Enhanced photodynamic efficacy and efficient delivery of Rose Bengal using nanostructured poly(amidoamine) dendrimers: potential application in photodynamic therapy of cancer
© Springer-Verlag 2011
Received: 28 June 2011
Accepted: 27 July 2011
Published: 13 August 2011
Photodynamic therapy (PDT) is a promising treatment methodology whereby diseased cells and tissues are destroyed by reactive oxygen species (ROS) by using a combination of light and photosensitizers (PS). The medical application of Rose Bengal (RB), photosensitizer with very good ROS generation capability, is limited due to its intrinsic toxicity and insufficient lipophilicity. In this report, we evaluate the potential of polyamidoamine (PAMAM) dendrimers in delivering RB and its phototoxic efficiency towards a model cancer cell line. The spherical, nanoscaled dendrimers could efficiently encapsulate RB and showed characteristic spectral responses. The controlled release property of dendrimer–RB formulation was clearly evident from the in vitro drug release study. ROS generation was confirmed in dendrimer–RB system upon white light illumination. Photosensitization of Dalton’s Lymphoma Ascite (DLA) cells incubated with dendrimer–RB formulation caused remarkable photocytotoxicity. Importantly, the use of dendrimer-based delivery system reduced the dark toxicity of RB.
Photodynamic therapy is a method of clinical treatment whereby diseased cells and tissues are destroyed by a combination of light and special drugs called photosensitizers (Lopez et al. 2010; Silva et al. 2009; Roy et al. 2003). In addition, the presence of adequate molecular oxygen in the tissue is also required. These components, tolerated singly by the diseased cells, generate cytotoxic oxygen-based molecular species when combined in proper dosage and concentration (Robertson et al. 2009; Guo et al. 2010). PDT is noninvasive and is recognized as a useful initial treatment for malignant tumors (Dolmans et al. 2003; Macdonald and Dougherty 2001). PDT using porfimer sodium (Photofrin®) has been approved for the treatment of esophageal cancer in the United States and Canada, early and late stage lung cancers in the Netherlands, bladder cancer in Canada, and early stage lung, esophageal, gastric and cervical cancers in Japan (Fisher et al. 1996). Photosensitizers can be divided into hydrophilic and hydrophobic compounds. The major drawbacks of the hydrophobic photosensitizers are that they cannot be simply injected intravenously since they form aggregates in solution that restricts their medical applications (Orenstein et al. 1996; Labouebe et al. 2006). Hence, hydrophobic photosensitizers need complex formulation for systematic delivery (Fenga et al. 2004; Shive and Anderson 1997). Hydrophilic photosensitizers are advantageous than hydrophobic photosensitizers since they can be easily delivered intravenously and significantly improve tumor killing (Moore et al. 2009; Vrouenraets et al. 2002). However, hydrophilic photosensitizers poorly accumulate in tumor cells as it finds difficulty in crossing cell membranes. This is mainly because the cellular transport systems in cancer cells are slowly accelerated for hydrophilic drugs to pass through when compared to normal cells (Kessel 1981).
Rose Bengal (RB) is a hydrophilic photosensitizer with a high absorption coefficient in the visible region of the spectrum at 552 nm showing good quantum yield of singlet oxygen (Kochevar et al. 1996). Although it has potential in photodynamic therapy of tumors, its tendency to aggregate in solution under physiological condition decreases the yield of reactive oxygen species (ROS) (Killig et al. 2004). Therefore, it is essential to have an appropriate formulation for the delivery of this hydrophilic photosensitizer in therapeutic levels. The ideal drug delivery system for carrying PDT should be biodegradable, have minimum toxicity, incorporate the photosensitizer without loss or alteration of the sensitizer activity and provide an environment where the photosensitizer can be administered in monomeric form (Konan et al. 2002). Importantly, the delivery system should enable selective accumulation of the PS within the diseased tissue in therapeutic concentrations with little or no uptake by nontarget cells (Chatterjee et al. 2008).
It is expected that charged or slightly lipophilic nanoscaled drug delivery systems can be used for efficient delivery of highly hydrophilic photosensitizers for PDT of cancer. In this regard, the use of dendrimer-based nanocarriers is a promising method for the tumor specific delivery of PS. Nanoparticles, such as nanospheres and nanocapsules, possess high impact in delivery system as PS carriers because they can meet all the requirements for an ideal PDT agent (Premanathan et al. 2011; Murday et al. 2009; Koo et al. 2005). Dendrimers can be considered as the most versatile, compositionally and structurally controlled synthetic nanoscale building blocks available today (Koda et al. 2008; Bechet et al. 2008). Dendrimers have high degree of molecular uniformity, loading capacity, biocompatibility and a highly functionalized terminal surface that facilitates modification of the solubility of drugs to help target the drug to its therapeutic sites, or to alter the release profile of the therapeutic agent (Svenson and Tomalia 2005; Jansen et al. 1994). With the aim of improving the drug delivery and release kinetics suitable for carrying PDT and diminishing the dark toxicity of RB, dendrimer-based delivery system could be a better choice.
The present study relies on polyamidoamine (PAMAM) dendritic nanostructures as an efficient drug delivery system for a well-known hydrophilic photosensitizer, Rose Bengal that was evaluated by investigating the interaction between the dendrimer and RB and the photodynamic efficacy. However, our studies explored the influence of G2.5 PAMAM + RB on Dalton’s Lymphoma Ascite (DLA) cancer cell lines. A potential application of PAMAM dendrimers as an efficient drug delivery system for a hydrophilic photosensitizer will provide new opportunities in nanomedicine for PDT of cancer.
2 Material and methods
Methanol was obtained from Spectrum Chemicals, India. Methyl acrylate was procured from Loba Chemicals, India. Ethylenediamine, ammonium molybdate, pyridine and diethyl ether were obtained from Merck, India. Ammonia and potassium iodide were obtained from SD Fine Chemicals, India. Rose Bengal was purchased from Aldrich, USA. Dialysis tubing (12–14 kDa cutoff size) was obtained from Himedia, India. All the chemicals were used as received except methanol, which was distilled twice before use.
2.2 Synthesis of PAMAM dendrimer
The first generation dendrimer G1.0 was purified by three times centrifugation and redispersion in methanol. Addition of MA in proper molar ratios with G1.0 under heating (50°C) for 48 h results in production of G1.5 PAMAM dendrimer. Subsequent addition of EDA generates G2.0 dendrimer. Likewise, the chain reaction is continued till the synthesis for G2.5 dendrimer. The dendrimer was purified at each step by centrifugation for 30 min at 16,000 g and resuspending in methanol solution. Finally, the dendrimer solution was dialyzed against methanol water (1:10) mixture for 24 h in order to remove any nonreacted chemical species.
2.3 Encapsulation of Rose Bengal into the PAMAM dendritic box
The G2.5 PAMAM dendrimer and RB were mixed in the ratio of 10:1 in a solution of 20 ml of methanol and 5 ml of water. The resulting solution was vigorously stirred at 500 rpm for 24 h using a magnetic stirrer. After 24 h, the solvent was removed under vacuum using a rotary evaporator. The final product obtained was purified by centrifugation at 16,000 g for 30 min followed by dialysis and stored at 4°C.
2.4 Characterization techniques
Surface morphology of G2.5 PAMAM dendrimer was analyzed using atomic force microscopy (AFM) in contact mode using XE 70, SPM, Park System, South Korea, in a scan area of 20 μm. The sample preparation was performed by taking 5 μl of G2.5 PAMAM and diluting 100 times. The zeta potential analysis was performed at 25°C, in MilliQ® water using Zetasizer Nano, Malvern Instruments, UK. A few drops of the prepared solution were allowed to spin coat on a glass substrate for 10 min and then dried before measurement. UV–vis spectra (Lambda 25, Perkin Elmer, USA) of PAMAM dendrimers were measured in methanol:water (50% v/v). Fourier transform infrared (FTIR) spectra of the samples were recorded in liquid mode using a modern Bruker optic GmbH-Alpha T spectrometer, Germany. Fluorescence spectra of were obtained in aqueous environment using a spectroflourimeter (Jasco FP-6300, Japan).
2.5 Estimation of drug loading and encapsulation efficiency
- W 1 :
weight of the drug present in dendrimer
- W 2 :
net weight of the dendrimer
- W 3 :
weight of the drug added, and
- W 4 :
weight of the drug released into the supernatant.
2.6 Measurement of drug release kinetics
The release of RB from the G2.5 PAMAM was measured spectrophotometrically as follows: 50 mg of RB encapsulated dendrimer was made up to 1 ml using a mixture of methanol:water (50:50, v/v). This solution was dialyzed using a dialysis tubing with a MW cutoff 12,000–14,000 Da (~2.4 nm) (Himedia, India) against phosphate buffer saline (PBS) of pH 7.4 at 37°C with mild stirring. This was continued for 72 h and at each time interval 1 ml was withdrawn from the PBS for spectrophotometric analysis at λmax 540 nm and was replaced by fresh PBS of the same amount. A graph was plotted with cumulative release% against time interval in hours representing the drug release profile.
2.7 Light source for PDT
A 150 W xenon arc lamp was used as a light source. The therapeutic window was adjusted by using 10% KI (5 cm path length) and pyridine (1 cm path length) as a filter for UV radiation. The specimen was kept in an open quartz cuvette and air saturated by magnetic stirring. It was irradiated at a distance of 12 cm from the light source.
2.8 Measurement of quantum yield of ROS generation by iodide method
The iodide assay was used for the evaluation of ROS generation of RB encapsulated G2.5 PAMAM dendrimers (Mosinger and Micka 1997). This assay is based on the reaction of singlet oxygen (1O2) (produced in the photodynamic reaction) with I− in the presence of ammonium molybdate as a catalyst. The reaction product is I 3 − , the amount of which (measured spectrophotometrically at λ = 351 nm) is directly proportional to the generated 1O2.
2.9 In vitro cell viability test—MTT assay
3 Results and discussion
3.1 Synthesis and characterization of PAMAM dendrimers
The PAMAM dendrimers of various generations (G0.5, G1.0, G1.5, G2.0 and G2.5) are synthesized using the Michael addition method as described in the previous section. The PAMAM dendrimers with ammonia as the core molecule possess an ester group (R-COO-R) and amine (R-CO-NH2) group as terminal surfaces in the successive half and full generation. The various generation dendrimers were characterized by UV–vis spectroscopy, FTIR spectroscopy, AFM and zeta potential. The UV–vis spectroscopy of all generations of PAMAM dendrimers shows a characteristic absorption at 275 to 290 nm (Figure S1). The FTIR spectra of the half generation (G0.5/G1.5/G2.5) shows a characteristic –C = O stretching vibrations around 1,729 cm−1 due to the presence of a free ester (C = O) group in the end surface (Kolhe et al. 2003). In addition to this, a band at 1,000 cm−1 to 1,200 cm−1 also appeared, which is assigned to the –C–O stretching mode [Fig. 3, 2.5 PAMAM]. The FTIR spectrum of the full generation dendrimers shows characteristic N–H stretching vibrations around 1,620–1,650 cm−1 because of the reaction of ester with amine making it as an amine end (Figure S2).
3.2 Dendrimer–Rose Bengal interaction
3.3 Drug loading and encapsulation efficiency of G2.5 PAMAM + RB nanocapsules
High drug loading and better encapsulation efficiency is expected for an ideal drug delivery agent, thereby, reducing the quantity of the matrix materials for drug administration (Mohanraj and Chen 2006). The drug loading in the G2.5 PAMAM + RB is through the absorption technique. Hence, the delivery system should be ideal in case of drug loading efficiency and drug release kinetics for carrying PDT, not suppressing the quantum yield of the PS after encapsulation. The amount of drug loaded into the dendrimer and the encapsulation efficiency of the G2.5 PAMAM dendrimers were measured spectrophotometrically during purification of G2.5 PAMAM + RB by centrifugation and are to be observed as 1.8% and 92.5% respectively.
3.4 In vitro drug release kinetics of G2.5 PAMAM + RB
3.5 ROS quantum efficiency of G2.5 PAMAM + RB
3.6 Phototoxicity and dark toxicity of G2.5 PAMAM + RB
PAMAM dendritic nanostructures could effectively deliver RB photosensitizers into cancer cells and produce enhanced photodynamic efficacy. The interaction between the PAMAM dendrimer and RB are investigated by UV–vis and FTIR spectra, fluorescence quenching and zeta potential measurements. Our systemic investigation of the dendrimer-based formulation of RB shows that it is feasible to encapsulate even a hydrophilic photosensitizer with excellent encapsulation efficiency and release kinetics. Results showed that drug release was quite faster in the initial hours and, above 80%, was released in 72 h, which was found to be more satisfactory for a water-soluble photosensitizer. It is observed that the PAMAM dendrimer-based delivery of RB could retain its ROS generation property upon irradiation and also could reduce its toxicity by holding the RB molecules in the internal cavities. Importantly, the dendritic formulation exhibited minimized dark toxicity within the concentration used and enhanced phototoxicity to DLA cells compared to the dark phototoxicity of free RB. The key findings of our work with the above-mentioned advantages ensure that PAMAM dendrimer-based nanocarriers of PS delivery should be a promising candidate for PDT.
The authors are thankful to the Management and the Principal of Mepco Schlenk Engineering College for providing the necessary facilities to carry out this work. S.-J. Kim acknowledges support from Korea Research Foundation (KRF) project number 2011–0015829.
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