Polymers as enhancers of photodynamic activity of chlorin photosensitizers for photodynamic therapy

DOI 10.1515/plm-2013-0011 Photon Lasers Med 2013; 2(3): 189–198

Nadezhda A. Aksenova, Timur M. Zhientaev, Anna A. Brilkina, Ljubov V. Dubasova, Andrey V. Ivanov*, Peter S. Timashev, Nicolay S. Melik-Nubarov and Anna B. Solovieva

Polymers as enhancers of photodynamic activity of chlorin photosensitizers for photodynamic therapy

Polymere als Verstärker der photodynamischen Aktivität von Chlorin-Photosensibilisatoren für die Photodynamische Therapie

Abstract: The impact of water-soluble and amphiphilic polymers with different structures, namely carboxymethyl cellulose (CMC), polyvinyl alcohol (PVA) and polyvi-nylpyrrolidone (PVP), was studied on the photoactivity of chlorin photosensitizers (PSs) in photodynamic therapy (PDT). It was shown that such polymers can cause a con-siderable increase in the PS activity, both in the process of singlet oxygen photogeneration in cell experiments, and in the model reaction of a substrate photooxidation in water. Amongst the studied polymers, CMC and PVP appeared to have the most significant influence on the photoactivity of PSs. The observed effect of the polymers on the photosensitizing activity of PSs can be attributed to the presence of chlorin-polymer interactions resulting in the porphyrin disaggregation in aqueous phase. The effect of the polymers on the photocytotoxicity of PSs is attrib-uted to the absence of interactions between chlorin and polypeptide or lipoproteins which results in a decrease of the photoactivity of chlorins in cell culture. The PS/poly-mer systems appear to be a new effective dosage form of PDT drugs.

Keywords: amphiphilic polymers; chlorin photosensitiz-ers; complexes; photodynamic therapy; photooxidation; cancer cells.

Zusammenfassung: Die Wirkung von wasserlöslichen und amphiphilen Polymeren mit unterschiedlichen Strukturen (Carboxymethylcellulose, CMC; Polyvinylal-kohol, PVA; Polyvinylpyrrolidon, PVP), auf die Photoak-tivität von Chlorin-Photosensibilisatoren (PS) in der photodynamischen Therapie (PDT) wurde untersucht. Es wurde gezeigt, dass solche Polymere eine erhebliche Steigerung der PS-Aktivität bewirken können, so wohl im Prozess der Singulett-Sauerstoff-Generation in Zell-experimenten als auch in der Modellreaktion einer

Substrat-Photooxidation in Wasser. Unter den unter-suchten Polymeren schienen CMC und PVP den größten signifikanten Einfluss auf die Photoaktivität der PS zu haben. Die beobachtete Wirkung der Polymere auf die photosensibilisierende Aktivität der PS kann auf das Vorhandensein von Chlorin-Polymer-Wechselwirkungen zurückgeführt werden, die in einer Porphyrin-Disaggre-gation in wässriger Phase resultieren. Die Wirkung der Polymere auf die Photozytotoxizität der PS wiederum ist auf das Fehlen von Wechselwirkungen zwischen Chlo-rin und Polypeptid bzw. Lipoproteinen zurückzuführen, die zu einer Abnahme der Photoaktivität von Chlorin in der Zellkultur führt. Die PS/Polymer-Systeme scheinen eine neue wirksame Darreichungsform von PDT-Medika-menten zu sein.

Schlüsselwörter: amphiphile Polymere; Chlorin-Photo-sensibilisatoren; Komplexe; photodynamische Therapie; Photooxidation; Krebszellen.

*Corresponding author: Andrey V. Ivanov, N. N. Blokhin Cancer Research Center, Russian Academy of Medical Sciences, Kashirskoe Shosse 24, 115478 Moscow, Russian Federation, e-mail: [email protected] A. Aksenova and Anna B. Solovieva: N. N. Semenov Institute of Chemical Physics, Russian Academy of Sciences, ul. Kosygina 4, 119911 Moscow, Russian FederationTimur M. Zhientaev: Polymer Science Department, School of Chemistry, Moscow State University, 1 Leninskie Gory, 119992 Moscow, Russian Federation Anna A. Brilkina and Ljubov V. Dubasova: N. I. Lobachevsky State University of Nizhny Novgorod, pr. Gagarina 23, 603950 Nizhny Novgorod, Russian FederationPeter S. Timashev: Institute of Laser and Information Technologies, Russian Academy of Sciences, Pionerskaya 2, 142190 Troitsk, Moscow, Russian FederationNicolay S. Melik-Nubarov: M. V. Lomonosov Moscow State University, Leniskiye Gory, Acad. Khokhlov 1 (build. 40), 119992 Moscow, Russian Federation

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190 N.A. Aksenova et al.: Polymers as enhancers of photodynamic activity of chlorin photosensitizers for photodynamic therapy

1 IntroductionPhotodynamic therapy (PDT) belongs to the more gentle methods for cancer treatment. It uses a nontoxic photoac-tive material and light for selective degradation of malig-nant tissues [1]. Porphyrin or chlorin photosensitizers (PSs) are some of the most effective types of photoactive material used in PDT. Many studies have already been published, proving the efficiency of PDT in the treatment of infectious diseases, skin diseases, atherosclerosis, rheu-matoid arthritis, and age-related macular degeneration [2–5]. The essence of malignant tissue destruction mech-anism during PDT is, that PSs are localized in the tumor and when treated with a suitable wavelength are converted to the excited triplet state, and after the interaction with molecular oxygen, transfer excitation energy to it. Singlet oxygen generated during this process is a strong oxidizer and leads to the destruction of abnormal cells [6, 7].

One of the most important advantages of PDT is that PSs have an affinity for hyperproliferative tissues, espe-cially for the tissues that are supplied by the neoplastic blood system. There is currently no unique explanation of the PSs’ tropism to the tumor. However, one of the pos-sible causes of this selectivity may be a tendency of non-covalent PSs to associate with the plasma lipoprotein that is more strongly contained in proliferating cells as com-pared to other cells [6]. At the same time, the PDT method has some disadvantages. After therapy the patient should avoid exposure to intense sunlight for several days and photodynamic exposure can cause pain [8]. In order to avoid the high photosensitivity of the patient’s skin and other possible side effects, the accurate dosage of PS and light power should be calculated [9].

Another approach to overcome these disadvan-tages is the utilization of polymeric carriers for effective increase and elimination of the PS acceleration [10–12]. For example, the inclusion of protoporphyrin IX (PpIX) in the diblock copolymer micelles methoxy-polyethyleneglycol-b-(polycaprolactam) shows a 10-fold increase in its pho-totoxicity compared to the phototoxicity of the initial PpIX [13]. Use of a chlorin e6 covalent binding with polycation increases the affinity for pathogenic bacteria cells and yeast, resulting in a significant increase in the bactericidal and fungicidal activity [14]. In [15], the polymeric micelles (con-jugates of polyethylene glycol-phosphatidylethanolamine) are an effective system for extreme hydrophobic meso-tet-raphenylporphyrin solubilization. It has been shown that such polymeric micelles with PS associates have a high pho-totoxicity for mouse and human cancer cells.

Various authors propose a different photodynamic activity of PSs improvement mechanisms when they

bind to polymers. In particular, in [16–19] it was sug-gested that the addition of PSs to hydrophilic and amphi-philic polymers improves their photophysical character-istics. At the same time in [20] the authors suggested that the polymeric carriers (for example, negatively charged nanoparticles of sodium alginate and dioctyl sodium sulphosuccinate) change the intracellular distribution of the PS (methylene blue) in pathological cells (breast adenocarcinoma).

Thus, the published data suggests that the inclusion of PSs in the compositions based on polymers can sig-nificantly increase their photodynamic activity. However, there is no consensus of opinion for the “polymer” effect in the literature [11, 13, 16–20]. The main idea of the pre-sented paper is to identify the basic feasibility of polymer influence on the physical and chemical properties of pho-tosensitizing activity in the reaction of photooxidation of tryptophan (Trp) in the aqueous phase, the photocytotox-icity against different cancer cells, and in particular, the accumulation and distribution of chlorin PSs inside the cells.

2 Materials and methods

2.1 Chemicals

The following chemicals were used.

2.1.1 Chlorin photosensitizers

– N-methyl-di-D-glucamine salt of chlorin e6 – “Photoditazine” (PD) (Veta-Grand, Russia),

– Sodium salt of chlorin e6 (ChL) (Frontier Scientific, USA).

The chemical structures of both chlorin PSs are given in Figure 1.

2.1.2 Polymers

– Polyvinylpyrrolidone (PVP) (Dr. Theodor Schuchardt, Germany),

– Sodium salt of carboxymethyl cellulose (CMC) (Acros, USA),

– Polyvinyl alcohol (PVA) (Sigma-Aldrich, USA).

The molecular weights (Mw) of all the polymers used are shown in Table 1.



N.A. Aksenova et al.: Polymers as enhancers of photodynamic activity of chlorin photosensitizers for photodynamic therapy 191

CH2

CH2CH3

CH3A B

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3CH3

CH3

NH

NHNH

NHOH

OH

OH

OH

OH

OH

OHOH

HO HO+H2N+H2N

O

O

O

O

O

O

O

O

-O

-O-O

Na+ Na+

-O

N

N

N

N

Figure 1 Chemical structures of (A) PD and (B) ChL.

2.1.3 Other chemicals

Penicillin/streptomycin solution, puromycin, dimethyl-sulfoxide (DMSO), salts and buffer components were purchased from Sigma-Aldrich Corp. (USA). Standard cell culture media – Dulbecco’s modified eagle medium (DMEM), 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetra-zolium bromide (MTT), buffer solution (PBS), trypsin and Versen solution were purchased from PanEco (Russia), newborn calf serum was from Invitrogen (USA). Cell orga-nelles dyes were used such as LysoTracker® Green, ER-Tracker™ dyes for live-cell endoplasmic reticulum labeling (Hoechst from PanEco, Russia), NBD- and BODIPY dye-labeled sphingolipids from Invitrogen (USA).

2.2 Preparation of PS/polymer formulations

For cell experiments corresponding amounts of the polymer and PS were dissolved in PBS (10 mm Na2HPO4, 150 mm NaCl, pH 7.4) and were then mixed and incubated at ambient temperature for 15 min. Addition to the cell cul-tures resulted in a 10-fold dilution of the preparations. For model reaction of Trp oxidation, corresponding amounts of the polymer and PS dissolved in water were mixed and incubated at ambient temperature for 15 min.

Table 1 Polymer molecular weights Mw.

Polymer Mw (Da)

PVP 25,000CMC 250,000PVA 30,000

2.3 Cell culturing

Transformed NIH-3T3-EWS-FLI1 mouse fibroblasts were immortalized by NIH-3T3 transfection with the EWS-FLI1 cDNA under the control of the retroviral long terminal repeat (LTR) retroviral promoter. This cell line displayed some characteristic features of tumor cells, such as inabil-ity to undergo apoptosis and uncontrollable growth. The cells were maintained in DMEM medium supplemented with 10% newborn calf serum, 4 mm glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2.5 μg/ml puro-mycin. The cells were transferred to the puromycin-free medium to avoid possible side effects of this antibiotic, and splitted twice a day before the experiment to ensure logarithmic growth.

Human breast adenocarcinoma HBL-100, human ovarian carcinoma Skov-3, human urinary bladder car-cinoma T-24 and human liver adenocarcinoma SK-HEP-1




cells were cultured in DMEM supplemented with 10% fetal calf serum, 4 mm glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. The cells were plated at a density of about 25,000/cm2 every third day.

All cell cultures were maintained at 37°C in a humidi-fied atmosphere containing 5% CO2 and were seeded for the experiment in 96-well plates at a density of about 10,000/well.

2.4 Experiments

2.4.1 Cell photodynamic treatment

The NIH-3T3-EWS-FLI1 cells were pretreated with PS solution in the complete puromycin-free medium for 3 h. The photo effect of PS was studied at a dose of 1 J/sample in 96-well plate (square of a well 0.28 cm2). Experiments on cells incubated with PS were carried out in nearly total darkness. A semi-conductor laser (Atkus-2; Space Instruments Engineering Ltd., Russia) with a wavelength of 661 nm and equipped with an optic fiber (diameter 0.5 mm) was used as a light source. The fiber was maintained at a distance of 65 mm from the sample to ensure uniform irradiation of the 2.6 cm2 area corre-sponding to a quadruplet of wells in 96-well cell cultur-ing plate. The wells of the plate neighboring with the irradiated sample were omitted from the measurements, since they could be irradiated by the light scattered from the illuminating wells. The irradiance (20 mW/cm2) was calibrated using laser powermeter LP-1 (Sanwa, Japan), and exposure time was adjusted to obtain 3.6 J/cm2 fluency. The plate was automatically shifted from one quadruplet to another every 3 min using a device pro-duced by Research Institute of Impulse Technique (Russia).

2.4.2 Photocytotoxicity

Photocytotoxicity was evaluated using MTT test accord-ing to [21]. Briefly, the cells in 96-well plates, at an initial density of about 20,000 cells/cm2, were treated with the polymers, PS or their mixtures and then 50 μl of sterile MTT solution in DMEM (1 mg/ml) were added to the wells. The plate remained in the humidified atmosphere containing 5% CO2 for 4 h, after which the medium was removed and formazan crystals were dissolved in 100 μl of DMSO. The absorbance of the samples at 550 nm was measured using a microplate reader (Multiscan Plus; Titertek, USA).

2.4.3 PS or PS/polymer kinetic of accumulation in cells

PS or PS/polymer kinetics of the accumulation in cells was performed using a flow cytofluorometer (FACSCalibur; BD Biosciences, USA). For experiments on formulation accu-mulation kinetics, the cells were seeded into 24-well plates at a density of 1 × 105 cells per well. Cells were incubated for at least 6 h (until they were attached to the substrate), which was followed by PS solution treatment under the temperature-controlled CO2 incubator conditions. The initial culture medium was changed to a PS containing medium with chlorin concentration 1 × 10-5 m. We used the following exposure times: 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 10, 12 h. After incubation of the cells with PS, the cell culture was removed from the substrate. The cells were washed with PBS, followed by addition of 0.1 ml trypsin solution into each well, to remove the cells from the substrate, (for T-24 cells: a mixture of 0.25% trypsin solution / Versen solution (1/1); for SK-HEP-1: the same mixture but with 1/3 ratio) and then placed for 5 min into CO2 incubator. After this, each well was washed three times with 1.5 ml PBS, washing the cells in a centrifuge tube and centrifuged for 5 min at 1000 rev/min. Then the cells were re-suspended in PBS (300 ml) at a concentration of 1 × 106 cells/ml, and the formulation accumulation level was observed with a flow cytofluorometer. PS was excited with a 633 nm diode laser (CristaLaser, USA) and the fluorescence was observed in the range 650–710 nm. A CeelQuest software was used for calculation, and each experimental point was performed by counting 10,000 cells.

2.4.4 Laser scanning microscopy

PS and PS/polymer visualization in the cells was carried out using a complex laser scanning microscope LSM 510 META (inverted microscope Axiovert 200M, laser scanning module Carl Zeiss LSM 510, Carl Zeiss spectral module 23 META), equipped with a femtosecond Ti: Sapphire, tunable wavelength laser system (Mai Tai® eHP, Spectra Physics). The temperature of the sample was maintained during the experiment. PS was excited at λ = 633 nm and the fluorescence was observed in the range 650–710 nm. For experiments to localize the intracellular PS and PS/polymer, the cells were seeded in 96-well plates at a density of 4 × 103 cells per well and incubated in 5% CO2 atmosphere at 37°C overnight. Then the culture medium was replaced with a mixture of medium and PS. Exposure time was 2 h. After this, the medium was replaced with a new drug-free medium followed by visualization with the LSM images.




The following was used to stain the cell organelles: lysosomes – LysoTracker® Green (50 nm), endoplasmic reticulum – ER-Tracker™ dyes for live-cell endoplasmic reticulum labeling (500 nm), and for the Golgi apparatus – NBD- and BODIPY dye-labeled sphingolipids (500 nm).

2.4.5 Photosensitizing activity of PS

The photosensitizing activity of PS was investigated using a model reaction of Trp oxidation. The photoactivity experiments were carried out in accordance with [22, 23]. The kinetics of the process was monitored through the change in the concentration of Trp, which was deter-mined from the optical density of the absorption band at λ∼280 nm in the UV spectra of Trp. The rate constant was determined from the linear portion of the kinetic curve at the initial moments in time. The effective rate constant keff (in l/mol s) for the photooxidation of Trp was calculated by the formula:

Trp

effPS Trp

Ck

C C t∆

=⋅ ⋅∆ (1),

where CPS and CTrp are the concentrations of PS and Trp, respectively, and ΔCTrp is the change in the concentration of the Trp during the time interval, Δt. The error of the effective rate constant measurements was estimated to be 10%.

2.4.6 UV/VIS spectroscopy

The UV and UV/VIS spectra of solutions were recorded on a Cary 50 spectrophotometer (Varian Medical Systems, Austria); the fluorescence spectra on CaryEclipse spectro-fluorometer (Varian Medical Systems, Austria).

3 Results

3.1 Polymer influence on the PS/polymer photodynamic activity in cells experiments

Figures 2 and 3 show the dependencies of the survived HBL-100, Skov-3 and NIH-3T3-EWS-FLI1 cell lines on PD and ChL concentrations, in the presence and absence of polymers. As it can be seen from both figures, the PS phototoxicity increases in the presence of polymers.

100

80

60

Frac

tion

of s

urvi

ving

cel

ls (

%)

40

53 4 2 1

0

20

0.1 1.0 10

lg (CPD×106) (M)

Figure 3 PS and PS/polymer system phototoxicity studied on a malignant tumor NIH-3T3-EWS-FLI1 cell line: PD (curve 1), ChL (curve 2), ChL-CMC (curve 3), PD-PVP (curve 4) and ChL-PVP (curve 5). The PVP concentration (per unit) = 9 × 10-3 m; the CMC concentration (per unit) = 4 × 10-3 m.

100

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60

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urvi

ving

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%)

40

4

3

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1

2

6

20

0.1 1.0 10

lg (CPD×106) (M)

Figure 2 PD phototoxicity studied on tumor cell lines HBL-100 (red lines; curves 1, 3, 5) and Skov-3 (green lines; curves 2, 4, 6): PD solution in water (curves 1, 2), with CMC (curves 3, 4) and with PVA (curves 5, 6). The PVA concentration (per unit) = 2 × 10-2 m; the CMC concentration (per unit) = 4 × 10-3 m.

Indeed, PS concentration to induce the death of 50% cells (IC50) in PD-CMC systems for the HBL-100 and Skov-3 cell lines varied from 10-7 to 2.5 × 10-7 m, while the PD solution in water with concentrations of 10-5 to 1.5 × 10-5 m cause the death of 50% cells (Figure 2). This means, that the PS concentration IC50 for the PD-CMC system decreased 100-fold. Under the same conditions,




the IC50 shifts were similar in cases where PVA was used. Indeed, PS concentration IC50 in PD-PVA systems varied within the limits of 2.5 × 10-7 to 10-6 m for both cell lines (IC50 decreased in ∼20 times). At the same time PD or ChL phototoxicity in the presence of CMC or PVP in NIH-3T3-EWS-FLI1 cells experiment increases too (Figure 3). The data presented here shows that PS concentration IC50 for systems based on CMC and PVP decreased 3–5-fold. Thus, the increase in the photocytotoxic effect, in the presence of the polymer, is determined not only by the cell’s nature, but also by the polymer structure. In this case the greatest influence on photocytotoxicity is exhibited by CMC and PVP, and the smallest by PVA. One possible explanation of this effect is the influence of the polymer on the photosensitizing activity of PS/polymer systems. To test this hypothesis, we studied the photo-activity of PS at concentration IC50 in the presence of the polymer.

3.2 Tryptophan photosensitized oxidation

Figure 4 shows the dependence keff for the photooxida-tion of Trp in the presence of PS/polymer systems on the polymer concentration at a constant concentration of PS (CPS = 1 × 10-5 m). It can be seen that utilization of the PVP and CMC had the strongest, and PVA the lowest influ-ence on keff. Such differences in polymer influence on the PS photosensitizing activity maybe associated with polymer interaction with chlorins’ features and their disaggregation. However, the principles identified in the

photosensitizing activity study of the PS and PS/polymer systems under model conditions, do not correspond to the laws identified in experiments on photocytotoxic-ity. In particular, the CMC significantly affects only the photosensitizing activity of PD and has no effect on the activity of ChL, but in cell experiments, CMC significantly reduces the IC50 value in the case of chlorin e6. In addi-tion, the CMC effect on ChL photocytotoxicity is compara-ble with the effect of PVP in case of cell experiments, and for PD with the influence of PVA, whereas experiments on photooxidation of Trp have other regularities. It can be assumed that the polymer effect on the photosensitizing activity of PS is not the determining factor of the described effect. One possible reason for the polymer influence on cells photocytotoxicity may be the polymer effect on PS penetration and its distribution within the cells.

3.3 Polymer influence on the PS accumulation and distribution inside cells

To illustrate the polymer influence on accumulation of PS in cells, experiments were carried out on the human urinary bladder carcinoma T-24 and human liver adeno-carcinoma SK-HEP-1 cells. The features of accumulation kinetic (the dependences of the fluorescence signal of ChL or ChL-PVA complex in cells on incubation time) of PS or PS/polymer systems at chlorin concentrations of 1 × 10-5 m were studied. Figure 5 shows the fluorescence signal kinet-ics of ChL and ChL-PVA system in SK-HEP-1 and T-24 cells.

k eff (

l/mol

s)

k eff (

l/mol

s)

680A B

660

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540

520

500

680

660

640

620

600

580

560

540

520

500

1 1

2 2

3 3

10-4 10-3 10-2 10-1

CPolymer (M)

10-4 10-3 10-2 10-1

CPolymer (M)

Figure 4 Tryptophan oxidation effective constant keff dependencies on polymer (1 – CMC, 2 – PVA, 3 – PVP) per unit concentration CPolymer at a constant concentration of PS (CPS = 1 × 10-5 m) in the presence of PD (A) or ChL (B). Dot on the Y-axis refers to constant keff in the presence of PS without polymer.




The presented data shows that the presence of PVA does not increase the photosensitizer content in cells. ChL and ChL-PVA visualization in the cells was carried out using a LSM. It was shown that all PS and PS/Polymer systems accumulated in the perinuclear area with thread-like and mesh distribution after incubation of 2 h. Also it was

Fluo

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04

39.4

73

49.3

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59.2

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69.0

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88.8

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98.6

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549

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418

128.

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min min min

Figure 6 LSM images and fluorescence profiles of ChL-PVA (1) and lysosomes (A), endoplasmic reticulum (B) or Golgi apparatus (C) dyes (2).

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resc

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(a.

u.)

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0 2

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4 6 8 10 12Time (h)

Figure 5 Fluorescence signal kinetics of ChL (curves 1, 2) or ChL-PVA (curves 3, 4) in SK-HEP-1 cell line (red lines; curves 1, 3) and in T-24 cell line (green lines; curves 2, 4). The PVA concentration (per unit) = 2 × 10-2 m, the constant concentration of PS CPS = 1 × 10-5 m.

shown that lysosomes don’t accumulate ChL or ChL-PVA formulations, but Golgi apparatus and endoplasmic retic-ulum intensively accumulated both PS and PS/polymer system. Figure 6 shows the fluorescence signal of ChL-PVA with fluorescence signal of the organelle dyes in T-24 cells; the LSM pictures also show corresponding images. These results demonstrate that there is no influence of polymer on the accumulation kinetics and distribution of ChL in cells.

3.4 Porphyrin-polymer interactions features

We studied the spectral properties of chlorin-PVP systems, taking account of the PVP maximum influence on the PS photosensitizing and photocytotoxicity activity. Both the PS disaggregation and interaction with the polymers were indicated by the changes in the UV/VIS absorption of chlorins in the presence of PVP. As shown in Figure 7, PVP affects the band intensities (increasing) and the posi-tions (bathochromic shifts) in the UV/VIS spectra of ChL (Figure 7A) or PD (Figure 7B). Such changes are usually associated with changes in the microenvironment of the porphyrin cycle and are the result of the interaction of PS with polymers and PS disaggregation.




4 Discussion

It was shown earlier [22–26] that the photosensitizing activity of PS/amphiphilic polymer systems is determined by the degree of interaction of photoactive component and the macromolecules, leading to a disaggregation of PS. The PS interaction with amphiphilic polymers was examined using the spectrophotometric method to detect the features of intermolecular interactions in photoactive systems. The validity of this approach is demonstrated by the results presented in this paper. In particular, the chlorin-PVP system absorption spectra analysis shows that association of PVP with ChL or PD is weaker than the previously studied porphyrin PS [23]. The absorption spectra of chlorins in the presence of PVP led to an increase in the optical density of the bands and bathochromic shift of 10 nm at polymer concentrations of ≥ 1 × 10-1 m (for ChL) or concentrations of ≥ 1 × 10-2 m (for PD). At the same time, the previously studied porphyrin PS absorption spectra show a significant increase in the optical density of the bands (rising to 2 times) and the bathochromic shift of 10 nm at polymer concentrations of ≥ 3 × 10-3 m [23]. Basically, the photosensitizing activity of ChL in the presence of PVP increased by 1.2 times, the PD by 1.6 times and the previously studied porphyrin PS by 2.2 times. Thus, we can assume that PD is likely (com-pared to ChL) to be associated with the amphiphilic and water-soluble polymers, the values of Trp photooxida-tion constants for PD systems increased in the presence

of CMC by 1.5 times, and the presence of CMC does not affect the photoactivity ChL (i.e., chlorin e6 do not bind with CMC) (Figure 3).

One of the reasons for the PD-polymer binding ability may be the presence of glucamine counterions. It was shown in [23] that the PS/polymer supramolecular asso-ciates are formed as a result of hydrophobic, dipolar and hydrogen interactions. It is obvious that bulk glucamine fragments also interact with macromolecules via hydro-phobic and hydrogen interactions, which leads to addi-tional PS binding and its disaggregation. At the same time, all utilized PS do not form associates with PVA, as indicated by a slight increase of the oxidation constant value for the Trp photoactive systems. However, experi-ments on PD photocytotoxicity showed that the effect of PVA on the photodynamic activity is comparable to the effect of CMC (Figure 2). At the same time, experiments on ChL photocytotoxicity revealed that the effect of CMC is comparable to the influence of PVP (Figure 3). Thus, the polymer does not increase the photosensitizing activity of PS, i.e., polymers which are substantially related to the PS significantly increase the photodynamic activity of chlo-rins in cell experiments.

It was also shown that polymer does not influence the accumulation and distribution of PS. In particular, PVA does not have any influence on the accumulation kinetics of ChL in the T-24 and SK-HEP-1 cells, and PVA does not change the distribution of ChL in such cells (Figure 5).

The ability of PS to bind with proteins or lipoproteins is the possible explanation of the low polymer influence

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Wavelength (nm) Wavelength (nm)

Figure 7 (A) UV/VIS spectra of 5 μm ChL (curve 1) and its mixtures with 0.09 mm (curve 2), 9 mm (curve 3), 90 mm (curve 4), and 450 mm (curve 5) of PVP and (B) UV/VIS spectra of 5 μm PD (curve 1) and its mixtures with 0.09 mm (curve 2), 0.90 mm (curve 3), 9 mm (curve 4), and 450 mm (curve 5) of PVP.




on the accumulation and distribution of PS and as a result of such binding, PS penetrates inside cells as PS/protein associates. For example, it is known that PS can interact with proteins, including bovine serum albumin [27]. Interestingly enough, the existence of such PS/protein associates may cause a rise in PS photodynamic activity in the presence of polymers. It is known that, depending on the type of interaction with polypeptides, the photoactivity of PS can decrease (in the case of elec-trostatic associates) or increase (in the case of hydro-phobic interactions between the components) [28]. Thus, assuming that photosensitizing activity of chlor-ins falls after interaction with proteins or lipoproteins; this could explain the restoration or enhancement of the functional activity of PS in the presence of polymers. However, the mechanism of PS/protein binding pre-vention by water soluble polymers CMC and PVA is still unclear. In this case it appears that the polymers could encapsulate the PS. It can be assumed that the polymers in aqueous solution, weakly-bonded in a mesh structure (grid formed by multiple hydrogen bonds) are encapsu-lated by PS as a solvate shells. This is indicated by the immutability of the photophysical properties of PS in aqueous solution in the presence of CMC and PVA. High-molecular proteins and lipoproteins can not be built in such a grid and be made to interact with a photoactive compound.

5 ConclusionIn summary, it was shown in this paper that the photody-namic impact on tumor cell lines in the presence of some polymers increases significantly, which allows the thera-peutic dosage of utilized PS (PD and ChL) to be decreased by more than 10-fold. The main reason for this effect is the interaction of PS with macromolecules (in the case of amphiphilic polymers) or encapsulation by PS water soluble polymers. So the PS/polymer systems appear to be a new effective dosage form of PDT drugs.

Acknowledgments: This work was supported by the Rus-sian Foundation for Basic Research (projects numbers 13-03-00429-a, 11-02-01090-a and 11-03-12074-ofi-m). The PS/polymer formulations were generously prepared by N. N. Glagolev (Semenov Institute of Chemical Physics, Russian Academy of Sciences, Moscow, Russia). NIH-3T3-EWS-FLI1 mouse fibroblasts were a generous gift of Prof. C. Malvy (Institut Gustave Roussy, Villejuif, France). The photodynamic activity of HBL-100 and Skov-3 cells was studied by E. Yu. Filinova (N. N. Blokhin Cancer Research Center, Russian Academy of Medical Sciences).

Received February 1, 2013; revised May 21 , 2013; accepted May 28, 2013

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Polymers as enhancers of photodynamic activity of chlorin photosensitizers for photodynamic therapy

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