Mountain Research Centre (CIMO), ESA, Polytechnic Institute of Bragança, Campus de

Santa Apolónia, 1172, 5301-855 Bragança, Portugal

c

Centro de Química - Vila Real, Universidade de Trás-os-Montes e Alto Douro, 5001-801

Vila Real, Portugal.

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Abstract In this work, a squaraine dye CS5 was characterized and evaluated for its potential in photodynamic therapy. The studies were performed in ethanol and also in a powdered biopolymer, in this case chitosan. Ground state absorption, absolute fluorescence quantum yields, fluorescence lifetimes and transient absorption were determined in order to evaluate the advantage of adsorbing the dye onto a biopolymer. Several concentrations of the dye, adsorbed onto chitosan, were prepared in order to evaluate the concentration effect on the photophysical parameters under study. A remarkable increase in the fluorescence quantum yield and lifetimes was detected when compared with the dye in solution. Also, a very clear dependence of the fluorescence quantum yield on the concentration range was found. A lifetime distribution analysis of these systems fluorescence evidenced the entrapment of the dye onto the chitosan environment with a monoexponential decay which corresponds to the monomer emission in slightly different environments. The transient absorption spectrum was obtained without sensitization indicating the existence of a triplet state which takes special importance in the generation of phototoxic species namely singlet oxygen. The subcellular localization of a photosensitizer is critical for efficient photoinduced cell death, in this way, co-localization studies were performed within HeLa cell line (human cervical carcinoma) through confocal microscopy. Toxicity in the dark and phototoxicity of CS5 were also evaluated for the same cellular model.


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1.

Introduction Photodynamic therapy of cancer (PDT) requires the selective incorporation of a drug,

i.e., a photosensitizer (PS), into the targeted cells/tissues, followed by the subsequent exposure of the target region to light of appropriate wavelengths, visible/near infrared light, which, in the presence of molecular oxygen, leads to the generation of cytotoxic species and consequently to cell death and tissue destruction.1 Photosensitizers for PDT should ideally meet several criteria: (1) high purity, (2) chemical stability, (3) water solubility, (4) high quantum yield of 1O2* generation (Φ∆), (5) absence cytotoxicity in the dark, (6) tumour selectivity, (7) rapid accumulation in cancer cells, (8) rapid clearance from patients and (9) a high molar absorption coefficient (ε) in the long wavelength region (600–800 nm), were light can penetrate deeper into the tissues. 2,3 A family of compounds which could be employed as PSs are squaraine dyes also called squaraines.4,5 Squaraines can be regarded as derivatives of cyanine dyes, in which a squaraine ring is introduced in the polymethine chain.6 The squaraine ring shifts the absorption and emission maxima to longer wavelengths, relative to the parent cyanine dye, and it is expected to increase the photostability of the dyes, through reduction of the photoisomerization very common in cyanine dyes.7 The PDT efficiency of these emerging non-porphyrin PSs still remains to be fully studied. These compounds present several advantages concerning water solubility, light-absorption ability at longer wavelengths region and possible higher photostability over other photosensitizers. In spite of all the advantages of these photosensitizers, squaraines present some issues regarding their application in PDT. The major drawbacks are: compound lost until targeting the localized tumour; absence of biocompatibility; low selectivity and solubility of the drug; and also difficulty in reaching certain depths in tissues.8 In spite of these difficulties, the



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possible occurrence of photoisomerization is another problem inherent to squaraine dyes since this non-radiative process - which is a pathway for the excited state deactivation competes with the triplet state formation essential for singlet oxygen generation.9 The adsorption of these dyes onto surfaces emerges as a solution to overtake the majority of the described disadvantages. The incorporation of compounds onto suitable surfaces may promote the molecules rigidification by diminishing the photoisomerization process and also other deactivation mechanisms; it may also increase the dye photostability and solubility, it can prevent molecular aggregation, it can protect the photosensitizer from external agents (oxidative attacks in the blood current) and can increase the bioaffinity of the dyes while prolonging circulation time in the blood current.10,11 Polymer-based drug delivery systems have been investigated over the last few decades as a mean to achieve high therapeutic concentrations of the drug at the site of malignant disease in cancer patients.12,13,14 The best example of a biopolymer that can be used for this purpose is Chitosan.15,16,17 This polymer has excellent properties such as hydrophilicity, biocompatibility, biodegradability, antibacterial and adsorption applications. It also presents very low toxicity.18,19 When adsorbed onto materials, sensitizers can be implanted locally increasing the tumour cell selectivity and increase the amount of sensitizer presented to the target cells, as well as minimizing side effects. In recent years, much progress has been made in the use of conjugates between cytotoxic drugs and polymeric carriers. In this work we report for the first time the photochemical characterization of a squaraine dye adsorbed onto powdered chitosan. The characterization of the behavior of the adsorbed photosensitizer is of great importance to estimate the potential of the dye/chitosan system as a drug delivery system for localized therapy.


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We choose for this study the squaraine: 3-hexyl-2-[3-(3-hexyl-3H-benzoselenazol-2ylidenemethyl)-2-(2-hydroxyethylamino)-4-oxocyclobut-2-enylidenemethyl]benzoselenazol3-ium iodide (CS5), which had been previously studied in solution. 20 Possible implementation of this dye as an imaging agent was also evaluated and the absolute fluorescence quantum yields and lifetimes of the CS5/biopolymer system were determined. The transient absorption spectrum was also obtained in order to evaluate the ability of this system in triplet state generation. The triplet-triplet absorption band was obtained as in solution but without sensitization allowing the possible formation of phototoxic species responsible for killing cells. In order to evaluate the possible use of this dye as PS for cancer photodynamic therapy, we studied the dark toxicity and also the phototoxicity of this dye in human cervical cancer cells (HeLa cells). As several reports emphasize, 21 the subcellular localization of the PS takes special importance in order to evaluate the efficiency of the therapy. In this way, we performed through confocal microscopy, an extensive comparison of colocalization of CS5 with subcellular markers in HeLa cells.

2. Materials and Methods

2.1. General Chitosan low molecular weight and solvents of analytical grade were purchased from Sigma-Aldrich Co. and used as received. The 3-hexyl-2-[3-(3-hexyl-3H-benzoselenazol-2ylidenemethyl)-2-(2-hydroxyethylamino)-4-oxocyclobut-2-enylidenemethyl]benzoselenazol3-ium iodide (CS5) was synthesized according to reference. 20



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2.2. Sample preparation Solutions of the dye were prepared in ethanol for all the studies. For the confocal microscopy experiments, these solutions were diluted in culture medium without phenol red. Squaraine dye adsorption onto chitosan was achieved by the solvent evaporation method, which consists in the gradually addition of an ethanolic solution of the dye (the solvent was previously dried with molecular sieves) to the previously very well dried powdered solid substrate. This mixture was maintained under stirring at room temperature for about 12h for slow solvent evaporation. Final solvent removal was performed for about 4h in an acrylic chamber with an electrically heated shelf (Heto, Model FD 1.0-110) with temperature control (30º) and under moderate vacuum (ca. 10-3 Torr). In this work we prepared several concentrations of squaraine onto chitosan: 0.025µmol/g, 0.05µmol/g, 0.1µmol/g, 0.25µmol/g, 0.5 µmol/g and 1 µmol/g.

2.3. UV-Visible absorption spectra and ground state diffuse reflectance absorption spectra (GSDR) Steady-state absorption spectra were recorded with the use of a Camspec M501 single beam scanning UV/Visible spectrophotometer at room temperature in the spectral range from 190 to 1100nm. The optical densities were measured using a UV quartz cuvette (1cm path length). Ground-state absorption studies were performed using a homemade diffuse reflectance laser flash photolysis setup, with a powerful 250W W-Hal lamp as monitoring lamps. A fixed monochromator coupled to an ICCD with time gate capabilities was used for detecting the reflectance signals. The reflectance, R, from each sample was obtained in the UV-Vis-NIR spectral regions and the remission function, F(R), was calculated using the Kubelka-Munk


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equation for optically thick samples. The remission function is F(R) = (1 – R)2 / 2R. Details regarding the data treatment can be found in reference 22 and references quoted therein.

2.4. Laser-induced luminescence and absolute fluorescence emission quantum yields The set-up for time resolved luminescence LIL is presented in reference 23. Time-resolved emission spectra were performed in the nanosecond to second time range with a N2 laser (excitation wavelength = 337 nm, PTI model 2000, ca. 600 ps FWHM, about 1 mJ per pulse) with a dye laser coupled to the N2 (excitation wavelength=640 nm, using the rhodamine 101 as laser dye). An integrating sphere for relative and absolute measurements was used in this work, as a way to obtain the values for the fluorescence emission quantum yields (ΦF) of the powdered samples of the squaraine under study, as EPA recommends.

24

Using this method,

the ΦF determination of a standard fluorophore, HITC dye (1,3,3,1',3',3',-hexamethyl-2,2'indotricarbocyanine iodide), with a known quantum yield was performed for validation purposes (in ethanolic solution ΦF = 0.28 ± 0.05 in argon saturated samples or airequilibrated conditions). The agreement found between the ΦF obtained here by the absolute method and the reported literature value

25

validate the photoluminescence quantum yields

determined here for the powdered samples of the squaraine dyes adsorbed onto powdered chitosan. The absolute fluorescence quantum yields were obtained by the use of the following equations 24: ΦF = (Pc − (1 − A) × Pb)/A × La with A = (1 − Lc/Lb) where A is the absorption coefficient, Pb is the light emitted by the sample after absorption of scattered excitation light, Pc is the light emitted by the sample after absorption of total laser



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light, La is the total amount of excitation laser light, Lb is the scattered laser light, and Lc is excitation light spectrum. In many cases Pb is negligible and the equation simply becomes 26: ΦF = Pc/(La − Lc)

2.5. Fluorescence lifetimes determination Fluorescence lifetimes were determined using Easylife VTM equipment from OBB (Lifetime range from 90 ps to 3 µs). This technique uses pulsed light sources from different LEDs (630 nm in this case) and measures fluorescence intensity at different time delays after the excitation pulse. In this case a 665 nm cut-off filter was used. The instrument response function was measured using a Ludox scattering solution. FelixGX software from OBB was used for fitting and analysis of the decay dynamics, 1 to 4 exponentials and also the exponential series method - ESM (lifetime distribution analysis).

2.6. Diffuse reflectance laser flash photolysis Diffuse-reflectance laser flash photolysis (DRLFP) experiments were carried out with the fourth harmonic of a Nd:YAG laser (266 nm, ca. 6 ns FWHM, 10–30 mJ/pulse) from B. M. Industries (Thomson-CSF, model Saga 12–10), in the diffuse reflectance mode. The light arising from the irradiation of solid samples by the laser pulse is collected by a collimating beam probe coupled to an optical fiber (fused silica) and is detected by a gated intensified charge coupled device (Andor ICCD detector). The ICCD is coupled to a fixed imaging compact spectrograph (Oriel, model FICS 77440). The system can be used either by capturing all light emitted by the sample or in a time-resolved mode by using a delay box (Stanford Research Systems, model D6535). The ICCD has high speed gating electronics (2.2 ns) and intensifier and covers the 200–900 nm wavelength range.


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Transient absorption data are reported as percentage of absorption (% Abs.), defined as 100∆Jt/Jo = (1-Jt/ Jo)100, where Jo and Jt are diffuse reflected light from sample before exposure to the exciting laser pulse and at time t after excitation, respectively.

2.7. Confocal Microscopy 2.7.1 Membrane probes 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene-p-toluenesulfonate (TMA-DPH) and [6-amino-9-(2-methoxycarbonylphenyl)xanthen-3-ylidene]azanium (Rho123) were purchased from Invitrogen (Carlsbad, CA, USA). The probes were quantified spectrophotometrically with absorbance and fluorescence data and absorption coefficients obtained from literature (TMA-DPH: εmax= 52000M-1cm-1, λ abs/excmax 365 nm, λemmax 427nm. Rho-123: εmax= 101000M-1cm-1, λ abs/excmax 505 nm, λemmax 525 nm). Stock solutions were kept in organic solvents at -26˚C.

2.7.2 Cell lines The HeLa cell line was cultured at the incubator with controlled temperature (37 °C) with 5% CO2. Culture was carried out in DMEM (Dulbecco's modified eagle medium) containing 10% heat inactivated FBS (fetal bovine serum), 200 mM L-glutamine, 1% penicillin and streptomycin. For imaging purposes, HeLa cells were grown in eight-well microscopic chambers (ibidi GmbH, Martinsried, Germany) previously coated with poly-L-lysine (Sigma-Aldrich) for improved adhesion.



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2.7.3 Confocal fluorescence microscopy All measurements were performed on a Leica TCS SP5 (Leica Microsystems CMS GmbH, Manheim, Germany) inverted confocal microscope (DMI600). Excitation lines were provided by Argon and HeNe lasers focused into the sample by an apochromatic water immersion objective (63x, NA 1.2; Zeiss, Jena Germany). A 111.4 µm diameter pinhole positioned in front of the image plane blocked out-of-focus signals. Two photon excitation microscopy of TMA-DPH was carried out using the same set-up coupled to a Ti:sapphire laser (Mai Tai, Spectra-Physics, Darmstadt, Germany) as the excitation source. The excitation wavelength was set to 780 nm and the fluorescence emission of TMA-DPH was collected at 450 ± 50 nm. Imaging of Rhodamine 123 fluorescence was performed with 476nm excitation and detection at 530 ± 25 nm. Fluorescence imaging of squaraine dye was performed with 633nm excitation and detection at 725 ± 75 nm.

2.8. Evaluation of cytotoxicity in HeLa cell line 2.8.1. Preparation of the compound solutions Stock solutions were prepared by dissolving the compound in DMEM with 20% DMSO and 0.1% acetic acid, and kept at −20°C. Prior to the assays, appropriate dilutions were prepared using the same solvent. The absence of DMSO toxicity was confirmed by treating cells with the maximum concentration of DMSO (20%) used in the assays. The compounds cytotoxicity was tested in the dark and under irradiation. For irradiation of the cells a Halogen/Tungsten lamp (24V and 250W; Osram, Carnaxide, Portugal) was used with a fluency of 23-24 µW/cm2 (measured with ILT-1400 radiometers with a SEL033 detector). A K2Cr2O7 solution (3%) was used between the irradiation source and the cells in order to select the radiation reaching the plates.


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2.8.2. Cytotoxic assay The human tumor cell line HeLa (cervical carcinoma) was purchased by DSMZ (Leibniz-Institut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH). Cells were routinely maintained as adherent cell cultures in RPMI-1640 medium containing 10% heat-inactivated FBS and 2 mM glutamine at 37ºC, in a humidified air incubator containing 5% CO2. It was plated at an appropriate density (1.0×104 cells/well) in 96-well plates and allowed to attach for 24h. Cells were then treated for 24h with various concentrations of the compounds. Following this incubation period, the cells tested under irradiation were irradiated for 30 minutes. The medium was changed and incubated again for 24 hours. After this time, the adherent cells were fixed by adding cold 10% trichloroacetic acid (TCA; 100 µL) and incubated for 60 min at 4ºC. Plates were then washed with deionized water and subsequently dried; Sulforhodamine B (SRB) solution (0.1% in 1% acetic acid, 100 µL) was then added to each plate well and incubated for 30 min at room temperature. Unbound SRB was removed by washing with 1% acetic acid. Plates were air dried, the bound SRB was solubilized with 10 mM Tris (200 µL) and the absorbance was measured at 540 nm in ELX800 Microplate Reader (Bio-Tek Instruments, Inc; Winooski, USA).27 The results were expressed in GI50 values (compound concentration that inhibited 50% of the net cell growth).

3. Results and discussion 3.1 Dye selection As referred in previous studies20,

28

the development of improved synthetic routes

allows structural modifications which can be reflected in dye’s properties. It was shown that including Selenium substituents and/or Iodide as counterion lead to an increase of singlet oxygen quantum yield of about 20% due to the heavy atom effect. The functionalization of



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the central four-membered ring strongly influenced the photophysical characteristics depending on the nature of the substituents. The rigidification introduced by the squaric ring reduced very efficiently the isomerization pathways of the dyes and finally, the increase of the alkyl chain length from ethyl to hexyl has very little influence on the photoisomerization process (so common in these compounds) because the fluorescence quantum yields remain reasonably constant. Taking into account all these findings from recent published work from our group20, 28 we decided to choose for this study the squaraine compound (CS5) presented in Figure 1.

Figure 1. Structure of the squaraine dye CS5.

This dye presents the most balanced properties between all the squaraines studied namely, high singlet oxygen quantum yield (0.36 in chloroform) and also a reasonable fluorescence quantum yield suitable for being used as photosensitizer and also as fluorescent marker (theranostic agent). The adsorption of this dye onto chitosan takes special importance in order to improve the photophysical parameters and photostability of this compound, reducing the possibility of photoproduct generation and also of the non-radiative pathways of deactivation. Such modifications are expected to facilitate the use of this dye/biopolymer system as a drug delivery system or as a localized therapeutic agent. 3.2 Photochemical Characterization 3.2.1. Ground state UV-visible and fluorescence in solution


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In ethanol (Figure 2), CS5 shows a strong absorption in the so called phototherapeutic window between ~600-700nm with a maximum absorption wavelength at 676nm. The fluorescence emission peaks at 699nm with a quite reasonable quantum yield of 0.14 and the fluorescence lifetime as about 0.8ns (χ2=1.0). These results of the dye in ethanolic solution will be used for comparison of the data obtained in solution and in the solid state - dye adsorbed onto powdered chitosan. Absorption Emission

1 Normalized Intensity (a.u.)

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0.8 0.6 0.4 0.2 0 500

600

700 800 Wavelength (nm)

900

Figure 2. Absorption and fluorescence emission normalized spectra of the squaraine dye CS5 in ethanol. The fluorescence emission spectrum was obtained with an excitation wavelength of 640nm.

3.2.2. Ground state diffuse reflectance of solid samples Figure 3 presents the Kubelka-Munk remission function, defined by the equations presented in reference

29

, for the squaraine adsorbed onto powdered chitosan. In the

wavelength range presented, the powdered chitosan, without dye adsorption, does not evidence any absorption band as the first curve shows. Samples with different concentrations between 0.025 and 1 µmol/g were prepared in order to understand the dependence of the photophysical parameters with concentration. In all the samples the absorption band peaks at ~679nm resembling the absorption obtained in ethanolic solution (Figure 2), apart the bathochromic shift of about 4-5 nm, quite common in solid powdered samples, as previously observed for several cyanine dyes 29. As the loading of the dye increases we do not noticed 13 ACS Paragon Plus Environment


the appearance of any new band. However, the prominence of the shoulder at about 630nm, the decrease of absorption intensity and the enlargement of the principal band at higher concentrations (1µmol/g) is strongly suggestive of dye aggregation within chitosan at higher loadings of dye as the inset in Figure 3 clearly shows. The normalized ground state diffuse reflectance spectra of sample 2, 4 and 6 shows the increase of the shoulder with the increase of the sample concentration. This new absorption at about 630nm is assigned to the formation of H aggregates (sandwich aggregates) absorbing hypsochromically and shifted from the monomer absorption. 0.05 Remission Function F(R)

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5 0.04 0.03

6

0.02

4 3 2

0.01

1 0 550

600

650 700 Wavelength (nm)

750

800

Figure 3. GSDR spectra of the dye adsorbed onto powdered chitosan with different concentrations. Sample 1: 0.025µmol/g, Sample 2: 0.05µmol/g, Sample 3: 0.1µmol/g, Sample 4: 0.25µmol/g, Sample 5: 0.5µmol/g and Sample 6: 1µmol/g. At the top-right corner we can see the normalized ground state diffuse reflectance spectra of sample 2, 4 and 6.

3.2.3. Fluorescence emission spectra, absolute fluorescence quantum yields and fluorescence lifetimes Figure 4 exhibits the corrected emission spectra of the CS5 adsorbed onto chitosan at different dye loadings and excited at 640nm. Taking into account the information in solution (Figure 1), the emission wavelength in the solid state was bathochromically shifted about


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10nm peaking between 707-712nm. In the low concentration range (0.025, 0.05, 0.1µmol/g) the fluorescence intensity increase with concentration is very obvious, however, following that, a strong concentration quenching effect was detected especially in the last sample, with a concentration of 1µmol/g. This quenching effect is due to the formation of aggregates, most probably dimers which are not emissive when compared with the monomer present at lower concentrations. The inset of figure 4 shows the total area under the corrected fluorescence emission spectrum (IF) as a function of the light absorbed by the dye at the excitation wavelength (1-R)fdye. It can be seen that below 0.25µmol/g a linear relation is observed and for higher loadings a strong decrease in IF was clearly shown, in accordance with non-

2000000

3

1600000 1200000

4 2

800000

5 1

400000

6

Fluorescence Intensity (a.u.)

emissive aggregates.


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0

3

4

5

2 6

1

0.05

0.1

0.15

0.2

(1-R) fdye

0 550

650

750 Wavelength (nm)

850

Figure 4. Luminescence corrected spectra of the dye adsorbed onto powdered chitosan using an excitation wavelength of 640nm. Sample 1: 0.025µmol/g, 2: 0.05µmol/g, 3: 0.1µmol/g, 4: 0.25µmol/g, 5: 0.5µmol/g and 6: 1µmol/g. The inset represents the fluorescence intensity of the squaraine onto chitosan, measured as the total area under the corrected emission spectrum, as a function of (1-R)fdye.

Taking into account the integrated emission area of the monomer and assuming that the dimer does not emit fluorescence with a detectable quantum yield, the absolute 15 ACS Paragon Plus Environment


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fluorescence quantum yields of the powdered samples were obtained utilizing an integrating sphere. The increase of the fluorescence quantum yield from solution (0.14 in ethanol) to powder is very evident and is about four times higher in the first three samples. Again, we can notice the quenching effect after the third sample, diminishing drastically the fluorescence quantum yield. Table 1.Concentration of each sample of squarylium dye adsorbed onto powdered chitosan with the respective fluorescence lifetime and absolute fluorescence quantum yield.

Sample

Concentration

τ (ns)

χ2

ΦF

(µmol/g) Sample 1

0.025

3.75

0.82

0.56

Sample 2

0.05

3.48

0.92

0.49

Sample 3

0.1

3.57

1.15

0.47

Sample 4

0.25

3.53

0.85

0.41

Sample 5

0.5

3.78

0.74

0.28

Sample 6

1

3.99

0.82

0.17

A lifetime determination was also performed for the dye adsorbed onto chitosan with our set-up, and Figure 5a presents the fluorescence lifetime decay of sample 0.1µmol/g. In this case a lifetime determination analysis was performed and only one emissive species was detected with a lifetime between 3.75 and 3.99ns. Regarding the value in solution (0.8ns) the fluorescence lifetime increase is in the order of the quantum yield increase. In Figure 5b it is shown the lifetime distribution analysis for the same sample, revealing the existence of a monoexponential fluorescence decay. Fluorescence lifetimes for the different CS5 concentrations are shown in Table 1.


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a)

Sample 3 Ludox Fitting

400

40 57

64

71 Time (ns)

78

85

b)

0.05 0.04 Amplitude (a.u.)

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0.03 0.02 0.01 0 0

2

4 6 Lifetime (ns)

8

10

Figure 5. a) Fluorescence decay of CS5 adsorbed onto powdered chitosan (0.1µmol/g). Ludox solution was used as scatter (blue) and the mono exponential fitting is marked in red using 1 to 4 exponential method. b) Exponential series method (ESM) analysis of the lifetime distribution of the dye adsorbed onto chitosan (0.1µmol/g).

The monoexponential fluorescence decays of CS5 adsorbed to chitosan suggest that the dye is emitting in a very ordered environment where the squaraine is well entrapped into the chitosan polymeric chains. Also, it proves that the possible aggregates formed are not 17 ACS Paragon Plus Environment


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emissive corresponding to a static quenching mechanism. In solid samples, the dye exhibits larger fluorescence lifetimes and quantum yields due to the high constrain imposed by the entrapment, resulting in the decrease of the non-radiative deactivation processes, in this case a decrease in the photoisomerization process.

3.2.4. Diffuse reflectance laser flash photolysis Due to the impossibility of determine the singlet oxygen quantum yields in solid samples we decided to perform diffuse reflectance laser flash photolysis experiments in order to evaluate the excited triplet state formation. Squaraine dyes are usually characterized by small triplet quantum yields, typically, with nano-, pico-, or femtosecond pulse durations the population of the triplet states is negligibly small. However, the triplet-triplet energy transfer process is an excellent method to characterize the triplet excited state of these dyes. Photosensitizers such as anthracene were able to generate detectable amounts of dye triplets according with the previous paper 20. With the sensitization process we detect the triplet-triplet absorption band of the squaraine dye in ethanol at 515nm. The transient absorption spectrum obtained by the diffuse reflectance laser flash photolysis of CS5 adsorbed onto chitosan is shown in Figure 6. A bleaching at ~670 nm (ground state depletion) and transient absorption maxima at 515nm is observed. The appearance of the T-T absorption at 515nm without sensitization is of extremely importance. This result suggests that the diminishing of the non-radiative pathways of deactivation due to the solid adsorption of the dye increases the population of the excited single state. This increase, apart from enable higher fluorescence quantum yields, improves the possibility of inter-systems crossing (ISC) occurrence from the single to the triplet state. From the triplet state the dye can react with molecular oxygen generating phototoxic species responsible for


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cells death. The presence of the triplet state in the spectrum of Figure 6 indicates the possibility of phototoxic species production, the most common - singlet oxygen.

Chitosan Sample 5 Sample 4 Sample 6

515nm

8.5

% Absorption

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3.5

-1.5 350

450

550

650

750

-6.5

-11.5

Wavelength (nm)

Figure 6: Transient absorption spectra of the dye onto chitosan (0.25, 0.5 and 1µmol/g) in air equilibrated sample. The excitation wavelength was 266nm.

3.2.5. Subcellular distribution of CS5 within HeLa cells with Confocal Microscopy The efficiency of photosensitizer entry into the cancer cell and its subcellular distribution in different organelles are of special importance for PDT. Intracellular localization of a PS has been shown to be critical to define the mechanism and efficiency of photoinduced cell death and is dictated by charge and amphiphilicity1. Determining the localization of a photosensitizer in a cell can help focus attention on the probable primary site(s) of initial damage by PDT with a specific PS. Studies from in vitro culture systems and in vivo animal models have indicated that both necrosis and apoptosis of the target cells or tissues represent the major therapeutic effect of PDT.30,31,32 It is very clear that the localization of the photosensitizers in the plasma membrane and in the mitochondria plays an important role to understand the cell death mechanisms. However,



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it is important to refer that sufficient damage to any cell organelle or essential component can kill the cell. Therefore, we were interested in determining the subcellular localization of the photosensitizers within the cells. To do so, we carried out colocalization experiments with specific organelle markers through confocal fluorescence microscopy. At first we can see in figure 7 the staining pattern of a) Rho-123 and b) TMA-DPH in the absence of squaraine dye. Rho-123 is a non-cytotoxic, cell-permeant, cationic, greenfluorescent dye that is rapidly sequestered by active mitochondria (as a result of the negative membrane potential across the mitochondrial inner membrane).33 The mitochondrial membrane and plasma membrane co-localization studies were performed through the incubation of HeLa cells with 3 µM of Rho-123 and 1 µM of TMA-DPH separately for 15 minutes. The pattern of Rho-123 distribution in the cytoplasm of HeLa cells (Fig. 7a), is typical of mitochondria, and confirms efficient uptake of Rho-123 by active mitochondria. TMA-DPH is a cationic and hydrophobic fluorescent membrane probe that interacts with living cells by instantaneous incorporation into the plasma membrane, where it becomes fluorescent34. Efficient plasma membrane staining by TMA-DPH is also evident from Fig. 7b.

Figure 7. Subcellular distribution of Rho-123 (A, green), and plasma membrane marker TMA-DPH (B, blue) in HeLa cells, overlay of both channels presented in C.


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Figure 8 shows the spatial distribution of CS5 within HeLa cells utilizing Rho-123 (Fig. 8b) and TMA-DPH (Fig. 8c) as mitochondrial and plasma membrane markers.

Figure 8. Subcellular distribution of CS5 (A), mitochondrial marker Rho-123 (B), TMA-DPH (C) and overlay of all channels (D).

Comparing the staining pattern of Rho-123 in the absence (Figure 7) and presence of CS5 (Figure 8), we can clearly identify that in the presence of the PS, Rho-123 distribution becomes non-specific within HeLa cells, as the result of a loss of mitochondrial uptake. This result strongly suggests that CS5 toxicity lead to a disruption in mitochondrial membrane potential.

On the other hand, the staining pattern of CS5 in HeLa cells shows reduced

nuclear accumulation and a heterogeneous distribution within the cytosol, with some cells exhibiting an absence of strong specific compartmentalization of the squaraine dye (Figure



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8a),

while

others

evidenced

a

punctated

distribution

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(Fig.

8d)

suggesting

endosome/lysosome entrapment. The subcellular distribution of CS5 adsorbed onto powdered chitosan after incubation with HeLa cells is shown on Figure 9.


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Figure 9. Distribution of powdered CS5 chitosan (A, red), within Hela cells. Mitochondria and plasma membrane were labelled with Rho-123 (B, green) and TMA-DPH (C, blue), respectively. The overlay image of all channels is also shown (D).

The release of the CS5/chitosan onto the HeLa cells is unmistakable, as we can see in Figure 9a). However, intracellular concentrations of CS5 were significantly lower than the one observed after incubation with of HeLa cells with the free form of the PS, and in this case, Rho-123 mitochondrial uptake (Fig. 9b) is maintained suggesting reduced impact of CS5/chitosan at these levels in mitochondrial membrane potentials. In agreement with previous observations for CS5 distribution, the dye has low affinity for the plasma membrane of HeLa cells, as we didn’t observe a colocalization between the squaraine and TMA-DPH (Fig.9c). As Figure 9d) shows, CS5 is distributed throughout the cytoplasm. Colocalization with mitochondria is also detected, as shown by the analysis performed with colocalization plugin WCIF from ImageJ software and presented in Figure 9e). This plugin recovers values for the Product of the Differences from the Mean (PDM) for each pixel.35 PDM values are useful for spatial inspection of areas with synchronous or asynchronous distribution of two fluorophores in a given image. These values are determined according to: PDM = (red intensity- mean red intensity)×(green intensity – mean green intensity). The values for each pixel were then displayed through a pseudocolor scale (Figure 9e). We can clearly see the yellow regions correspondent to the overlap of both dyes (Rhod-123 and CS5). The Pearson’s correlation coefficient (Rr),36 which quantifies the extent of correlation between two fluorescence signals in an image was also determined. The values for Rr can range from 1 to 1 and a value near 1 corresponds to perfect correlation, while values around 0 correspond to completely independent signals. For this study the value obtained was about 0.45 which suggests partial mitochondrial uptake of CS5. Adventitious overlap of mitochondrial and



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non-mitochondrial spaces in the same confocal volume is unlikely as confocal resolution is higher than typical mitochondria dimensions.37

3.3 Cytotoxicity studies in HeLa cell line The dark toxicity and phototoxicity of the squarylium dye in solution and adsorbed onto chitosan incubated with HeLa cells are presented in Table 2. Regarding the GI50 (50% growth inhibitory concentration) values obtained in the dark and in irradiated samples, we can conclude that the squaraine dye is more active when irradiated than in the dark conditions (GI50=0.16±0.01 µM and 0.96±0.08 µM, respectively). Moreover when adsorbed onto powdered chitosan the GI50 values slightly increased, but still the sample subjected to irradiation presenting higher activity (GI50= 0.28±0.01 µM) than the sample in the dark (GI50= 1.01±0.03 µM). We also performed the cytotoxicity studies utilizing only chitosan without any adsorbed dye. The studies revealed that chitosan by their own presents cancer cell growth inhibition. In this way, we are very convinced that we have the system composed by the squarylium dye adsorbed onto chitosan acting as cancer cells destroying agent. Therefore, the squaraine dye and the squaraine/chitosan system seems to be very promising agents to be used in cancer photodynamic therapy due to the lower toxicity exhibited to the cells in the dark and the destruction ability of cancer cells induced by irradiation. Table 2. Dark toxicity and phototoxicity of the dye incubated with Hela cells.

GI 50 (µM)

Sample Dark

Irradiated

CS5

0.96±0.08

0.16±0.01

CS5/Chitosan

1.01±0.03

0.28±0.01


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4. Conclusions CS5 in solution has been shown to have a high singlet oxygen formation quantum yield of about 0.36. This efficiency in singlet oxygen generation is not common for squaraine dyes and suggests that this molecule could be useful in PDT. Ground state absorption and fluorescence studies of the squaraine dye were performed in solution and adsorbed onto powdered chitosan to evaluate the potential of the squaraine/chitosan as a system for localized therapy. Differences in fluorescence properties of the squaraine dye between both environments are substantial. An increase in the fluorescence quantum yields and lifetimes of about four times was determined, which is a very promising result for future application of as theranostic agents. Also, it is important to refer that the results evidence an enhancement of the stability of the photosensitizer due to the entrapment in the polymer environment, which reduces the deactivation processes which interfere with the radiative ones. The results of diffuse reflectance laser flash photolysis substantiate this statement. The reduction of the non-radiative pathways increases the population of the excited single state permitting the occurrence of ISC. From the triplet state the dye can react with molecular oxygen generating phototoxic species responsible for cells death. The transient absorption spectrum was also obtained in order to evaluate the ability of this system in triplet state generation. The triplet-triplet absorption band was obtained as in solution but without sensitization allowing the possible formation of phototoxic species responsible for killing cells. Confocal microscopy colocalization studies of CS5 with subcellular markers showed that the free squaraine dye in solution is distributed throughout the cytosol, without a clear cellular target, although in some cases it was found within endosomal and/or lysosomal particles. Also, this dye (under the concentration employed) disrupts mitochondrial membrane potential, influencing Rhodamine 123 staining. The dye adsorbed onto powdered



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chitosan is also found distributed throughout the cytosol, but at significantly lower intracellular concentrations which did not lead to disruption of mitochondrial membrane potential as evaluated by the staining pattern of Rho-123. Partial mitochondrial uptake of CS5 was also observed, which might contribute to the mitochondrial toxicity evidenced by this PS at high concentrations. Mitochondrial insertion by photosensitizers has been shown to be a very important determinant for efficient photodynamic therapy of cancer. Importantly, the compound is found to be non-toxic in the dark and highly toxic under radiation, which is one of the most important requirements of PDT. These results suggest that this dye, both in solution and adsorbed onto chitosan could be suitable for application as a photosensitizer in localized therapy and cancer photodynamic therapy. For squaraine adsorbed onto powdered chitosan, the enhancement of the fluorescence quantum yield and lifetimes, the great singlet oxygen quantum yield, the favorable localization of the sensitizers onto cancer cells, the absence of toxicity in the dark and the ability of killing cells under light, strongly suggest that we are in the presence of a system with a great therapeutic potential.


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Corresponding author * To whom correspondence should be addressed. E-mail: [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Acknowledgements Thanks are due to FCT (Portugal’s Foundation for Science and Technology) for the support by scientific projects (RECI/CTM-POL/0342/2012). FCT also provides PhD fellowships for Diana Ferreira (SFRH/BD/95359/2013), Tânia Sousa (SFRH/BD/92398/2013), and David Conceição (SFRH/BD/95358/2013), as well as a post-doctoral fellowship to Fábio Fernandes (SFRH/BPD/64320/2009) and R.C.Calhelha (SFRH/BPD/68334/2010).



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TOC graphic

HeLa Cells


Chitosan System for Photodynamic

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