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Phosphorus dendrimers and photodynamic therapy. Spectroscopic studies on two dendrimer-photosensitizer complexes: Cationic phosphorus dendrimer with rose bengal and anionic phosphorus dendrimer with methylene blue
International Journal of Pharmaceutics 492 (2015) 266–274
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Phosphorus dendrimers and photodynamic therapy. Spectroscopic studies on two dendrimer-photosensitizer complexes: Cationic phosphorus dendrimer with rose bengal and anionic phosphorus dendrimer with methylene blue Monika Dabrzalskaa , Maria Zablockab , Serge Mignanic , Jean Pierre Majorald,e,* , Barbara Klajnert-Maculewicza,f a
Department of General Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, Pomorska 141/143, 90-236 Lodz, Poland Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland c Université Paris Descartes, PRES Sorbonne Paris Cité, CNRS UMR 860, Laboratoire de Chimie et de Biochimie pharmacologiques et toxicologique, 45 rue des Saints Pères, 75006 Paris, France d Laboratoire de Chimie de Coordination CNRS, 205 route de Narbonne, 31077 Toulouse, France e Université de Toulouse, UPS, INPT, 31077, Toulouse Cedex 4, France f Leibniz-Institut für Polymerforschung Dresden e.V., Hohe Strasse 6, 01069 Dresden, Germany b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 May 2015 Received in revised form 9 June 2015 Accepted 12 June 2015 Available online 24 June 2015
Dendrimers due to their unique architecture may play an important role in drug delivery systems including chemotherapy, gene therapy and recently, photodynamic therapy as well. We investigated two dendrimer-photosensitizer systems in context of potential use of these systems in photodynamic therapy. The mixtures of an anionic phosphorus dendrimer of the second generation and methylene blue were studied by UV–vis spectroscopy while that of a cationic phosphorus dendrimer (third generation) and rose bengal were investigated by spectrofluorimetric methods. Spectroscopic analysis of these two systems revealed the formation of dendrimer-photosensitizer complexes via electrostatic interactions as well as p stacking. The stoichiometry of the rose bengal-cationic dendrimer complex was estimated to be 7:1 and 9:1 for the methylene blue-anionic dendrimer complex. The results suggest that these polyanionic or polycationic phosphorus dendrimers can be promising candidates as carriers in photodynamic therapy. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Phosphorus dendrimer Photosensitizer Rose bengal Methylene blue Photodynamic therapy Drug delivery system
1. Introduction Dendrimers are well-defined hyperbranched polymers characterized by low polydispersity. The structure of a dendrimer consists of a core molecule, branches, internal cavities and many terminal groups. Due to such an architecture, these polymers are attractive carriers of drugs, imaging or transfection agents and can be used for diverse ways of administration (Klajnert and Bryszewska, 2001; El Kazzouli et al., 2012; Mignani et al., 2013a,b).
Abbreviations: PDT, photodynamic therapy; ROS, reactive oxygen species; PS, photosensitizer; PAMAM, polyamidoamine dendrimer; PPI, polypropyleneimine dendrimer; PEG, polyethylene glycol; PpIX, protoporphyrin IX; ALA, aminolevulinic acid; MB, methylene blue; RB, rose bengal; 1cat, cationic phosphorus dendrimer of generation 3; 1an, anionic phosphorus dendrimer of generation 2. * Corresponding author. Fax: +33 561 553 003. E-mail address: [email protected] (J.P. Majoral). http://dx.doi.org/10.1016/j.ijpharm.2015.06.014 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
Photodynamic therapy (PDT) is an alternative cancer treatment method whereby cancer cells are destroyed by reactive oxygen species (ROS) and singlet oxygen (1O2) generated via a photodynamic effect. The photodynamic effect is a result of use of photosensitizer (PS) which under the influence of a specific wavelength of visible light induces a cascade of reactions leading to oxidative stress and cell death (Dolmans et al., 2003). However, PDT has limits similar to conventional chemotherapy. It involves, among others, poor PS selectivity, long-lasting skin sensitivity to light and rapid PS destruction. It is related to inappropriate biodistribution of photosensitizers (O’Connor et al., 2009). Therefore, to overcome these problems the challenge of modern PDT is creation of more selective and effective PS. This can be achieved by use of drug delivery systems. Recently dendrimers have gained some attention as promising transport systems for PS that potentially improve photodynamic therapy efficiency (Klajnert et al., 2012).
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PS molecules can be covalently bound to the core of the dendrimer, to the interior of the molecule or attached to the terminal groups. The presence of internal cavities allows for encapsulation of PS molecules in the dendrimer (Ihre et al., 2002; Patri et al., 2005; Bhadra et al., 2003). In several studies dendrimers were used as photosensitizer carriers. Polyamidoamine (PAMAM) and polypropyleneimine (PPI) dendrimers modified with polyethylene glycol (PEG) have been reported to be efficient in encapsulation of rose bengal and protoporphyrin IX (PpIX). PpIX encapsulated in PEG-PPI dendrimers revealed higher phototoxicity in comparison with a free photosensitizer (Kojima et al., 2007). A different approach to use dendrimers in PDT is based on the synthesis of photosensitizer dendrimers possessing porphyrin or phtalocyanine molecules in the core. Dendritic photosensitizers were also incorporated into polyion complex micelles through PEG-polyelectrolyte block copolymers. Polymeric micelles encapsulating dendrimer phthalocyanine showed high phytotoxicity in vitro and in vivo (Herlambang et al., 2011; Nishiyama et al., 2009; Zhang et al., 2003). Dendrimer conjugates containing 18 molecules of aminolevulinic acid (ALA) have been reported to efficiently transport ALA to tumor cells and induce porphyrin production in vitro (Battah et al., 2007). As mentioned above, several types of dendrimers, including PPI and PAMAM, have been studied in the context of use in PDT. In this paper, we propose a novel approach to use phosphorus dendrimers as potential photosensitizer carriers. Phosphorus dendrimers exhibit interesting biological properties that make them promising drug carriers, transfection, anti-prion, imaging (Caminade et al., 2010), anti-inflammatory (Poupot et al., 2006) or anti-tumoral agents (El Brahmi et al., 2015). However, phosphorus dendrimers have not been studied as potential photosensitizer carriers. In designing new drug delivery systems it is crucial to determine the potential interactions between drug molecules and nanocarriers. In this paper we present studies on the interaction of two dendrimer-photosensitizer systems in order to evaluate whether phosphorus dendrimers can be used as carriers in PDT. We chose two phosphorus dendrimers that differ in generation and electrostatic charge and two photosensitizers: methylene blue (MB) and rose bengal (RB). Methylene blue is a monocationic phenothiazine dye with potential to be a promising
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photosensitizer (Tardivo et al., 2005). Rose bengal, on the other hand, is an dianionic xanthene dye that is also reported to possess good PDT efficacy (Wachter et al., 2003). Both PSs upon irradiation generate singlet oxygen (Tardivo et al., 2005; Wachter et al., 2003). The aim of the study was therefore to check whether dendrimers are capable to form complexes with PSs possessing opposite charges using absorption and fluorescence spectroscopy in the case of methylene blue and rose bengal, respectively. The results allow estimating the potential role of phosphorus dendrimers in PDT as carriers of photosensitizers. 2. Materials and methods 2.1. Materials Two phosphorus dendrimers were used: cationic phosphorus dendrimer of generation 3 (MW = 15150.25 g/mol) possessing 48 ammonium terminal groups (1cat), and anionic phosphorus dendrimer (1an) of generation 2 (MW = 7851 g/mol) with 24 carboxyl terminal groups. Both dendrimers possess cyclotriphosphazene core. Dendrimers were synthesized in Laboratoire de Chimie de Coordination du CNRS, Toulouse, France. Rose bengal (RB) and methylene blue (MB) were purchased from Sigma–Aldrich. All solutions were made in phosphate-buffered saline (PBS), pH 7.4 using distilled water from Mili-Q system (Millipore). Scheme 1 depicts chemical characterization of the compounds used in this study. 2.2. Methods 2.2.1. UV–vis spectroscopy: study on the interaction between anionic phosphorus dendrimer (1an) and methylene blue (MB) Absorption spectra were recorded on a Jasco V-650 spectrophotometer. All measurements were performed in 100 mM PBS pH 7.4, at room temperature. Spectra were recorded in a wavelength range from 550 nm to 700 nm. Optical path length was 1 cm. PBS was used as a reference for all measurements. In titration experiments methylene blue (MB) was used at constant concentration of 10 mM. The MB solution was titrated with anionic dendrimer using different stocks of dendrimer in order to maintain specific molar ratios (1–20) of MB molecules per
Scheme 1. Chemical structures of dendrimers and photosensitizers used in this study. (A) anionic phosphorus dendrimer of the second generation—1an; (B) cationic phosphorus dendrimer of the third generation—1cat; (C) rose bengal; (D) methylene blue; (E) schematic structure of the second generation of phosphorus dendrimer; (F) schematic structure of the third generation of phosphorus dendrimer.
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dendrimer at each titration point. Dendrimer did not absorb light in this region. Next, the influence of sodium salt concentration on the interaction between MB and the dendrimer was checked. In this experiment MB:1an solutions at molar ratios equal to 3, 5, 7 and 10, were prepared in PBS buffer. Then NaCl was added. Final concentrations of NaCl were equal to 0.01; 0.1; 1; 10 and 100 mM. Additionally, the time-dependent stability of MB:1an complex was investigated. MB:1an complexes at molar ratio 5:1 and 10:1 were chosen for this experiment. All spectrophotometric measurements were repeated three times. 2.2.2. Spectrofluorimetric study on the interaction between cationic phosphorus dendrimer (1cat) and rose bengal (RB) Fluorescence experiments were performed using a PerkinElmer LS-50B spectrofluorimeter. All measurements were performed in 100 mM PBS, pH 7.4, at room temperature. The excitation wavelength was set to 525 nm and spectra were collected in a wavelength range from 540 nm to 650 nm. Excitation and emission slits were 5 nm and 7 nm, respectively. Dendrimer did not show any emission in this wavelength range. Rose bengal (RB) at concentration of 1 mM was titrated with cationic dendrimer in a concentration range from 0.05 mM to 1 mM using different stock solutions to maintain molar ratio of RB molecules per dendrimer from 1 to 20. Each experiment was repeated three times. The influence of sodium salt concentration was checked by recording fluorescence spectra of RB:1cat complexes at molar ratios 3, 5, 7 and 10, as a function of salt concentration (0.01, 0.1, 1, 10 and 100 mM). Additionally, RB was titrated with dendrimer in PBS buffer containing 100 mM NaCl. Next, time stability of 5:1 (RB:1cat) complex was studied. 3. Results 3.1. UV–vis spectroscopy: study on the interaction between anionic phosphorus dendrimer (1an) and methylene blue (MB). Firstly we checked whether the dendrimer is able to interact with methylene blue. Fig. 1 shows MB absorption spectra upon addition of the dendrimer. Solution of MB exhibits a strong band at
664 nm and a shoulder at approximately 610 nm. These bands are assigned to the absorption of monomeric and dimeric form of methylene blue, respectively (Bergman and O'Konski, 1963). MB in its monomeric and dimeric form is depicted in Scheme 2. Dendrimer was added in different concentrations to maintain specific molar ratios of the compounds. The first titration point was at a MB:1an molar ratio equal to 20 and the last one was at 1:1 MB:1an ratio. The dendrimer caused significant changes in absorption spectra of methylene blue. Three stages were distinguished during the titration. Firstly, upon addition of the dendrimer (molar ratios MB:1an 20–11) absorption significantly decreased. Secondly, there was a red shift of the main absorption band of MB from 664 nm to 672 nm. Thirdly, in the range of MB:1an molar ratios from 9 to 1 absorption increased. Additionally, in the case of lower MB:1an ratios there was an isosbestic point at 651 nm. Significant influence of the dendrimer on the MB absorption indicates that there is an interaction between the photosensitizer and the dendrimer. The fact that the dimer band decreases together with a red shift and an increase of the monomer band for lower MB:1an ratios suggests that the addition of the dendrimer caused a decrease in the dimeric form of MB. Hence, the red shifted band corresponds to the complex MB-dendrimer. Fig. 2 depicts changes in the position of absorption maximum (lmax) of methylene blue upon addition of the dendrimer. As the concentration of the dendrimer increased the changes in the positions of lmax were more pronounced. The highest band shift was observed for MB:1an 1:1 molar ratio. In order to reveal the changes in MB absorption spectra upon interaction with the dendrimer, a plot of A664/A610 versus MB:1an molar ratio was made (Fig. 3A). MB shows an absorption peak at 664 nm and a shoulder at 610 nm that correspond to monomer and dimer, respectively. MB complexed with dendrimer showed red shifted absorption band, therefore a plot of A672/A664 versus corresponding MB:1an ratio was made to determine the stoichiometry of the complex according to the ratio method (Fig. 3B) (Nandinii and Vishalaksi, 2010). The stoichiometry of the complex was determined from the point where the curve changed its slope. As it can be seen in Fig. 3A the ratio of the monomer band (A664) to dimer band (A610) depends on the MB:1an ratio. At molar ratio
Fig. 1. The absorption spectra of methylene blue (10 mM) upon addition of the anionic dendrimer in a molar ratio (MB:1an ranging from 20 to 1). Dendrimer concentration ranged from 0.5 mM to 10 mM. (A) The first stage: decrease in absorption (MB:1an from 20 to 11); (B) The second stage: red shift of lmax; (C) The third stage: increase in absorption (MB:1an from 9 to (1); a—pure MB spectrum; b—20:1 MB:1an ratio; c—10:1 MB:1an ratio; d—5:1 MB:1an ratio; e—1:1 MB:1an ratio.
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Scheme 2. Structures of the monomeric and the dimeric form of methylene blue (MB).
equal to approx. 10 the A664/A610 parameter achieved the lowest value and then it progressively increased as the MB:1an ratio decreased. It clearly indicates the interaction between methylene blue and dendrimer molecules. The A664/A610 ratio significantly increased in the case of MB:1an molar ratios above 10. Higher values of the A664/A610 ratio indicate that the fraction of MB dimers decreased upon the addition of the dendrimer. For lower dendrimer concentrations the A664/A610 values are low and stay at the plateau. Though, above the dendrimer concentration of 1 mM (MB:1an molar ratio 10:1), there are enough dendrimer molecules to easily interact with methylene blue molecules and therefore the MB dimer formation is suppressed by the formation of MB:1an complexes. Figure 3B shows changes in the ratio of A672–A664. It can be observed that A672/A664 values also increased for lower MB:1an ratios. Such changes correspond to the red shift and increase of intensity for the monomer band at 672 nm. Two different slops of the lines were observed that crossed when MB:1an ratio was approx. 9–10. Therefore, the stoichiometry of MB:1an complex was determined to be either 9:1 or 10:1. Four different MB:1an molar ratios (3, 5, 7 and 10) were chosen for the investigation of the character of the interactions between the photosensitizer and the anionic dendrimer. Absorption spectra of all solutions were measured as a function of NaCl concentration ranging from 0 mM to 100 mM. For all tested systems the absorbance slightly decreased with increasing salt concentration. However, the detailed analysis of ratios A664/A610 and A672/A664 revealed no effect of a salt concentration on interactions between MB and the dendrimer (Fig. 4A and B).
To determinate time stability of the complexes, MB:1an 5:1 and 10:1 complexes were chosen to study over the course of time. Plots of A664/A610 and A672/A664 versus time are depicted in Fig. 5. The absorbance of the complexes was measured up to 24 h. The lack of changes indicates that both complexes were stable for 24 h.
Fig. 2. Changes in the position of lmax of methylene blue absorption spectra upon addition of the dendrimer at different MB:1an molar ratio. MB = 10 mM.
Fig. 3. Changes in MB absorption ratios A664/A610 (A) and A672/A664 (B) as a function of MB:1an molar ratio.
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Fig. 6. Fluorescence spectra of RB (1 mM) upon addition of cationic dendrimer in molar ratio of RB molecules per dendrimer (RB:1cat ranging from 20 to 1). Concentration of the dendrimer ranged from 0.05 to 1 mM. (A) Rose bengal spectrum; b—RB:1cat in 20:1 molar ratio; c—RB:1cat in 10:1 molar ratio; d— RB:1cat in 5:1 molar ratio; e—RB:1cat in 3:1 molar ratio; f —RB:1cat in 2:1 molar ratio; (B) RB:1cat in 1:1 molar ratio.
Fig. 4. (A) A672/A664 ratio as a function of NaCl concentration; (B) A664/A610 as a function of NaCl concentration.
However, A664/A610 and A672/A664 values are lower for MB:1an 10:1 complex than for MB:1an 5:1 complex. Though, the fraction of MB dimers is lower in the case of MB:1an 5:1 complex. 3.2. Spectrofluorimetric study on the interaction between cationic phosphorus dendrimer (1cat) and rose bengal (RB). The interaction between rose bengal and the cationic dendrimer was investigated using spectrofluorimetric method due to sensitivity of this technique and a strong fluorescence signal given by rose bengal. Similarly as it was done in the case of spectrophotometric experiments, rose bengal was titrated with the dendrimer, maintaining specific RB:1cat molar ratios at each titration point. Fig. 6 shows fluorescence spectra of RB upon addition of the cationic dendrimer. With increasing concentration of the dendrimer, quenching of RB fluorescence was observed. Then a significant red shift from 564 nm to 578 nm occurred and the fluorescence intensity started
Fig. 7. Stoichiometry of the RB:1cat complex.
to rise again. Such an effect indicates that RB forms complexes with the cationic dendrimer. Different spectral characteristics were observed for pure RB and for the formed complex. In order to estimate the stoichiometry of the RB:1cat complex, the fluorescence intensity for two fluorescence maxima (F564/F578) was calculated (Fig. 7). The stoichiometry was determined to be 7:1 (RB:1cat).
Fig. 5. Time stability of MB:1an 5:1 complex and MB:1an 10:1 complex.
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Fig. 8. Fluorescence spectra of RB:1cat complexes (RB:1cat = 3, 5, 7 and 10) in absence and presence of different concentrations of NaCl (0.01; 0.1; 1; 10 and 100 mM).
For molar ratios from 10 to 20 F564/F578 values were on the same level as for free RB solution. For molar ratios less than 10, the values of F564/F578 decreased significantly and then reached a plateau. Next, the NaCl effect on the interaction of RB with cationic dendrimer was studied. Fluorescence spectra of four complexes (RB:1cat = 3, 5, 7 and 10) were measured in presence of NaCl (0.01 mM–100 mM). Fig. 8 shows changes in fluorescence spectra caused by increasing ionic strength.
Fig. 9. Changes in RB fluorescence ratio F564/F578 in PBS and in PBS containing additional 100 mM of NaCl.
It was found that upon addition of NaCl the fluorescence maximum shifted to 580 nm. However, only slight changes were observed in the case of RB:1cat 3:1. The lowest intensity was observed in presence of 100 mM of NaCl. For 5:1 complex, the fluorescence intensity was found to increase progressively upon increasing NaCl concentration, except for 100 mM of NaCl. In this case fluorescence signal was significantly diminished. Interestingly, for 7:1 complex in presence of 100 mM NaCl the fluorescence intensity significantly increased and the fluorescence maximum was localized at 564 nm which is characteristic for free RB molecules. In the case of 10:1 RB:1cat solution, the fluorescence intensity was very low in presence of 0.1 mM but in presence of 100 mM of NaCl the intensity was only slightly lower than that in absence of NaCl. For all samples (RB:1cat 10:1) the fluorescence maximum was at 564 nm. The influence of NaCl on the interaction between RB and cationic dendrimer is presented in Fig. 9 which depicts changes in F564/F578 ratio in PBS and in PBS containing 100 mM NaCl. Fluorimetric titration of RB with the cationic dendrimer in PBS containing 100 mM NaCl showed the same effect as the titrations performed in PBS. However, high salt concentration did influence on the rate of complex formation. In the presence of NaCl complexes were formed slower than in PBS buffer (Fig. 9). Interestingly, the stoichiometry of RB:1cat complex upon addition of 100 mM NaCl to PBS decreased from 7:1 to 5:1.
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Fig. 10. (A) RB:1cat 5:1 fluorescence decay in time stability experiment; (B) decrease of the maximum fluorescence intensity of RB:1cat complex (5:1) in the course of time.
Study on the time stability of RB:1cat complex (5:1) revealed very fast decay of fluorescence signal (Fig. 10A). However, the fluorescence maximum did not shift to wavelength specific for free RB, so the complex did not release free RB molecules. The maximum fluorescence intensity of RB:1cat complex decreased within 4 h (Fig. 10B) but it is clear that such an effect was caused by photobleaching. 4. Discussion Methylene blue (MB) and rose bengal (RB) are dyes with promising applications in photodynamic therapy (PDT). Thus we tested potential uses of polyanionic and polycationic phosphorus dendrimers as carriers of MB and RB. In order to design good therapeutic complexes it is important to check if their potential as carriers allows the binding of the guest molecules, the characterization of the type of the interaction and the determination of stoichiometry of such complexes. In this paper we applied spectroscopic methods to investigate dendrimer-photosensitizer complexes. As far as we know this is the first study on the complexation of MB and RB with phosphorus dendrimers in the context to use such complexes in PDT. Two separate dendrimer-photosensitizer systems were chosen on the basis of opposite charges: an anionic phosphorus dendrimer (1an) with a cationic dye—MB and a cationic phosphorus dendrimer (1cat) with an anionic dye—RB. UV-VIS spectroscopy study on the interaction between the anionic dendrimer and MB showed substantial influence of the dendrimer on MB absorption spectrum. There were three different effects in MB absorption upon addition of dendrimer. Both, monomer and dimer bands showed significant changes: MB momoner band was firstly diminished by the dendrimer, and then, as the MB:1an molar ratio was diminishing, intensity of monomer band increased. However, the dimer band constantly decreased. These two effects resulted in an isosbestic point characteristic to lower MB:1an ratios. According to the literature, the interaction of cationic dyes with polymers often leads to aggregation and a shift of the absorption band of the dye that confirms the formation of dye-polymer complexes (Fradj et al., 2014; Gadde et al., 2009). An absorption shift when accompanied with change of colour is called metachromasy or metachromatic effect. It is a common effect related to MB adsorption on anionic
polymers or clay minerals (Schoonheydt and Heughebaert, 1972; Otsuki and Adachi, 1993). Investigation of adsorption of MB on Starburst dendrimer pointed out the formation of dimers and higher-order aggregates (dependent on dendrimer generation), which were characterized mainly by an absorption decrease and a blue shift (Jockusch et al., 1995). In our experiment we discovered the opposite effect: under the influence of the dendrimer a red shift (about 6 nm) was observed for MB:1an molar ratios lower than 10. Similarly, a slight bathochromic shift was observed in the investigation on binding MB molecules to a single stranded polyribonucleic acid (Hossain et al., 2012). Methylene blue when aggregates can form either so called Haggregates or J-aggregates. According to Kasha’s exciton theory, a blue shift is characteristic for H-aggregates (face-to-face molecular arrangement), whether a red shift corresponds to a formation of Jaggregates (head-to-tail alignment) (Kasha, 1963). However, a J-aggregates band is usually observed above 700 nm, thus we assume that the red shift in our study may be related to MB monomers bound to the dendrimer. The presence of an isosbestic point clearly confirms the formation of MB:1an complexes. Changes in absorption of MB upon addition of dendrimer allowed to determine the stoichiometry of the complex as MB:1an equals to 10:1. In the next experiment no influence of ionic strength on complex formation was observed. This indicates that electrostatic interactions are involved in forming MB:1an complexes, but also that p stacking between the terminal aromatic rings in 1an and in MB are formed. The p–p interactions between aromatic ring of MB and internal aromatic rings of the dendrimer 1an seems to be less probable or more negligible due to the strong hydrophobicity of the interior of 1an and even if, in general, the second generation of a dendrimer has a relatively open structure with internal cavities available for encapsulation of dye molecules as it was reported for PAMAM dendrimers with a hydrophilic interior (Klajnert and Bryszewska, 2001). Taking into account the complexity of dendrimer structures, more than one type of interactions involved in complexation are observed for many types of molecules combined with dendrimers (Gupta et al., 2007; Zhang et al., 2014; D'Emanuele and Attwood, 2005). Time stability study showed that both, MB:1an 5:1 and MB:1an 10:1 complexes were stable over the time. Nevertheless, the MB:1an 5:1 complex presented higher values of the ratios A664/A610 and A672/A664. Though, the fraction of MB dimers was lower in the case of MB:1an 5:1 complex. It is an important result taking into account a potential therapeutic use. In the case of the interaction between rose bengal and a cationic phosphorus dendrimer we obtained similar results to those of MB:1an interactions. Upon addition of cationic dendrimer the fluorescence intensity was almost completely quenched and afterwards for lower RB:1cat ratios fluorescence maximum of RB moved to longer wavelengths that was accompanied by an increase of a fluorescence intensity. The similar red shift in absorption spectrum of RB was earlier obtained in the study on RB interaction with cyclodextrins and was confirmed as an effect of forming complexes with cyclodextrins (Fini et al., 2004). In our study, we also concluded that the change in fluorescence maximum position confirmed the formation of RB:1cat complexes. The stoichiometry of the complexation was determined to be RB:1cat 7:1. In marked contrast with what was observed in the case of MB:1an interactions, RB:1cat complexes were affected by the NaCl concentration. For RB:1cat 7:1 complex, 100 mM of NaCl resulted in changing the position of fluorescence maximum back to free RB band, with additional increase of fluorescence. Such a strong effect indicated that high salt concentration caused a release of bound RB molecules from the
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dendrimer. According to earlier studies, RB molecules have a tendency to aggregate at high salt concentration (Fini et al., 2004). In the case of RB:1cat 10:1 the fluorescence band was located at 564 nm—thus it confirmed the results of stoichiometry determination. The results of titration of RB with the dendrimer in presence of high salt concentration (100 mM NaCl) showed lower stoichiometry of the complex and slower rate of complex formation. Thus we assume that the electrostatic forces dominate the character of RB:1cat complexation, that is to say interactions may occur between the carboxylic groups of RB and ammonium groups present at the periphery of the dendrimer. Interestingly, different results were obtained in the case of interaction of RB with PAMAM dendrimers: RB was encapsulated in the cavities of PAMAM dendrimers of generation 2.5 possessing ester terminal groups. 2.5 PAMAM dendrimers were electrostatically neutral (but easily protonated): the spectroscopic study showed that RB was encapsulated in the dendritic box through electrostatic interactions with the hydrophilic interior of the PAMAM dendrimer (Karthikeyan et al., 2011). Our result is though probably connected to dendrimer structure: phosphorus dendrimer of third generation 1cat possess 48 terminal groups and presents more dense surface and a hydrophobic interior and thus, probably RB molecules cannot interact so efficiently with the internal cavities of the dendrimer. RB:1cat 5:1 complex showed fast decay of a fluorescence intensity over the time, though there was no shift of fluorescence band toward shorter wavelength characteristic to free RB molecules. It is possible that strong photobleaching of RB bound to the dendrimer occurred. Theoretically when comparing the number of terminal groups present on anionic and cationic dendrimer, the cationic one should be capable to bind more photosensitizer molecules, because 1cat possesses twice more cationic groups (48) than 1an possess anionic ones. However, MB has a smaller size than RB. Our results showed that 1an dendrimer interacts with MB through electrostatic and p–p interactions and RB:1cat interactions were only due to electrostatic forces. This might be due to the fact that the 1an dendrimer has a more open structure (generation 2) than 1cat (generation 3) and possesses terminal groups favoring p–p interactions. In conclusion our study showed the formation of two complexes of phosphorus dendrimers with photosensitizers: cationic dendrimer with rose bengal and anionic dendrimer with methylene blue. Spectroscopic analysis allowed us to conclude that different forces were responsible for complexation: electrostatic forces and p–p stacking in the case of anionic dendrimer and methylene blue and electrostatic interactions in the case of cationic dendrimer and rose bengal. MB:1an and RB:1cat stoichiometries were estimated to be 9:1 and 7:1, respectively. The MB:1an complex was more stable, while in the case of RB:1cat the complex was prone to a photobleaching effect, although there was no destruction of the RB:1cat complex over the time. Therefore, phosphorus dendrimers used in this study present different behaviors than that of other dendrimers as PAMAM for example, illustrating their own specificity which allows to consider them as promising candidates for carriers in photodynamic therapy. Further investigations in this direction are under active investigation as well as experiments concerning the role of the size of dendrimers (generations 1 to 4; the so-called “dendritic effect”) on their complexing properties. Acknowledgements This study was funded by the project “Phosphorus dendrimers as carriers of photosensitizers in photodynamic therapy and its combination with hyperthermia in in vitro studies” operated
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within the Foundation for Polish Science VENTURES Programme (Project VENTURES number VENTURES/2013-11/3) co-financed by the EU European Regional Development Fund and by the grant HARMONIA “Studying phosphorus dendrimers as systems transporting photosensitizers” no. UMO-2013/08/M/NZ1/00761 supported by National Science Centre. References Battah, S., Balaratnam, S., Casas, A., O'Neill, S., Edwards, C., Batlle, A., Dobbin, P., MacRobert, A.J., 2007. Macromolecular delivery of 5-aminolaevulinic acid for photodynamic therapy using dendrimer conjugates. Mol. Cancer Ther. 6, 876– 885. Bergman, K., O’Konski, C.T., 1963. A spectroscopic study of methylene blue’ monomer, dimer, and complexes with montmorillonite. J. Phys. Chem. 67, 2169– 2177. Bhadra, D., Bhadra, S., Jain, S., Jain, N.K., 2003. A PEGylated dendritic nanoparticulate carrier of fluorouracil. Int. J. Pharm. 257, 111–124. Caminade, A.M., Turrin, C.O., Majoral, J.P., 2010. Biological properties of phosphorus dendrimers. New J. Chem. 34, 1512–1524. D'Emanuele, A., Attwood, D., 2005. Dendrimer–drug interactions. Adv. Drug Deliv. Rev. 57, 2147–2162. Dolmans, D.E., Fukurama, D., Jain, R.K., 2003. Photodynamic therapy for cancer. Nat. Rev. Cancer 3, 380–387. El Brahmi, N., Mignani, S., Caron, J., El Kazzouli, S., Bousmina, M., Caminade, A.M., Cresteil, T., Majoral, J.P., 2015. Investigations on dendrimer space reveal solid and liquid tumor growth-inhibition by original phosphorus-based dendrimers and the corresponding monomers and dendrons with ethacrynic acid motifs. Nanoscale 7, 3915–3922. El Kazzouli, S., El Brahmi, N., Mignani, S., Bousmina, M., Zablocka, M., Majoral, J.P., 2012. From metallodrugs to metallodendrimers for nanotherapy in oncology: a concise overview. Curr. Med. Chem. 19, 4995–5010. Fini, P., Castagnolo, M., Catuccia, L., Cosma, P., Agostiano, A., 2004. Inclusion complexes of Rose Bengal and cyclodextrins. Thermochim. Acta 418, 33–38. Fradj, A.B., Lafi, R., Hamouda, S.B., Gzara, L., Hamzaoui, A.H., Hafiane, A., 2014. Effect of chemical parameters on the interaction between cationicdyes and poly (acrylic acid). J. Photochem. Photobiol. A 284, 49–54. Gadde, S., Batchelor, E.K., Kaifer, A.E., 2009. Controlling the formation of cyanine dye H- and J-aggregates with cucurbituril hosts in the presence of anionic polyelectrolytes. Chem. Eur. J. 15, 6025–6031. Gupta, U., Agashe, H.B., Jain, N.K., 2007. Polypropylene imine dendrimer mediated solubility enhancement: effect of ph and functional groups of hydrophobes. J. Pharm. Pharmaceut. Sci. 10, 358–367. Herlambang, S., Kumagai, M., Nomoto, T., Horie, S., Fukushima, S., Oba, M., Miyazaki, K., Morimoto, Y., Nishiyama, N., Kataoka, K., 2011. Disulfide crosslinked polyion complex micelles encapsulating dendrimer phthalocyanine directed to improved efficiency of photodynamic therapy. J. Control. Release 155, 449–457. Hossain, M., Kabir, A., Kumar, G.S., 2012. Binding of the phenothiazinium dye methylene blue with single stranded polyriboadenylic acid. Dyes Pigments 92, 1376–1383. Ihre, H.R., Padilla De Jesús, O., Szoka Jr., F.C., Fréchet, J.M.J., 2002. Polyester dendritic systems for drug delivery applications: design synthesis, and characterization. Bioconjug. Chem. 13, 443–452. Jockusch, S., Turro, N.J., Tomalia, D.A., 1995. Aggregation of methylene blue adsorbed on starburst dendrimers. Macromolecules 28, 7416–7418. Karthikeyan, K., Babu, A., Kim, S.J., Murugesan, R., Jeyasubramanian, K., 2011. Enhanced photodynamic efficacy and efficient delivery of Rose Bengal using nanostructured poly(amidoamine) dendrimers: potential application in photodynamic therapy of cancer. Cancer Nano. 2, 95–103. Kasha, M., 1963. Energy transfer mechanisms and the molecular exciton model for molecular aggregates. Radiat. Res. 20, 55–71. Klajnert, B., Bryszewska, M., 2001. Dendrimers: properties and applications. Acta Biochim. Pol. 48, 199–208. Klajnert, B., Rozanek, M., Bryszewska, M., 2012. Dendrimers in photodynamic therapy. Curr. Med. Chem. 19, 4903–4912. Kojima, C., Toi, Y., Harada, A., Kono, K., 2007. Preparation of poly(ethylene glycol)attached dendrimers encapsulating photosensitizers for application to photodynamic therapy. Bioconjug. Chem. 18, 663–670. Mignani, S., El Kazzouli, S., Bousmina, M., Majoral, J.P., 2013a. Dendrimer space concept for innovative nanomedicine: a futuristic vision for medicinal chemistry. Prog. Polym. Sci. 38, 993–1008. Mignani, S., El Kazzouli, S., Bousmina, M., Majoral, J.P., 2013b. Expand classical drug administration ways by emerging routes using dendrimer drug delivery systems: a concise overview. Adv. Drug Deliv. Rev. 65, 1316–1330. Nandinii, R., Vishalaksi, B., 2010. A study of interaction of cationic dyes with anionic polyelectrolytes. Spectrochim. Acta Mol. Biomol. Spectros. 75, 14–20. Nishiyama, N., Nakagishi, Y., Morimoto, Y., Lai, P.S., Miyazaki, K., Urano, K., Horie, S., Kumagai, M., Fukushima, S., Cheng, Y., Jang, W.D., Kikuchi, M., Kataoka, K., 2009. Enhanced photodynamic cancer treatment by supramolecular nanocarriers charged with dendrimer phthalocyanine. J. Control. Release 133, 245–251. O’Connor, A.E., Gallagher, W.M., Byrne, A.T., 2009. Porphyrin and nonporphyrin photosensitizers in oncology: preclinical and clinical advances in photodynamic therapy. Photochem. Photobiol. 85, 1053–1074.
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Otsuki, S., Adachi, K., 1993. Metachromasy in polymer films. Changes in the absorption spectrum of methylene blue in nafion films by hydration. Polym. J. 25, 1107–1112. Patri, A.K., Kukowska-Latallo, J.F., Baker Jr., J.R., 2005. Targeted drug delivery with dendrimers: comparison of the release kinetics of covalently conjugated drug and non-covalent drug inclusion complex. Adv. Drug Deliv. Rev. 57, 2203– 2214. Poupot, M., Griffe, L., Marchand, P., Maraval, A., Rolland, O., Martinet, L., L’FaqihiOlive, F.E., Turrin, C.O., Caminade, A.M., Fournié, J.J., Majoral, J.P., Poupot, R., 2006. Design of phosphorylated dendritic architectures to promote human monocyte activation. Faseb J. 20, 2339–2351. Schoonheydt, R.A., Heughebaert, L., 1972. Clay adsorbed dyes; methylene blue on laponite. Clay Miner. 27, 91–100.
Tardivo, J.P., Giglio, A.D., de Oliveira, C.S., Gabrielli, D.S., Junqueira, H.C., Tada, D.B., Severino, D., de Fátima Turchiello, R., Baptista, M.S., 2005. Methylene blue in photodynamic therapy: frombasic mechanisms to clinical applications. Photodiagn. Photodyn Ther. 2, 175–191. Wachter, E., Dees, C., Harkins, J., Scott, T., Petersen, M., Rush, R.E., Cada, A., 2003. Topical Rose Bengal: preclinical evaluation of pharmacokinetics and safety. Lasers Surg. Med. 32, 101–110. Zhang, G.D., Harada, A., Nishiyama, N., Jiang, D.L., Koyama, H., Aida, T., Kataoka, K., 2003. Polyion complex micelles entrapping cationic dendrimer porphyrin: effective photosensitizer for photodynamic therapy of cancer. J. Control. Release 93, 141–150. Zhang, Y., Xu, M.Y., Jiang, T.K., Huang, W.Z., Wu, J.Y., 2014. Low generational polyamidoamine dendrimers to enhance the solubility of folic acid: a dendritic effect investigation. Chin. Chem. Lett. 25, 815–818.
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Phosphorus dendrimers and photodynamic therapy. Spectroscopic studies on two dendrimer-photosensitizer complexes: Cationic phosphorus dendrimer with rose bengal and anionic phosphorus dendrimer with methylene blue Monika Dabrzalskaa , Maria Zablockab , Serge Mignanic , Jean Pierre Majorald,e,* , Barbara Klajnert-Maculewicza,f a
Department of General Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, Pomorska 141/143, 90-236 Lodz, Poland Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland c Université Paris Descartes, PRES Sorbonne Paris Cité, CNRS UMR 860, Laboratoire de Chimie et de Biochimie pharmacologiques et toxicologique, 45 rue des Saints Pères, 75006 Paris, France d Laboratoire de Chimie de Coordination CNRS, 205 route de Narbonne, 31077 Toulouse, France e Université de Toulouse, UPS, INPT, 31077, Toulouse Cedex 4, France f Leibniz-Institut für Polymerforschung Dresden e.V., Hohe Strasse 6, 01069 Dresden, Germany b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 May 2015 Received in revised form 9 June 2015 Accepted 12 June 2015 Available online 24 June 2015
Dendrimers due to their unique architecture may play an important role in drug delivery systems including chemotherapy, gene therapy and recently, photodynamic therapy as well. We investigated two dendrimer-photosensitizer systems in context of potential use of these systems in photodynamic therapy. The mixtures of an anionic phosphorus dendrimer of the second generation and methylene blue were studied by UV–vis spectroscopy while that of a cationic phosphorus dendrimer (third generation) and rose bengal were investigated by spectrofluorimetric methods. Spectroscopic analysis of these two systems revealed the formation of dendrimer-photosensitizer complexes via electrostatic interactions as well as p stacking. The stoichiometry of the rose bengal-cationic dendrimer complex was estimated to be 7:1 and 9:1 for the methylene blue-anionic dendrimer complex. The results suggest that these polyanionic or polycationic phosphorus dendrimers can be promising candidates as carriers in photodynamic therapy. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Phosphorus dendrimer Photosensitizer Rose bengal Methylene blue Photodynamic therapy Drug delivery system
1. Introduction Dendrimers are well-defined hyperbranched polymers characterized by low polydispersity. The structure of a dendrimer consists of a core molecule, branches, internal cavities and many terminal groups. Due to such an architecture, these polymers are attractive carriers of drugs, imaging or transfection agents and can be used for diverse ways of administration (Klajnert and Bryszewska, 2001; El Kazzouli et al., 2012; Mignani et al., 2013a,b).
Abbreviations: PDT, photodynamic therapy; ROS, reactive oxygen species; PS, photosensitizer; PAMAM, polyamidoamine dendrimer; PPI, polypropyleneimine dendrimer; PEG, polyethylene glycol; PpIX, protoporphyrin IX; ALA, aminolevulinic acid; MB, methylene blue; RB, rose bengal; 1cat, cationic phosphorus dendrimer of generation 3; 1an, anionic phosphorus dendrimer of generation 2. * Corresponding author. Fax: +33 561 553 003. E-mail address: [email protected] (J.P. Majoral). http://dx.doi.org/10.1016/j.ijpharm.2015.06.014 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
Photodynamic therapy (PDT) is an alternative cancer treatment method whereby cancer cells are destroyed by reactive oxygen species (ROS) and singlet oxygen (1O2) generated via a photodynamic effect. The photodynamic effect is a result of use of photosensitizer (PS) which under the influence of a specific wavelength of visible light induces a cascade of reactions leading to oxidative stress and cell death (Dolmans et al., 2003). However, PDT has limits similar to conventional chemotherapy. It involves, among others, poor PS selectivity, long-lasting skin sensitivity to light and rapid PS destruction. It is related to inappropriate biodistribution of photosensitizers (O’Connor et al., 2009). Therefore, to overcome these problems the challenge of modern PDT is creation of more selective and effective PS. This can be achieved by use of drug delivery systems. Recently dendrimers have gained some attention as promising transport systems for PS that potentially improve photodynamic therapy efficiency (Klajnert et al., 2012).
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PS molecules can be covalently bound to the core of the dendrimer, to the interior of the molecule or attached to the terminal groups. The presence of internal cavities allows for encapsulation of PS molecules in the dendrimer (Ihre et al., 2002; Patri et al., 2005; Bhadra et al., 2003). In several studies dendrimers were used as photosensitizer carriers. Polyamidoamine (PAMAM) and polypropyleneimine (PPI) dendrimers modified with polyethylene glycol (PEG) have been reported to be efficient in encapsulation of rose bengal and protoporphyrin IX (PpIX). PpIX encapsulated in PEG-PPI dendrimers revealed higher phototoxicity in comparison with a free photosensitizer (Kojima et al., 2007). A different approach to use dendrimers in PDT is based on the synthesis of photosensitizer dendrimers possessing porphyrin or phtalocyanine molecules in the core. Dendritic photosensitizers were also incorporated into polyion complex micelles through PEG-polyelectrolyte block copolymers. Polymeric micelles encapsulating dendrimer phthalocyanine showed high phytotoxicity in vitro and in vivo (Herlambang et al., 2011; Nishiyama et al., 2009; Zhang et al., 2003). Dendrimer conjugates containing 18 molecules of aminolevulinic acid (ALA) have been reported to efficiently transport ALA to tumor cells and induce porphyrin production in vitro (Battah et al., 2007). As mentioned above, several types of dendrimers, including PPI and PAMAM, have been studied in the context of use in PDT. In this paper, we propose a novel approach to use phosphorus dendrimers as potential photosensitizer carriers. Phosphorus dendrimers exhibit interesting biological properties that make them promising drug carriers, transfection, anti-prion, imaging (Caminade et al., 2010), anti-inflammatory (Poupot et al., 2006) or anti-tumoral agents (El Brahmi et al., 2015). However, phosphorus dendrimers have not been studied as potential photosensitizer carriers. In designing new drug delivery systems it is crucial to determine the potential interactions between drug molecules and nanocarriers. In this paper we present studies on the interaction of two dendrimer-photosensitizer systems in order to evaluate whether phosphorus dendrimers can be used as carriers in PDT. We chose two phosphorus dendrimers that differ in generation and electrostatic charge and two photosensitizers: methylene blue (MB) and rose bengal (RB). Methylene blue is a monocationic phenothiazine dye with potential to be a promising
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photosensitizer (Tardivo et al., 2005). Rose bengal, on the other hand, is an dianionic xanthene dye that is also reported to possess good PDT efficacy (Wachter et al., 2003). Both PSs upon irradiation generate singlet oxygen (Tardivo et al., 2005; Wachter et al., 2003). The aim of the study was therefore to check whether dendrimers are capable to form complexes with PSs possessing opposite charges using absorption and fluorescence spectroscopy in the case of methylene blue and rose bengal, respectively. The results allow estimating the potential role of phosphorus dendrimers in PDT as carriers of photosensitizers. 2. Materials and methods 2.1. Materials Two phosphorus dendrimers were used: cationic phosphorus dendrimer of generation 3 (MW = 15150.25 g/mol) possessing 48 ammonium terminal groups (1cat), and anionic phosphorus dendrimer (1an) of generation 2 (MW = 7851 g/mol) with 24 carboxyl terminal groups. Both dendrimers possess cyclotriphosphazene core. Dendrimers were synthesized in Laboratoire de Chimie de Coordination du CNRS, Toulouse, France. Rose bengal (RB) and methylene blue (MB) were purchased from Sigma–Aldrich. All solutions were made in phosphate-buffered saline (PBS), pH 7.4 using distilled water from Mili-Q system (Millipore). Scheme 1 depicts chemical characterization of the compounds used in this study. 2.2. Methods 2.2.1. UV–vis spectroscopy: study on the interaction between anionic phosphorus dendrimer (1an) and methylene blue (MB) Absorption spectra were recorded on a Jasco V-650 spectrophotometer. All measurements were performed in 100 mM PBS pH 7.4, at room temperature. Spectra were recorded in a wavelength range from 550 nm to 700 nm. Optical path length was 1 cm. PBS was used as a reference for all measurements. In titration experiments methylene blue (MB) was used at constant concentration of 10 mM. The MB solution was titrated with anionic dendrimer using different stocks of dendrimer in order to maintain specific molar ratios (1–20) of MB molecules per
Scheme 1. Chemical structures of dendrimers and photosensitizers used in this study. (A) anionic phosphorus dendrimer of the second generation—1an; (B) cationic phosphorus dendrimer of the third generation—1cat; (C) rose bengal; (D) methylene blue; (E) schematic structure of the second generation of phosphorus dendrimer; (F) schematic structure of the third generation of phosphorus dendrimer.
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dendrimer at each titration point. Dendrimer did not absorb light in this region. Next, the influence of sodium salt concentration on the interaction between MB and the dendrimer was checked. In this experiment MB:1an solutions at molar ratios equal to 3, 5, 7 and 10, were prepared in PBS buffer. Then NaCl was added. Final concentrations of NaCl were equal to 0.01; 0.1; 1; 10 and 100 mM. Additionally, the time-dependent stability of MB:1an complex was investigated. MB:1an complexes at molar ratio 5:1 and 10:1 were chosen for this experiment. All spectrophotometric measurements were repeated three times. 2.2.2. Spectrofluorimetric study on the interaction between cationic phosphorus dendrimer (1cat) and rose bengal (RB) Fluorescence experiments were performed using a PerkinElmer LS-50B spectrofluorimeter. All measurements were performed in 100 mM PBS, pH 7.4, at room temperature. The excitation wavelength was set to 525 nm and spectra were collected in a wavelength range from 540 nm to 650 nm. Excitation and emission slits were 5 nm and 7 nm, respectively. Dendrimer did not show any emission in this wavelength range. Rose bengal (RB) at concentration of 1 mM was titrated with cationic dendrimer in a concentration range from 0.05 mM to 1 mM using different stock solutions to maintain molar ratio of RB molecules per dendrimer from 1 to 20. Each experiment was repeated three times. The influence of sodium salt concentration was checked by recording fluorescence spectra of RB:1cat complexes at molar ratios 3, 5, 7 and 10, as a function of salt concentration (0.01, 0.1, 1, 10 and 100 mM). Additionally, RB was titrated with dendrimer in PBS buffer containing 100 mM NaCl. Next, time stability of 5:1 (RB:1cat) complex was studied. 3. Results 3.1. UV–vis spectroscopy: study on the interaction between anionic phosphorus dendrimer (1an) and methylene blue (MB). Firstly we checked whether the dendrimer is able to interact with methylene blue. Fig. 1 shows MB absorption spectra upon addition of the dendrimer. Solution of MB exhibits a strong band at
664 nm and a shoulder at approximately 610 nm. These bands are assigned to the absorption of monomeric and dimeric form of methylene blue, respectively (Bergman and O'Konski, 1963). MB in its monomeric and dimeric form is depicted in Scheme 2. Dendrimer was added in different concentrations to maintain specific molar ratios of the compounds. The first titration point was at a MB:1an molar ratio equal to 20 and the last one was at 1:1 MB:1an ratio. The dendrimer caused significant changes in absorption spectra of methylene blue. Three stages were distinguished during the titration. Firstly, upon addition of the dendrimer (molar ratios MB:1an 20–11) absorption significantly decreased. Secondly, there was a red shift of the main absorption band of MB from 664 nm to 672 nm. Thirdly, in the range of MB:1an molar ratios from 9 to 1 absorption increased. Additionally, in the case of lower MB:1an ratios there was an isosbestic point at 651 nm. Significant influence of the dendrimer on the MB absorption indicates that there is an interaction between the photosensitizer and the dendrimer. The fact that the dimer band decreases together with a red shift and an increase of the monomer band for lower MB:1an ratios suggests that the addition of the dendrimer caused a decrease in the dimeric form of MB. Hence, the red shifted band corresponds to the complex MB-dendrimer. Fig. 2 depicts changes in the position of absorption maximum (lmax) of methylene blue upon addition of the dendrimer. As the concentration of the dendrimer increased the changes in the positions of lmax were more pronounced. The highest band shift was observed for MB:1an 1:1 molar ratio. In order to reveal the changes in MB absorption spectra upon interaction with the dendrimer, a plot of A664/A610 versus MB:1an molar ratio was made (Fig. 3A). MB shows an absorption peak at 664 nm and a shoulder at 610 nm that correspond to monomer and dimer, respectively. MB complexed with dendrimer showed red shifted absorption band, therefore a plot of A672/A664 versus corresponding MB:1an ratio was made to determine the stoichiometry of the complex according to the ratio method (Fig. 3B) (Nandinii and Vishalaksi, 2010). The stoichiometry of the complex was determined from the point where the curve changed its slope. As it can be seen in Fig. 3A the ratio of the monomer band (A664) to dimer band (A610) depends on the MB:1an ratio. At molar ratio
Fig. 1. The absorption spectra of methylene blue (10 mM) upon addition of the anionic dendrimer in a molar ratio (MB:1an ranging from 20 to 1). Dendrimer concentration ranged from 0.5 mM to 10 mM. (A) The first stage: decrease in absorption (MB:1an from 20 to 11); (B) The second stage: red shift of lmax; (C) The third stage: increase in absorption (MB:1an from 9 to (1); a—pure MB spectrum; b—20:1 MB:1an ratio; c—10:1 MB:1an ratio; d—5:1 MB:1an ratio; e—1:1 MB:1an ratio.
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Scheme 2. Structures of the monomeric and the dimeric form of methylene blue (MB).
equal to approx. 10 the A664/A610 parameter achieved the lowest value and then it progressively increased as the MB:1an ratio decreased. It clearly indicates the interaction between methylene blue and dendrimer molecules. The A664/A610 ratio significantly increased in the case of MB:1an molar ratios above 10. Higher values of the A664/A610 ratio indicate that the fraction of MB dimers decreased upon the addition of the dendrimer. For lower dendrimer concentrations the A664/A610 values are low and stay at the plateau. Though, above the dendrimer concentration of 1 mM (MB:1an molar ratio 10:1), there are enough dendrimer molecules to easily interact with methylene blue molecules and therefore the MB dimer formation is suppressed by the formation of MB:1an complexes. Figure 3B shows changes in the ratio of A672–A664. It can be observed that A672/A664 values also increased for lower MB:1an ratios. Such changes correspond to the red shift and increase of intensity for the monomer band at 672 nm. Two different slops of the lines were observed that crossed when MB:1an ratio was approx. 9–10. Therefore, the stoichiometry of MB:1an complex was determined to be either 9:1 or 10:1. Four different MB:1an molar ratios (3, 5, 7 and 10) were chosen for the investigation of the character of the interactions between the photosensitizer and the anionic dendrimer. Absorption spectra of all solutions were measured as a function of NaCl concentration ranging from 0 mM to 100 mM. For all tested systems the absorbance slightly decreased with increasing salt concentration. However, the detailed analysis of ratios A664/A610 and A672/A664 revealed no effect of a salt concentration on interactions between MB and the dendrimer (Fig. 4A and B).
To determinate time stability of the complexes, MB:1an 5:1 and 10:1 complexes were chosen to study over the course of time. Plots of A664/A610 and A672/A664 versus time are depicted in Fig. 5. The absorbance of the complexes was measured up to 24 h. The lack of changes indicates that both complexes were stable for 24 h.
Fig. 2. Changes in the position of lmax of methylene blue absorption spectra upon addition of the dendrimer at different MB:1an molar ratio. MB = 10 mM.
Fig. 3. Changes in MB absorption ratios A664/A610 (A) and A672/A664 (B) as a function of MB:1an molar ratio.
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Fig. 6. Fluorescence spectra of RB (1 mM) upon addition of cationic dendrimer in molar ratio of RB molecules per dendrimer (RB:1cat ranging from 20 to 1). Concentration of the dendrimer ranged from 0.05 to 1 mM. (A) Rose bengal spectrum; b—RB:1cat in 20:1 molar ratio; c—RB:1cat in 10:1 molar ratio; d— RB:1cat in 5:1 molar ratio; e—RB:1cat in 3:1 molar ratio; f —RB:1cat in 2:1 molar ratio; (B) RB:1cat in 1:1 molar ratio.
Fig. 4. (A) A672/A664 ratio as a function of NaCl concentration; (B) A664/A610 as a function of NaCl concentration.
However, A664/A610 and A672/A664 values are lower for MB:1an 10:1 complex than for MB:1an 5:1 complex. Though, the fraction of MB dimers is lower in the case of MB:1an 5:1 complex. 3.2. Spectrofluorimetric study on the interaction between cationic phosphorus dendrimer (1cat) and rose bengal (RB). The interaction between rose bengal and the cationic dendrimer was investigated using spectrofluorimetric method due to sensitivity of this technique and a strong fluorescence signal given by rose bengal. Similarly as it was done in the case of spectrophotometric experiments, rose bengal was titrated with the dendrimer, maintaining specific RB:1cat molar ratios at each titration point. Fig. 6 shows fluorescence spectra of RB upon addition of the cationic dendrimer. With increasing concentration of the dendrimer, quenching of RB fluorescence was observed. Then a significant red shift from 564 nm to 578 nm occurred and the fluorescence intensity started
Fig. 7. Stoichiometry of the RB:1cat complex.
to rise again. Such an effect indicates that RB forms complexes with the cationic dendrimer. Different spectral characteristics were observed for pure RB and for the formed complex. In order to estimate the stoichiometry of the RB:1cat complex, the fluorescence intensity for two fluorescence maxima (F564/F578) was calculated (Fig. 7). The stoichiometry was determined to be 7:1 (RB:1cat).
Fig. 5. Time stability of MB:1an 5:1 complex and MB:1an 10:1 complex.
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Fig. 8. Fluorescence spectra of RB:1cat complexes (RB:1cat = 3, 5, 7 and 10) in absence and presence of different concentrations of NaCl (0.01; 0.1; 1; 10 and 100 mM).
For molar ratios from 10 to 20 F564/F578 values were on the same level as for free RB solution. For molar ratios less than 10, the values of F564/F578 decreased significantly and then reached a plateau. Next, the NaCl effect on the interaction of RB with cationic dendrimer was studied. Fluorescence spectra of four complexes (RB:1cat = 3, 5, 7 and 10) were measured in presence of NaCl (0.01 mM–100 mM). Fig. 8 shows changes in fluorescence spectra caused by increasing ionic strength.
Fig. 9. Changes in RB fluorescence ratio F564/F578 in PBS and in PBS containing additional 100 mM of NaCl.
It was found that upon addition of NaCl the fluorescence maximum shifted to 580 nm. However, only slight changes were observed in the case of RB:1cat 3:1. The lowest intensity was observed in presence of 100 mM of NaCl. For 5:1 complex, the fluorescence intensity was found to increase progressively upon increasing NaCl concentration, except for 100 mM of NaCl. In this case fluorescence signal was significantly diminished. Interestingly, for 7:1 complex in presence of 100 mM NaCl the fluorescence intensity significantly increased and the fluorescence maximum was localized at 564 nm which is characteristic for free RB molecules. In the case of 10:1 RB:1cat solution, the fluorescence intensity was very low in presence of 0.1 mM but in presence of 100 mM of NaCl the intensity was only slightly lower than that in absence of NaCl. For all samples (RB:1cat 10:1) the fluorescence maximum was at 564 nm. The influence of NaCl on the interaction between RB and cationic dendrimer is presented in Fig. 9 which depicts changes in F564/F578 ratio in PBS and in PBS containing 100 mM NaCl. Fluorimetric titration of RB with the cationic dendrimer in PBS containing 100 mM NaCl showed the same effect as the titrations performed in PBS. However, high salt concentration did influence on the rate of complex formation. In the presence of NaCl complexes were formed slower than in PBS buffer (Fig. 9). Interestingly, the stoichiometry of RB:1cat complex upon addition of 100 mM NaCl to PBS decreased from 7:1 to 5:1.
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Fig. 10. (A) RB:1cat 5:1 fluorescence decay in time stability experiment; (B) decrease of the maximum fluorescence intensity of RB:1cat complex (5:1) in the course of time.
Study on the time stability of RB:1cat complex (5:1) revealed very fast decay of fluorescence signal (Fig. 10A). However, the fluorescence maximum did not shift to wavelength specific for free RB, so the complex did not release free RB molecules. The maximum fluorescence intensity of RB:1cat complex decreased within 4 h (Fig. 10B) but it is clear that such an effect was caused by photobleaching. 4. Discussion Methylene blue (MB) and rose bengal (RB) are dyes with promising applications in photodynamic therapy (PDT). Thus we tested potential uses of polyanionic and polycationic phosphorus dendrimers as carriers of MB and RB. In order to design good therapeutic complexes it is important to check if their potential as carriers allows the binding of the guest molecules, the characterization of the type of the interaction and the determination of stoichiometry of such complexes. In this paper we applied spectroscopic methods to investigate dendrimer-photosensitizer complexes. As far as we know this is the first study on the complexation of MB and RB with phosphorus dendrimers in the context to use such complexes in PDT. Two separate dendrimer-photosensitizer systems were chosen on the basis of opposite charges: an anionic phosphorus dendrimer (1an) with a cationic dye—MB and a cationic phosphorus dendrimer (1cat) with an anionic dye—RB. UV-VIS spectroscopy study on the interaction between the anionic dendrimer and MB showed substantial influence of the dendrimer on MB absorption spectrum. There were three different effects in MB absorption upon addition of dendrimer. Both, monomer and dimer bands showed significant changes: MB momoner band was firstly diminished by the dendrimer, and then, as the MB:1an molar ratio was diminishing, intensity of monomer band increased. However, the dimer band constantly decreased. These two effects resulted in an isosbestic point characteristic to lower MB:1an ratios. According to the literature, the interaction of cationic dyes with polymers often leads to aggregation and a shift of the absorption band of the dye that confirms the formation of dye-polymer complexes (Fradj et al., 2014; Gadde et al., 2009). An absorption shift when accompanied with change of colour is called metachromasy or metachromatic effect. It is a common effect related to MB adsorption on anionic
polymers or clay minerals (Schoonheydt and Heughebaert, 1972; Otsuki and Adachi, 1993). Investigation of adsorption of MB on Starburst dendrimer pointed out the formation of dimers and higher-order aggregates (dependent on dendrimer generation), which were characterized mainly by an absorption decrease and a blue shift (Jockusch et al., 1995). In our experiment we discovered the opposite effect: under the influence of the dendrimer a red shift (about 6 nm) was observed for MB:1an molar ratios lower than 10. Similarly, a slight bathochromic shift was observed in the investigation on binding MB molecules to a single stranded polyribonucleic acid (Hossain et al., 2012). Methylene blue when aggregates can form either so called Haggregates or J-aggregates. According to Kasha’s exciton theory, a blue shift is characteristic for H-aggregates (face-to-face molecular arrangement), whether a red shift corresponds to a formation of Jaggregates (head-to-tail alignment) (Kasha, 1963). However, a J-aggregates band is usually observed above 700 nm, thus we assume that the red shift in our study may be related to MB monomers bound to the dendrimer. The presence of an isosbestic point clearly confirms the formation of MB:1an complexes. Changes in absorption of MB upon addition of dendrimer allowed to determine the stoichiometry of the complex as MB:1an equals to 10:1. In the next experiment no influence of ionic strength on complex formation was observed. This indicates that electrostatic interactions are involved in forming MB:1an complexes, but also that p stacking between the terminal aromatic rings in 1an and in MB are formed. The p–p interactions between aromatic ring of MB and internal aromatic rings of the dendrimer 1an seems to be less probable or more negligible due to the strong hydrophobicity of the interior of 1an and even if, in general, the second generation of a dendrimer has a relatively open structure with internal cavities available for encapsulation of dye molecules as it was reported for PAMAM dendrimers with a hydrophilic interior (Klajnert and Bryszewska, 2001). Taking into account the complexity of dendrimer structures, more than one type of interactions involved in complexation are observed for many types of molecules combined with dendrimers (Gupta et al., 2007; Zhang et al., 2014; D'Emanuele and Attwood, 2005). Time stability study showed that both, MB:1an 5:1 and MB:1an 10:1 complexes were stable over the time. Nevertheless, the MB:1an 5:1 complex presented higher values of the ratios A664/A610 and A672/A664. Though, the fraction of MB dimers was lower in the case of MB:1an 5:1 complex. It is an important result taking into account a potential therapeutic use. In the case of the interaction between rose bengal and a cationic phosphorus dendrimer we obtained similar results to those of MB:1an interactions. Upon addition of cationic dendrimer the fluorescence intensity was almost completely quenched and afterwards for lower RB:1cat ratios fluorescence maximum of RB moved to longer wavelengths that was accompanied by an increase of a fluorescence intensity. The similar red shift in absorption spectrum of RB was earlier obtained in the study on RB interaction with cyclodextrins and was confirmed as an effect of forming complexes with cyclodextrins (Fini et al., 2004). In our study, we also concluded that the change in fluorescence maximum position confirmed the formation of RB:1cat complexes. The stoichiometry of the complexation was determined to be RB:1cat 7:1. In marked contrast with what was observed in the case of MB:1an interactions, RB:1cat complexes were affected by the NaCl concentration. For RB:1cat 7:1 complex, 100 mM of NaCl resulted in changing the position of fluorescence maximum back to free RB band, with additional increase of fluorescence. Such a strong effect indicated that high salt concentration caused a release of bound RB molecules from the
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dendrimer. According to earlier studies, RB molecules have a tendency to aggregate at high salt concentration (Fini et al., 2004). In the case of RB:1cat 10:1 the fluorescence band was located at 564 nm—thus it confirmed the results of stoichiometry determination. The results of titration of RB with the dendrimer in presence of high salt concentration (100 mM NaCl) showed lower stoichiometry of the complex and slower rate of complex formation. Thus we assume that the electrostatic forces dominate the character of RB:1cat complexation, that is to say interactions may occur between the carboxylic groups of RB and ammonium groups present at the periphery of the dendrimer. Interestingly, different results were obtained in the case of interaction of RB with PAMAM dendrimers: RB was encapsulated in the cavities of PAMAM dendrimers of generation 2.5 possessing ester terminal groups. 2.5 PAMAM dendrimers were electrostatically neutral (but easily protonated): the spectroscopic study showed that RB was encapsulated in the dendritic box through electrostatic interactions with the hydrophilic interior of the PAMAM dendrimer (Karthikeyan et al., 2011). Our result is though probably connected to dendrimer structure: phosphorus dendrimer of third generation 1cat possess 48 terminal groups and presents more dense surface and a hydrophobic interior and thus, probably RB molecules cannot interact so efficiently with the internal cavities of the dendrimer. RB:1cat 5:1 complex showed fast decay of a fluorescence intensity over the time, though there was no shift of fluorescence band toward shorter wavelength characteristic to free RB molecules. It is possible that strong photobleaching of RB bound to the dendrimer occurred. Theoretically when comparing the number of terminal groups present on anionic and cationic dendrimer, the cationic one should be capable to bind more photosensitizer molecules, because 1cat possesses twice more cationic groups (48) than 1an possess anionic ones. However, MB has a smaller size than RB. Our results showed that 1an dendrimer interacts with MB through electrostatic and p–p interactions and RB:1cat interactions were only due to electrostatic forces. This might be due to the fact that the 1an dendrimer has a more open structure (generation 2) than 1cat (generation 3) and possesses terminal groups favoring p–p interactions. In conclusion our study showed the formation of two complexes of phosphorus dendrimers with photosensitizers: cationic dendrimer with rose bengal and anionic dendrimer with methylene blue. Spectroscopic analysis allowed us to conclude that different forces were responsible for complexation: electrostatic forces and p–p stacking in the case of anionic dendrimer and methylene blue and electrostatic interactions in the case of cationic dendrimer and rose bengal. MB:1an and RB:1cat stoichiometries were estimated to be 9:1 and 7:1, respectively. The MB:1an complex was more stable, while in the case of RB:1cat the complex was prone to a photobleaching effect, although there was no destruction of the RB:1cat complex over the time. Therefore, phosphorus dendrimers used in this study present different behaviors than that of other dendrimers as PAMAM for example, illustrating their own specificity which allows to consider them as promising candidates for carriers in photodynamic therapy. Further investigations in this direction are under active investigation as well as experiments concerning the role of the size of dendrimers (generations 1 to 4; the so-called “dendritic effect”) on their complexing properties. Acknowledgements This study was funded by the project “Phosphorus dendrimers as carriers of photosensitizers in photodynamic therapy and its combination with hyperthermia in in vitro studies” operated
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within the Foundation for Polish Science VENTURES Programme (Project VENTURES number VENTURES/2013-11/3) co-financed by the EU European Regional Development Fund and by the grant HARMONIA “Studying phosphorus dendrimers as systems transporting photosensitizers” no. UMO-2013/08/M/NZ1/00761 supported by National Science Centre. References Battah, S., Balaratnam, S., Casas, A., O'Neill, S., Edwards, C., Batlle, A., Dobbin, P., MacRobert, A.J., 2007. Macromolecular delivery of 5-aminolaevulinic acid for photodynamic therapy using dendrimer conjugates. Mol. Cancer Ther. 6, 876– 885. Bergman, K., O’Konski, C.T., 1963. A spectroscopic study of methylene blue’ monomer, dimer, and complexes with montmorillonite. J. Phys. Chem. 67, 2169– 2177. Bhadra, D., Bhadra, S., Jain, S., Jain, N.K., 2003. A PEGylated dendritic nanoparticulate carrier of fluorouracil. Int. J. Pharm. 257, 111–124. Caminade, A.M., Turrin, C.O., Majoral, J.P., 2010. Biological properties of phosphorus dendrimers. New J. Chem. 34, 1512–1524. D'Emanuele, A., Attwood, D., 2005. Dendrimer–drug interactions. Adv. Drug Deliv. Rev. 57, 2147–2162. Dolmans, D.E., Fukurama, D., Jain, R.K., 2003. Photodynamic therapy for cancer. Nat. Rev. Cancer 3, 380–387. El Brahmi, N., Mignani, S., Caron, J., El Kazzouli, S., Bousmina, M., Caminade, A.M., Cresteil, T., Majoral, J.P., 2015. Investigations on dendrimer space reveal solid and liquid tumor growth-inhibition by original phosphorus-based dendrimers and the corresponding monomers and dendrons with ethacrynic acid motifs. Nanoscale 7, 3915–3922. El Kazzouli, S., El Brahmi, N., Mignani, S., Bousmina, M., Zablocka, M., Majoral, J.P., 2012. From metallodrugs to metallodendrimers for nanotherapy in oncology: a concise overview. Curr. Med. Chem. 19, 4995–5010. Fini, P., Castagnolo, M., Catuccia, L., Cosma, P., Agostiano, A., 2004. Inclusion complexes of Rose Bengal and cyclodextrins. Thermochim. Acta 418, 33–38. Fradj, A.B., Lafi, R., Hamouda, S.B., Gzara, L., Hamzaoui, A.H., Hafiane, A., 2014. Effect of chemical parameters on the interaction between cationicdyes and poly (acrylic acid). J. Photochem. Photobiol. A 284, 49–54. Gadde, S., Batchelor, E.K., Kaifer, A.E., 2009. Controlling the formation of cyanine dye H- and J-aggregates with cucurbituril hosts in the presence of anionic polyelectrolytes. Chem. Eur. J. 15, 6025–6031. Gupta, U., Agashe, H.B., Jain, N.K., 2007. Polypropylene imine dendrimer mediated solubility enhancement: effect of ph and functional groups of hydrophobes. J. Pharm. Pharmaceut. Sci. 10, 358–367. Herlambang, S., Kumagai, M., Nomoto, T., Horie, S., Fukushima, S., Oba, M., Miyazaki, K., Morimoto, Y., Nishiyama, N., Kataoka, K., 2011. Disulfide crosslinked polyion complex micelles encapsulating dendrimer phthalocyanine directed to improved efficiency of photodynamic therapy. J. Control. Release 155, 449–457. Hossain, M., Kabir, A., Kumar, G.S., 2012. Binding of the phenothiazinium dye methylene blue with single stranded polyriboadenylic acid. Dyes Pigments 92, 1376–1383. Ihre, H.R., Padilla De Jesús, O., Szoka Jr., F.C., Fréchet, J.M.J., 2002. Polyester dendritic systems for drug delivery applications: design synthesis, and characterization. Bioconjug. Chem. 13, 443–452. Jockusch, S., Turro, N.J., Tomalia, D.A., 1995. Aggregation of methylene blue adsorbed on starburst dendrimers. Macromolecules 28, 7416–7418. Karthikeyan, K., Babu, A., Kim, S.J., Murugesan, R., Jeyasubramanian, K., 2011. Enhanced photodynamic efficacy and efficient delivery of Rose Bengal using nanostructured poly(amidoamine) dendrimers: potential application in photodynamic therapy of cancer. Cancer Nano. 2, 95–103. Kasha, M., 1963. Energy transfer mechanisms and the molecular exciton model for molecular aggregates. Radiat. Res. 20, 55–71. Klajnert, B., Bryszewska, M., 2001. Dendrimers: properties and applications. Acta Biochim. Pol. 48, 199–208. Klajnert, B., Rozanek, M., Bryszewska, M., 2012. Dendrimers in photodynamic therapy. Curr. Med. Chem. 19, 4903–4912. Kojima, C., Toi, Y., Harada, A., Kono, K., 2007. Preparation of poly(ethylene glycol)attached dendrimers encapsulating photosensitizers for application to photodynamic therapy. Bioconjug. Chem. 18, 663–670. Mignani, S., El Kazzouli, S., Bousmina, M., Majoral, J.P., 2013a. Dendrimer space concept for innovative nanomedicine: a futuristic vision for medicinal chemistry. Prog. Polym. Sci. 38, 993–1008. Mignani, S., El Kazzouli, S., Bousmina, M., Majoral, J.P., 2013b. Expand classical drug administration ways by emerging routes using dendrimer drug delivery systems: a concise overview. Adv. Drug Deliv. Rev. 65, 1316–1330. Nandinii, R., Vishalaksi, B., 2010. A study of interaction of cationic dyes with anionic polyelectrolytes. Spectrochim. Acta Mol. Biomol. Spectros. 75, 14–20. Nishiyama, N., Nakagishi, Y., Morimoto, Y., Lai, P.S., Miyazaki, K., Urano, K., Horie, S., Kumagai, M., Fukushima, S., Cheng, Y., Jang, W.D., Kikuchi, M., Kataoka, K., 2009. Enhanced photodynamic cancer treatment by supramolecular nanocarriers charged with dendrimer phthalocyanine. J. Control. Release 133, 245–251. O’Connor, A.E., Gallagher, W.M., Byrne, A.T., 2009. Porphyrin and nonporphyrin photosensitizers in oncology: preclinical and clinical advances in photodynamic therapy. Photochem. Photobiol. 85, 1053–1074.
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M. Dabrzalska et al. / International Journal of Pharmaceutics 492 (2015) 266–274
Otsuki, S., Adachi, K., 1993. Metachromasy in polymer films. Changes in the absorption spectrum of methylene blue in nafion films by hydration. Polym. J. 25, 1107–1112. Patri, A.K., Kukowska-Latallo, J.F., Baker Jr., J.R., 2005. Targeted drug delivery with dendrimers: comparison of the release kinetics of covalently conjugated drug and non-covalent drug inclusion complex. Adv. Drug Deliv. Rev. 57, 2203– 2214. Poupot, M., Griffe, L., Marchand, P., Maraval, A., Rolland, O., Martinet, L., L’FaqihiOlive, F.E., Turrin, C.O., Caminade, A.M., Fournié, J.J., Majoral, J.P., Poupot, R., 2006. Design of phosphorylated dendritic architectures to promote human monocyte activation. Faseb J. 20, 2339–2351. Schoonheydt, R.A., Heughebaert, L., 1972. Clay adsorbed dyes; methylene blue on laponite. Clay Miner. 27, 91–100.
Tardivo, J.P., Giglio, A.D., de Oliveira, C.S., Gabrielli, D.S., Junqueira, H.C., Tada, D.B., Severino, D., de Fátima Turchiello, R., Baptista, M.S., 2005. Methylene blue in photodynamic therapy: frombasic mechanisms to clinical applications. Photodiagn. Photodyn Ther. 2, 175–191. Wachter, E., Dees, C., Harkins, J., Scott, T., Petersen, M., Rush, R.E., Cada, A., 2003. Topical Rose Bengal: preclinical evaluation of pharmacokinetics and safety. Lasers Surg. Med. 32, 101–110. Zhang, G.D., Harada, A., Nishiyama, N., Jiang, D.L., Koyama, H., Aida, T., Kataoka, K., 2003. Polyion complex micelles entrapping cationic dendrimer porphyrin: effective photosensitizer for photodynamic therapy of cancer. J. Control. Release 93, 141–150. Zhang, Y., Xu, M.Y., Jiang, T.K., Huang, W.Z., Wu, J.Y., 2014. Low generational polyamidoamine dendrimers to enhance the solubility of folic acid: a dendritic effect investigation. Chin. Chem. Lett. 25, 815–818.