Cationic Phosphorus Dendrimer Enhances Photodynamic Activity of

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Cationic phosphorus dendrimer enhances photodynamic activity of rose bengal against basal cell carcinoma cell lines Monika Dabrzalska, Anna Janaszewska, Maria Zablocka, Serge Mignani, Jean Pierre Majoral, and Barbara Klajnert-Maculewicz Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00108 • Publication Date (Web): 28 Mar 2017 Downloaded from http://pubs.acs.org on March 31, 2017

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Molecular Pharmaceutics

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Cationic

phosphorus

dendrimer

enhances

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photodynamic activity of rose bengal against basal

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cell carcinoma cell lines

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Monika Dabrzalska1, Anna Janaszewska1, Maria Zablocka2, Serge Mignani3, Jean Pierre

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Majoral4,5, Barbara Klajnert-Maculewicz1,6*

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1

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University of Lodz, Pomorska 141/143, 90-236 Lodz, Poland

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2

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112, 90-363 Lodz, Poland

Department of General Biophysics, Faculty of Biology and Environmental Protection,

Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza

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Chimie et de Biochimie pharmacologiques et toxicologique, 45 rue des Saints Pères, 75006

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Paris, France

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Laboratoire de Chimie de Coordination CNRS, 205 route de Narbonne, 31077 Toulouse, France

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Université de Toulouse, UPS, INPT, 31077, Toulouse Cedex 4, France

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Leibniz-Institut für Polymerforschung Dresden e.V., Hohe Strasse 6, 01069 Dresden, Germany

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* Corresponding author: Barbara Klajnert-Maculewicz, email: [email protected]

Université Paris Descartes, PRES Sorbonne Paris Cité, CNRS UMR 860, Laboratoire de

ACS Paragon Plus Environment

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KEYWORDS: phosphorus dendrimer, rose bengal, photosensitizer, photodynamic therapy,

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basal cell carcinoma, phototoxicity.

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ABSTRACT

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In the last couple of decades, photodynamic therapy emerged as a useful tool in the treatment of

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basal cell carcinoma. However, it still meets limitations due to unfavorable properties of

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photosensitizers, such as poor solubility or lack of selectivity. Dendrimers - polymers widely

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studied in biomedical field - may play a role as photosensitizer carriers and improve the efficacy

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of photodynamic treatment. Here, we describe the evaluation of an electrostatic complex of

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cationic phosphorus dendrimer and rose bengal in such aspects as singlet oxygen production,

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cellular uptake and phototoxicity against three basal cell carcinoma cell lines. Rose bengal-

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cationic dendrimer complex in molar ratio 5:1 was compared to free rose bengal. Obtained

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results showed that the singlet oxygen production in aqueous medium was significantly higher

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for the complex than for free rose bengal. The cellular uptake of the complex was 2-7-fold higher

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compared to a free photosensitizer. Importantly, rose bengal, rose bengal-dendrimer complex and

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dendrimer itself showed no dark toxicity against all three cell lines. Moreover, we observed that

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phototoxicity of the complex was remarkably enhanced presumably due to high cellular uptake.

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Based on the obtained results we conclude that rose bengal-cationic dendrimer complex has a

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potential in photodynamic treatment of basal cell carcinoma.

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1. INTRODUCTION

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Basal cell carcinoma (BCC) is currently the most common type of skin cancer and has the

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highest incidence among all type of cancers in Caucasians.1 The treatment of BCC cancer

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is often based on surgical excision, although other therapies, such as photodynamic therapy

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(PDT), may be applied as well.2 As PDT is rather a topical treatment method, it seems to be a

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useful tool to treat topical diseases such as skin cancer, especially BCC tumors characterized by

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very low rate of metastasis. Particularly, PDT offers excellent cosmetic outcomes.3-5

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Photodynamic therapy has emerged as a treatment method nearly 40 years ago. This approach

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is based on three elements: a photosensitizer (PS), molecular oxygen and visible light. The

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mechanism of a photodynamic effect relies on a photosensitizer, which absorbs the energy after

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illumination with a specific wavelength of visible light. This cause the excitation of PS from

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the ground state (S0) to the first excited singlet state (S1). The excited photosensitizer can lose

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the energy by fluorescence emission, conversion into heat or via intersystem crossing forming

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long-lived excited triplet state (T1). A photosensitizer in an excited triplet state can react with

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other molecules via two types of reactions. A Type I mechanism is based on reactions of

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photosensitizer with surrounding substrates producing radicals and reactive oxygen species

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(ROS). A Type II mechanism involves direct energy transfer between photosensitizer and the

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molecular oxygen in ground state generating highly reactive singlet oxygen. Both types of

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reactions lead to the oxidative stress and a cascade of reactions that ultimately cause cell death

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(Scheme 1).6


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Scheme 1. Schematic representation of PDT mechanisms.

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Although many compounds have been identified as photoactive, there are still few

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photosensitizers approved in clinics. Unfortunately, the photodynamic therapy has a number of

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limitations including poor PS solubility, lack of selectivity and prolonged sensitivity to light.

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Hence, there is a need to improve the efficacy of photodynamic treatment.7 The challenge may

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be achieved by designing drug delivery systems for photosensitizers to overcome above

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mentioned disadvantages. Many different nanoparticles have been proposed as PS delivery

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systems, e.g. liposomes,8,9 polymeric nanoparticles10 or silica nanoparticles.11 Among many

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types of nanosystems, dendrimers have been proven to be promising platforms for drug delivery,

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especially because of their regular, hyperbranched architecture, which specifically enables

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several different ways to attach drug molecules to a carrier.12-14 PS molecules can be attached

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either to the internal or surface groups. In addition, PS can be located at the core of the


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dendrimer, or it is possible to encapsulate PS molecules in internal cavities between dendrimer

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branches.15 The majority of the studies on dendrimers as carriers of photosensitizers involved

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porphyrins and phthalocyanines,16-20 and in one case authors demonstrated enhanced PDT

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efficacy in vivo in comparison to clinically used PS –Photofrin®.21

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Dendrimers may also deliver hydrophilic photosensitizers, such as 5-aminolewulinic acid (5-

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ALA)22 or rose bengal.23 Rose bengal is a xanthene dye exhibiting high quantum yield of singlet

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oxygen generation.24,25 It is a hydrophilic molecule with tendency to aggregate in aqueous

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solutions. Therefore, the cellular uptake of rose bengal is often suppressed.26,27 It has a negative

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impact on the reactive oxygen species generation and a cytotoxic effect caused by rose bengal.

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Complexing rose bengal molecules with a dendrimer may significantly prevent the aggregation

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of PS molecules. Moreover, an effective delivery of rose bengal through the cell membrane may

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be enhanced by the use of a dendrimer as a nanocarrier of rose bengal molecules.

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Recently we have prepared and characterized a dendrimer-photosensitizer complex using a

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phosphorus dendrimer possessing cationic charges (1cat) and rose bengal (RB). Studies on the

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interactions between RB and 1cat using Fourier transform infrared spectroscopy (FTIR)

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demonstrated that 1cat formed a stable complex with RB molecules due to electrostatic

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interactions. The stoichiometry of the complex RB:1cat was determined to be approximately 7:1.

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We concluded that the cationic phosphorus dendrimer might be a promising candidate as a

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carrier for RB.28,29 Therefore, it encouraged us to further investigation of the system in vitro.

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The objective of this present study was to evaluate photodynamic activity of RB-1cat

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dendrimer complex. We checked the ability of the complex to produce singlet oxygen in aqueous

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environment and we compared a cellular uptake and phototoxicity of a free photosensitizer and

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of the RB-1cat complex. As a biological model for the studies, we chose basal cell carcinoma


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cell lines. We used three long-term, murine basal cell carcinoma cell lines (ASZ, BSZ and CSZ)

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that resemble the genetic features of human basal cell nevus syndrome and basal cell carcinoma

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tumors.30

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2. EXPERIMENTAL SECTION

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2.1. Materials

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Phosphorus dendrimer of the third generation possessing 48 ammonium surface groups

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(referred as 1cat) was synthesized in the Laboratoire de Chimie de Coordination du CNRS.31

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Rose bengal, Fetal Bovine Serum, Penicillin-Streptomycin solution, Trypsin-EDTA solution and

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MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) were purchased from

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Sigma-Aldrich. Dulbecco’s Phosphate Buffered Saline with no calcium and no magnesium

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(DPBS) was purchased from Biowest. 154 CF culture medium was obtained from Gibco®.

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Chelex® 100 Resin was obtained from Bio-Rad. Singlet Oxygen Sensor Green (SOSG) and

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2’,7’- dichlorodihydrofluorescein diacetate (H2DCFDA) were purchased from Molecular

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Probes™. SYTOX® Dead Cell Stain Kit and Trypan Blue Stain were obtained from

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Invitrogen™. Dimethyl Sulphoxide (DMSO) was purchased from POCH (Poland). Annexin V-

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fluorescein isothiocyanate (FITC) and binding buffer were obtained from BD Biosciences. The

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structure of rose bengal (RB), 1cat dendrimer and schematic structure of the dendrimer is

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depicted in Scheme 2.


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Scheme 2. Structures of the components used in this study: a) chemical structure of rose bengal

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(RB); b) schematic structure of the cationic phosphorus dendrimer of third generation (1cat); c)

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chemical structure of 1cat dendrimer.

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2.2. Methods

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2.2.1. Singlet Oxygen (1O2) generation in aqueous medium

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To determine singlet oxygen production we used Singlet Oxygen Sensor Green reagent

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(SOSG). SOSG is a commercially available, cell impermeable, fluorescent probe that is highly

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selective for 1O2. SOSG was used in 1 µM concentration. All solutions of RB and RB-1cat

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complex and 1cat were prepared in 10 mM DPBS buffer. Three concentrations of RB were used:

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0.1, 0.25 and 0.5 µM. A molar ratio RB:1cat was 5:1. Respectively, the dendrimer concentrations

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in a complex were 0.02, 0.05 and 0.1 µM. After sample preparation, 100 uL of solutions were

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transferred into 96-well black plate. All measurements were recorded with fluorescence


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microplate reader (Fluoroscan Ascent FL). The excitation wavelength was 500 nm and the

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excitation wavelength was 538 nm. Samples were shaken prior to every measurement. The first

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measurement was recorded with no SOSG probe in order to determine whether RB or RB-1cat

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emits fluorescence in this region. Both, RB and RB-1cat did not show any emission upon

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excitation at 500 nm. Following the first measurement, SOSG was added to each well and the

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fluorescence of SOSG without irradiation was checked. Next, the plate were immediately placed

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under the Q.Light Pro Unit lamp equipped with a filter emitting visible light from 385 nm to 780

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nm. All samples were irradiated and the fluorescence was measured in the course of irradiation

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time, from 1 to 60 minutes.

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2.2.2. Cell culture

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Three murine basal cell carcinoma lines were kindly provided by Dr. Ervin Epstein (Children’s

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Oakland Research Institute). ASZ, BSZ and CSZ cells were cultured in 154-CF mediumwith

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antibiotics, 0.05 mM calcium and 2% chelexed, heat-inactivated FBS. Cells were maintained at

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37°C in a humidified incubator and under a 5% CO2 atmosphere. After harvesting, cells were

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counted using trypan blue exclusion assay for further experiments (Countess Automated Cell

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Counter, Invitrogen). For harvesting the cells a 0.25% (w/v) trypsin–0.03% (w/v) EDTA solution

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was used.

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2.2.3. Cellular uptake

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ASZ, BSZ and CSZ cell lines were seeded in 24-well plates and incubated for 24 hours in a

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humified incubator under 5% CO2 atmosphere. Next, RB (10 µM) and RB-1cat were added to

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the cells in 154 CF medium. The cells were incubated with the compounds up to 7 hours.

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Following the incubation the compounds were discarded and cells were washed with DPBS

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buffer. Next, the cells were detached using Trypsin-EDTA solution. Then, fresh culture medium


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was added to the cells and the samples were gently mixed and collected for measurements. For

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estimating the cellular uptake, the fluorescence of the samples was measured using flow

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cytometry (LSRII, Becton Dickinson). The excitation and the emission filters were 520 nm and

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570 nm, respectively.

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2.2.4. Photodynamic activity of RB and RB-1cat complex in vitro

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To measure cytotoxicity cells were seeded in 96-well plates at density 3 × 104 cells well−1.

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After 24h incubation, the culture medium was aspirated from the wells. Then, 100 µL of free

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photosensitizer (RB) or complex (RB-1cat) were added to the cells. All components were

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prepared in fresh culture medium. RB was used at concentrations of 0.1, 0.25, 0.5 and 0.75 µM.

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The molar ratio RB:1cat was 5:1, therefore the dendrimer was used at concentrations of 0.02,

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0.05, 0.1 and 0.15 µM. The control cells were untreated, instead fresh culture medium was

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added. Cells were incubated with components for 5 hours in 37°C. Then, the medium containing

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tested components was replaced with fresh DPBS buffer and cells were irradiated with visible

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light using a lamp emitting broad spectrum light equipped with a filter (Q.Light Pro Unit,

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Q.Products AG, Switzerland). The filter used for photosensitizing RB emits visible light from

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385 nm to 780 nm. The time of irradiation was 30 minutes. Immediately after irradiation DPBS

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buffer was replaced with fresh culture medium and cells were incubated for 24 hours as post

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PDT incubation. Then, cell viability was measured using MTT assay. MTT was added to the

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wells at a concentration of 0.5 mg/mL and incubated in 37°C for 3 hours. The formazan crystals

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were dissolved with DMSO and the absorbance was read at 570 nm on the PowerWave HT

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Microplate Spectrophotometer (BioTek, USA). Also, the dark toxicity (without irradiation) of

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RB, RB-1cat and 1cat was checked. The protocol of these experiments was identical but in this

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case cells were not irradiated.


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2.2.5. Detection of apoptotic and necrotic cells after the irradiation

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Annexin V-Fluorescein Isothiocyanate (FITC)/Sytox staining was used to determine apoptotic

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and necrotic cells. The double staining method allows detecting apoptotic cells in which

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phosphatydylserine is translocated to outer layer of the plasma membrane. Annexin V possesses

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high affinity to phosphatydylserine. Apoptotic cells stained with Annexin V-FITC exclude

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Sytox, and necrotic cells are permeable to Sytox. Thus, the double staining allows differentiating

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between viable, apoptotic and necrotic cell populations. Cells were seeded in 12-well plates at

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the density of 2.5 x 105 cells/well and incubated for 24 hours. Then, RB and RB-1cat were

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added. The concentration of RB and 1cat was 0.25 µM and 0.05 µM, respectively. After 5 hours

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incubation, cells were irradiated as it was described in the previous experiments. Next, cells were

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washed with DPBS, detached and centrifuged at 2500 rpm. Then cells were suspended in 200 µL

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of binding buffer (delivered by the producer) containing 4 µL of Annexin V-FITC and 0.2 µL of

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Sytox. Samples were incubated for 15 min at 4°C in dark. Fluorescence intensity was measured

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with a Becton Dickinson LSR II flow cytometer. Apoptotic and necrotic cells were used as

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compensation controls. Apoptosis was induced using 80 µM camptothecin and necrosis was

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induced using frozen ethanol (data not shown).

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2.2.6. Reactive oxygen species

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We used H2DCFDA molecular probe (2’, 7’- dichlorodihydrofluorescein diacetate, Molecular

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Probes™) in order to investigate the intracellular production of ROS. Cells were seeded in 96-

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well plates at density 3 × 104 cells well−1and solutions of RB, RB1-cat and 1cat were added. The

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concentration of free and complexated RB was 0.25 µM and 1cat concentration was 0.05 µM.

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DPBS was used as a control. Cells were incubated with components for 5 hours at 37°C in a

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humified atmosphere containing 5% CO2. After washing the cells with DPBS, solution of 2 µM


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H2DCFDA in DPBS was added to each wells and plates were incubated for 15 min in dark.

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Next, the cells were washed with DPBS and 100 µL of DPBS was added. 485/530 nm

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fluorescence of DCF was measured as a background using BIOTEK PowerWave HT Microplate

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reader (Biotek). Then cells were irradiated for 30 min and the fluorescence was measured

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immediately after irradiation. Every measurement was corrected by subtraction of the

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background fluorescence intensity.

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2.2.7. Statistical analysis

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Statistical analysis was performed using Sigma Plot version 12.5 (Systat Software, USA).

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Results are presented as a mean ± SD of three independent experiments. One-way analysis of

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variance (ANOVA) was used for testing statistical differences between groups followed by post-

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hoc Tukey test. A P-value of

Cationic Phosphorus Dendrimer Enhances Photodynamic Activity of

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