Materials Science and Engineering C 51 (2015) 362–368
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Nanostructured delivery system for Suberoylanilide hydroxamic acid against lung cancer cells Renu Sankar a, Selvaraju Karthik a, Natesan Subramanian b, Venkateshwaran Krishnaswami b, Jürgen Sonnemann c, Vilwanathan Ravikumar a,⁎ a b c
Department of Biochemistry, School of Life Sciences, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India Laboratory for Lipid Based Systems, Anna University-BIT Campus, Tiruchirappalli 620 024, Tamil Nadu, India Jena University Hospital, Children's Clinic, Department of Pediatric Hematology and Oncology, Jena, Germany
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Article history: Received 27 April 2014 Received in revised form 8 January 2015 Accepted 23 February 2015 Available online 25 February 2015 Keywords: Histone deacetylase inhibitor Suberoylanilide hydroxamic acid Poly-D, L-lactide-co glycolide Nanoprecipitation A549 cells Antineoplastic
a b s t r a c t With the objective to provide a potential approach for the treatment of lung cancer, nanotechnology based Suberoylanilide hydroxamic acid (SAHA)-loaded Poly-D, L-lactide-co glycolide (PLGA) nanoparticles have been formulated using the nanoprecipitation technique. The acquired nanoparticles were characterized by various throughput techniques and the analyses showed the presence of smooth and spherical shaped SAHA-loaded PLGA nanoparticles, with an encapsulation efficiency of 44.8% and a particle size of 208 nm. The compatibility between polymer and drug in the formulation was tested using FT-IR, Micro-Raman spectrum and DSC thermogram analyses, revealing a major interaction between the drug and polymer. The in vitro drug release from the SAHAloaded PLGA nanoparticles was found to be biphasic with an initial burst followed by a sustained release for up to 50 h. In experiments using the lung cancer cell line A549, SAHA-loaded PLGA nanoparticles demonstrated a superior antineoplastic activity over free SAHA. In conclusion, SAHA-loaded PLGA nanoparticles may be a useful novel approach for the treatment of lung cancer. © 2015 Published by Elsevier B.V.
1. Introduction Cancer is the second most frequent cause of death in developed countries and the foremost cause of death in developing countries. Lung cancer is the most common cancer-related death, accounting for 13% (1.6 million) of the total cases and 18% (1.4 million) of the deaths in 2008 [1]. The equilibrium between histone acetylation and deacetylation appears to be essential for normal cell growth, and perturbation of the histone acetylation status has been associated with several diseases including lung cancer [2]. The acetylation status of histone is governed by two opposite enzymatic activates, histone acetyltransferases [3] and histone deacetylases (HDACs) [4]. Carcinogenesis has been associated with histone acetyltransferase (HATs) inactivation, whereas aberrant HDAC activity has been linked to the development and maintenance of the transformed state of human tumors, which is at least in part due to transcriptional repression of tumor suppressor gene expression [5]. In addition to histones, HDAC enzymes are also known to deacetylate numerous non-histone targets, such as transcription factors and proteins involved in cell cycle progression, indicating a complex multifunctional role for HDACs in health and disease [6]. Given these insights into the function of HDACs, it is not surprising that HDAC inhibitors are emerging as promising new agents for cancer therapy ⁎ Corresponding author. E-mail address:
[email protected] (V. Ravikumar).
http://dx.doi.org/10.1016/j.msec.2015.02.037 0928-4931/© 2015 Published by Elsevier B.V.
[7–11]. Indeed, HDAC inhibitors have been shown to induce potent anti-cancer effects including apoptosis, cytostasis, differentiation and inhibition of tumor angiogenesis in cultured cell lines [12,13]. Consequently, clinical trials with various HDAC inhibitors have been conducted and Suberoylanilide hydroxamic acid (SAHA; also known as vorinostat) has received FDA approval for the treatment of cutaneous T-cell lymphoma [14]. SAHA is a second generation polar–planar compound, which induces cell cycle arrest, differentiation and apoptosis in several transformed cells, including mouse xenograft models [2,5,14]. It has also been shown to be effective against chemotherapy-resistant patient-derived cancer cells [15]. Recently, SAHA drug is loaded with solid lipid nanoparticles to target the multidrug-resistant cancer cells [16]. Nanoparticles have recently gained considerable interest in pharmaceutical applications [17,18]. Meanwhile, nanoparticles synthesized by chemical methods are widely used for target drug delivery, likewise nanoparticles synthesized by some physical methods, such as electroporation are also used for drug/gene delivery [19]. Polymeric nanoparticles-based drug delivery is being increasingly investigated as delivery route to overcome many obstacles associated with the delivery of free drugs. Several drugs have been successfully encapsulated with biodegradable polymer to enhance their bioavailability, bioactivity and controlled delivery [20,21]. Poly-D, L-lactide-co glycolide (PLGA) [22] is one of the typically used biodegradable polymers for the development of nanomedicines. The human organism effectually hydrolyzed PLGA
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polymer and produced two degradable monomeric metabolites like lactic acid and glycolic acid [23]. In our study, PLGA polymer was chosen as drug delivery vehicle, due to their better encapsulation efficiency, bioavailability, less toxicity and controlled release. In our current approach, SAHA-loaded PLGA nanoparticles were formulated by the nanoprecipitation method and characterized for their encapsulation efficiency, yield percentage, drug release kinetics, particle size, surface properties, morphology, stability and drug polymer interaction. Furthermore, the antineoplastic effect of SAHA-loaded PLGA nanoparticles and SAHA were compared in the human lung cancer cell line A549. 2. Materials and methods 2.1. Materials Poly-D, L-lactide-co-glycolide (PLGA, L/G = 50:50) was a gift sample from Prof. Dr. Dragan Uskokovic, Institute of Technical Sciences of SASA, Serbia. SAHA was purchased from Sigma Aldrich, India. Tween 80, Span 80, acetone, methanol (HPLC grade) and DMSO (cell culture grade) were purchased from Merck, India. Dulbecco's modified Eagle's medium, fetal bovine serum, penicillin/streptomycin, MTT (dimethyl thiazolyltetrazolium bromide), acridine orange and ethidium bromide were purchased from Hi Media Laboratories, Mumbai, India. Doubledistilled deionized water was used throughout the experiments. 2.2. Methods 2.2.1. Preparation of SAHA-loaded PLGA nanoparticles SAHA-loaded PLGA nanoparticles were prepared by the nanoprecipitation method with modifications [24]. In brief, individual 10 mg of PLGA polymer was dissolved in 5 ml of acetone; 2 mg of SAHA was dissolved in 1 ml of ethanol and then mixed together for 10 min. Fifty milligrams of Span 80 was added over it and vortexed for 5 min. The formed solution was poured in 20 mg of Tween 80 solution under magnetic stirring for 12 h; the resulting nanoemulsion was evaporated overnight to remove the organic solvents. Further it was centrifuged at 28,000 rpm for 15 min and the nanoparticulate solution was freeze dried using 5% glucose as a cryoprotectant, stored in 4 °C until further use.
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2.2.4. Characterization of SAHA-loaded PLGA nanoparticles 2.2.4.1. Size and zeta potential. The particle size and zeta potential of SAHA-loaded PLGA nanoparticles were measured by dynamic light scattering (Malvern Instruments Ltd., Malvern, UK). 10 μg of particles was dispersed in 1 ml of deionized water sonicated for 5 s before measurement and performed triplicate. 2.2.4.2. Scanning electron microscopy (SEM). The size and surface morphology changes of the SAHA-loaded PLGA nanoparticles were achieved using scanning electron microscopy (Hitachi, Model: S-3400N). The nanoparticles were prepared on aluminum stubs and coated with gold prior to examination. 2.2.4.3. Transmission electron microscopy (TEM). SAHA-loaded PLGA nanoparticles were imaged using transmission electron microscopy performed on a Philips, Tecnai 10, Japan. The nanoparticles were placed on a copper grid and dried under vacuum, and the image was recorded at an accelerated voltage of 80 kV. 2.2.4.4. Differential scanning calorimetry (DSC). DSC thermogram of SAHA, PLGA polymer and SAHA-loaded nanoparticles were recorded. The physical status of SAHA inside the nanoparticles was investigated by differential scanning calorimetry (DSC 200F3 Maia). The samples were purged with dry nitrogen. The heating rate of 10 K/min was applied. 2.2.4.5. Fourier transform infra red spectroscopy (FTIR). FTIR studies were carried out to confirm the nanoparticles. The spectra were recorded on a Perkin Elmer FTIR spectrometer scanned over a range of 500–4000 cm− 1. Test samples were mixed with KBr pressed into a disk. FTIR spectra of drug, polymer and drug-loaded nanoparticles were recorded. 2.2.4.6. Micro-Raman spectroscopy. The Micro-Raman spectra of drug, polymer and drug-loaded nanoparticles were recorded at room
2.2.2. Encapsulation efficiency SAHA entrapped in PLGA nanoparticles was estimated using RP-HPLC (Shimadzu prominence consisting of LC 20AD). The chromatographic separation was achieved on Phenomenex Luna C18 (250 × 4.6 mm, 5 μ) analytical column. Accurately 0.1 ml (25 μg) of SAHA-loaded PLGA nanoparticles were dissolved in acetone:ethanol (1:1) and further dilution made with 0.1% Tween 80 containing phosphate buffered saline (PBS) pH 7.4 buffer. The mobile phase of methanol:0.1% trifluoro acetic acid (50:50% v/v) was used at a flow rate of 1 ml/min. The UV detection was carried out at 242 nm. The retention time of SAHA was found to be 4.2. The method was found to be linear at 5–25 μg/ml. 2.2.3. In vitro release studies The in vitro release study of SAHA-loaded PLGA nanoparticles was carried out using the dialysis bag method. One milliliter of nanoparticulate suspension was instilled in dialysis bag (cellophane molecular weight cutoff), tied and placed into 100 ml of 0.1% Tween 80 containing PBS pH 7.4 buffer solution at 37 °C with continuous stirring. At predetermined time intervals, aliquots were withdrawn and replaced with the same amount of (0.1% Tween 80 containing PBS pH 7.4) fresh buffer. The samples were analyzed using RP-HPLC using the same chromatographic condition of the encapsulation efficiency test.
Fig. 1. FTIR spectra of (a) SAHA, (b) PLGA polymer and (c) SAHA-loaded PLGA nanoparticles.
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2.2.7. Acridine orange-ethidium bromide double staining The A549 lung cancer cellular and nuclear morphological alterations were confirmed by means of fluorescence microscopy with acridine orange (AO) and ethidium bromide (EB) staining (1 mg/ml for both AO and EB in PBS) [25]. In brief, 5 × 105 cells/well were cultured on a coverslip in a 6-well plate and incubated overnight for attachment. On the next day, the old medium was replaced with fresh medium containing SAHA or SAHA-loaded PLGA nanoparticles. After 48 hour incubation, coverslip was removed and stained with AO/EB (10 μl) for 30 min and washed with PBS for removing excess staining dye. Coverslip was mounted on objective glass and cells were viewed using a Nikon Eclipse inverted fluorescence microscope at 20× magnification.
Fig. 2. Micro-Raman spectrum of SAHA, PLGA polymer and SAHA-loaded PLGA nanoparticles.
temperature using a laser Raman confocal microprobe (LabRam HR 800). A He–Ne laser (λ = 633 nm) was used as the excitation source with an output power of 17 mW and was focused to a spot of 1 μm. Before measurement, the system was calibrated using the 521 cm− 1 Raman line of a silicon (Si) wafer. The confocal hole of the spectrograph was 100 μ min in diameter. All Raman spectra were recorded in the region 100–3500 cm−1 in the backscattering geometry. The optical images were taken using the 50× objective in LabRam HR 800. 2.2.5. Cell culture A549 human non-small cell lung cancer cells were acquired from the National Center for Cell Science, Pune, India. They were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. 2.2.6. Cell viability assay Cells were seeded in 96-well plate at a cell density of 1 × 104/well and incubated for 24 h at 37 °C in a humidified atmosphere of 95% air and 5% CO2 for cell attachment. The cells were incubated with SAHA or SAHA-loaded PLGA nanoparticles at a concentration of 1–7.5 μM for 48 h. At the end of the incubation period, 20 μl of MTT (dimethyl thiazolyltetrazolium bromide) was added and the cultures were further incubated for 4 h. Then, MTT was aspirated and 200 μl of DMSO was added to dissolve the formazan crystals. The absorbance was measured at 570 nm using a microplate reader. The control with 100% viability was noted from the untreated cells.
2.2.8. Detection of intracellular reactive oxygen species levels using DCFH-DA The 2′,7′-dichlorofluorescein-diacetate (DCFH-DA) dye was used to measure the intracellular reactive oxygen species (ROS) generation in A549 cells. For quantifying the intracellular ROS, 5 × 105 cells were seeded on a coverslip in a 6-well plate and incubated overnight for attachment. On the next day, the old medium was replaced with fresh medium containing SAHA or SAHA-loaded PLGA nanoparticles. At the end of incubation, the coverslip was removed from the culture plate and stained with 40 μM DCFH-DA for 30 min. The stained coverslip was washed with PBS for removing extra dye. The coverslip was fixed on a glass slide and images of the cells were captured using a 20× objective under a fluorescence microscope [25]. For measuring total ROS level in spectrofluorometry, 5 × 105 cells were seeded in a 96-well fluorimetry plate and allowed to overnight for attachment. On the next day, the medium was replaced with fresh medium containing SAHA or SAHA-loaded PLGA nanoparticles incubated for 48 h. After incubation, 10 μM of DCFH-DA was added to each well and incubated at 37 °C for 45 min. The plate was read in a spectrofluorometer (Horiba, Fluoromax-4, Germany) using an excitation of 485 nm and an emission of 520 nm.
2.2.9. Assessment of mitochondrial membrane potential For measuring the mitochondrial membrane potential (Δψm), 5 × 105 cells were seeded in a 6-well plates and incubated overnight for attachment. After overnight incubation the old medium was replaced with fresh medium containing SAHA or SAHA-loaded PLGA nanoparticles. At the end of incubation the cells were stained with 50 μl of Rhodamine-123 dye (10 μg/ml) for 30 min, and excess dye was removed by washing with PBS [25]. The plate was read in a spectrofluorometer (Horiba, Fluoromax-4, Germany) using an excitation of
Fig. 3. Dynamic light scattering measurements. (a) Particle size distribution of SAHA-loaded PLGA nanoparticles: The particle diameter was found to be 208 nm; (b) zeta potential of SAHAloaded PLGA nanoparticles stable at −41.2 mV.
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3. Results and discussion 3.1. Characterization of SAHA-loaded nanoparticles
Fig. 4. In vitro release profiles of SAHA-loaded PLGA nanoparticles (S1—2 mg drug with 5 mg PLGA polymer; S2—2 mg drug with 10 mg PLGA polymer; S3—2 mg drug with 15 mg PLGA polymer).
488 nm and an emission of 525 nm, and the cell images were captured using a 40× objective under a fluorescence microscope. 2.2.10. Statistical analysis One-way ANOVA was used for testing the significance. The computations were performed using SPSS software version 16 (SPSS Inc., Chicago, IL, USA). Statistical significance was accepted at a level of P b 0.05.
The polymeric nanoparticles were prepared by the comparatively easiest technique and the nanoprecipitation method with three different polymer ratios. The partitioning and evaporation of organic solvents led to the polymeric nanoparticles formation. The entrapment efficiency was mainly affected by the polymer and drug ratios. Improvement in encapsulation efficiency and minimal drug wastage is mainly due to the high polymer concentrations used when compared to formulation S1, where 5 mg of polymer is used, which is relatively low. The confirmation of the chemical stability of SAHA in polymeric nanoparticles was assessed by the FTIR spectra of pure SAHA, PLGA polymer and SAHA-loaded PLGA nanoparticles as shown in Fig. 1. The characteristic peaks of PLGA are (888–3796 cm−1), amine C–N stretch (1315 cm−1) and anhydride C_O stretch (1792 cm−1). The peaks of SAHA and PLGA polymer were compared with the resulting peaks from SAHA-loaded PLGA nanoparticles. The major characteristic peaks of SAHA (805–3752 cm−1) and alkyl C–H stretch (2923 cm−1) are present both in free and SAHA-loaded PLGA nanoparticles, which confirms the presence of surface-bound drug over the polymeric surface also apart from encapsulation [26]. The qualitative composition of SAHAloaded nanoparticles was investigated by Micro-Raman spectroscopy. The characteristic peaks obtained from SAHA, PLGA polymer and SAHA nanoparticles are shown in Fig. 2. The spectrum of SAHA is characterized by the strong intensity C–H alkane stretching at 2989 cm−1,
Fig. 5. Antineoplastic activity. (a) Cytotoxicity of SAHA and SAHA-loaded PLGA nanoparticles in A549 cells, (b) morphological changes in A549 lung cancer cells after 48 h treatment of (a) control, (b) SAHA and (c) SAHA-loaded PLGA nanoparticles (scale bar = 80 μm).
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variable intensity–C–H alkane bending at 1415 cm−1, strong intensity C–O ether stretch at 1046 cm−1 and strong intensity _C–H alkene bending at 891 cm− 1. The PLGA spectrum exhibited bands at 2974, 2832 and 2703 cm−1 corresponding to alkane C–H stretching, a peak at 1708 cm−1 due to carbonyl C_O bond stretching, and peaks at 882 and 810 cm−1, due to alkene _C–H bending. The characteristic absorption bands of C–H alkane stretching at 2989 cm−1 and _C–H alkene bending at 891 cm− 1 of SAHA appeared in the SAHA-loaded PLGA nanoparticles, which probably indicates that SAHA molecules were fitted into the polymeric network. The SAHA-loaded PLGA nanoparticles possess a size range of 208 nm as shown in Fig. 3(a) with a narrow size distribution of polydispersity. The hydrophilic and lipophilic surfactants used in the formulation greatly influence the particle size, size distribution and encapsulation efficiency of the nanoparticles [27]. The particle stability as determined by the zeta potential of −41.2 mV (Fig. 3(b)) indicates the high surface charge of SAHA-loaded PLGA nanoparticles and its strong repellent interactions between the nanoparticles [28]. Our DLS analysis results were well correlated with the report of Marslin et al. which indicates that the cancer drug loaded PLGA nanoparticles average particle size distribution was 217 nm [20]. De Jong and Borm described that the above 100 nm in size nanoparticles is much suitable for biological applications especially in the area of drug delivery [29]. The particle size, size distribution and drug encapsulation efficiency of the nanoparticles have been significantly influenced by the surfactants used in the fabrication process of the formulation (Supplementary Table 1). The lipophilic surfactant aligns at the oil–water interface and enhances the particle stability. The enhanced nanoparticle stability devoid of coalescence and flocculation can be obtained by comparing the formulation without lipophilic surfactant. The physical state of SAHA
inside the nanoparticles was investigated by differential scanning calorimetry. The DSC thermogram of pure SAHA powder, PLGA polymer and SAHA-loaded PLGA nanoparticles are shown in Supplementary Fig. 1. SAHA displayed an endothermic peak at 93.7 °C. This peak has been slightly shifted to 111.8 °C in SAHA-loaded PLGA nanoparticles. The broad peaks shifted may be due to the ionic interaction between polymer and drug [30]. The SEM photograph of SAHA-loaded PLGA nanoparticles are shown in Supplementary Fig. 2(a). The nanoparticles were found to be smooth and spherical in shape, and the slower drug release may be due to the presence of smooth surface. The surface morphology confirmed by TEM image as shown in Supplementary Fig. 2(b) shows similar results; magnifying a single nanoparticle shows that particles are uniformly dispersed throughout the polymeric matrix [31]. 3.2. In vitro drug release The in vitro drug release profile of SAHA-loaded PLGA nanoparticles depicts a biphasic with an initial burst release (Fig. 4) due to the presence of drug on the surface of particles, followed by a sustained pattern owing to the presence of drug inside the core of particles. The drug release from the SAHA nanoparticles was found to be 60% in the first hour and 80% in the 50 h respectively (formulation code—S2). Whereas, the remaining formulations (formulation codes—S1 and S3) show a lesser drug release profile as compared with formulation-S2. Previously Zhang and Feng had prepared paclitaxel-loaded PLA–TPGS copolymeric nanoparticles and found a biphasic release pattern with a 51% release after 31 days. By comparing the release behavior, the SAHA-loaded PLGA nanoparticles elicit the release mechanism of drug diffusion, polymer matrix swelling or erosion [32].
Fig. 6. Intracellular reactive oxygen species level. (a) The A549 cells treated with (a) control, (b) SAHA and (c) SAHA-loaded PLGA nanoparticles for 48 h and captured using a fluorescence microscope (scale bar = 80 μm); (b) the level of reactive oxygen species in A549 cells for 48 h as measured by fluorimetry (*P b 0.05).
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3.3. Antineoplastic activity The in vitro cytotoxicity of SAHA-loaded PLGA nanoparticles, PLGA nanoparticles and pure SAHA was assessed in A549 lung cancer cells using MTT assay. The cytotoxic effect was concentration dependent, the cell viability decreased from 96% to 22% for pure SAHA, whereas for SAHA-loaded PLGA nanoparticles, the viability decreased from 85% to 10% (Fig. 5(a)). Non-loaded PLGA nanoparticles had no cytotoxic effect (data not shown). At 5 μM (Lethal dose), SAHA-loaded PLGA nanoparticles reduced cell viability by 55% while free SAHA reduced cell viability by only 40%. With an increase in SAHA concentration more effective in vitro cytotoxicity was observed. This may be elucidated by the fact that SAHA specially persuades apoptosis in highly proliferating cells, evidence to noticeable cancer cell death. Due to the PLGA nanoparticles enhanced permeability and retention effect specifically accumulate the SAHA drug in cancer cells, showed enhanced cytotoxic activity as compared with free drug [33]. Furthermore, the sustained release profile of SAHA-loaded PLGA nanoparticles can increase the drug efficacy through improving pharmacokinetic and pharmacodynamic profiles as compared with free drug [21]. Cellular and morphological changes that are indicative of apoptosis were studied by AO/EB staining by means of a fluorescence microscope [34]. AO stains both viable and dead cells and produces green fluorescence, whereas EB emits red or orange fluorescence only in nonviable cells. Cells treated with free SAHA or SAHA-loaded PLGA nanoparticles (5 μM) revealed clear signs of apoptosis, such as cellular shrinkage and nuclear condensation (Fig. 5(b)). SAHA-loaded PLGA nanoparticles produced a stronger effect than free SAHA, which may be due to the
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targeted drug delivery property of nanoparticles [35]. The results suggested that SAHA and SAHA-loaded nanoparticles could induce cell death through apoptosis. HDAC inhibitor-induced apoptosis can involve the generation of ROS [15]. Thus, intracellular ROS levels were evaluated using the fluorescent probe DCFH-DA. Fluorescence microscopy analysis shows that cells treated with SAHA or SAHA-loaded nanoparticles (5 μM) exhibited increased fluorescence intensity, indicative of ROS generation (Fig. 6(a)). Total cellular ROS levels were also quantified by a spectrofluorometer, supporting the results obtained by fluorescence microscopy (Fig. 6(b)). The excessive accumulation of ROS in cancer cells induced oxidative stress and loss of cell functioning and leads to cancer cell death. The enhanced ROS level in cancer cells alters the mitochondrial functions and plays a key role in apoptosis stimulation. The permeabilization of the mitochondrial membrane concomitant with the loss of the mitochondrial membrane potential is an early step in apoptosis. It can be determined by the lipophilic cationic fluorescent dye Rhodamine 123, that specifically accumulates in the intact mitochondria. Our analyses show, both by fluorescence microscopy (Fig. 7(a)) and spectrofluorometry (Fig. 7(b)), that treatment with free SAHA or SAHA-loaded nanoparticles (5 μM) induced a significant loss of the Δψm. Our study results clearly emphasize enhanced generation of ROS and the downregulation of Δψm in cancer cells is a key pathway of apoptosis development that leads to low cancer cell viability. Shaikh et al. revealed that PLGA nanoparticles encapsulated curcumin improved drug oral bioavailability in rats when compared with free curcumin [36]. Tran et al. proved that vorinostat loaded solid lipid nanoparticles effectively improved the oral bioavailability, circulation
Fig. 7. Mitochondrial membrane potential. (a) The A549 cells treated with (a) control, (b) SAHA and (c) SAHA-loaded PLGA nanoparticles for 48 h and captured using a fluorescence microscope (scale bar = 100 μm); (b) mitochondrial membrane potential alterations in A549 cells for 48 h as measured by fluorimetry (*P b 0.05).
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half-life, and chemotherapeutic promising of vorinostat [16]. Marslin et al. reported that the Erlotinib-HCl loaded PLGA nanoparticles have less toxic effect than the free drug in experimental rats [20]. Taken together, our results suggest that SAHA and SAHA-loaded nanoparticles elicit apoptosis in A549 lung cancer cells. They also suggest that SAHA-loaded nanoparticles are more effective than free SAHA. 4. Conclusion Here, we have described for the first time preparation of SAHA-loaded PLGA nanoparticles. We have shown that SAHA-loaded PLGA nanoparticles could be effectively prepared by the modified nanoprecipitation method. The successfully formulated SAHA-loaded PLGA nanoparticles were characterized by using cutting-edge techniques like RP-HPLC, FT-IR, Micro-Raman spectrum, DLS, DSC, SEM and TEM. In an in-vitro assay using the lung cancer cell line A549, SAHA-loaded PLGA nanoparticles revealed a superior antineoplastic activity over free SAHA. Thus, our results presented here establish the potential of SAHA-loaded PLGA nanoparticles for the treatment of cancer. Acknowledgements We are grateful to the University Grant Commission (UGC), Government of India for funding this project [Grant no. 40-208/2011 (SR) dated.29.06.2011]. We acknowledge the Department of Science and Technology—Fund for Improvement of S & T Infrastructure in Universities and Higher Educational Institutions (DST-FIST) for their financial support to promote the infrastructure facility of our department [Grant no. SR/FST/LSI-075/2011 dated.20.12.2011]. The authors are grateful to Prof. M. Krishnan, Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli for his help with fluorescence microscopic. We also thank Dr. V. Ramakrishnan, School of Physics, Madurai Kamaraj University, Madurai for his help in MicroRaman spectroscopy studies and Dr. S.N. Jaisankar, Polymer Division, Central Leather Research Institute, Chennai for his help in FT-IR studies. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2015.02.037. References [1] A. Jemal, F. Bray, M.M. Center, J. Ferlay, E. Ward, D. Forman, CA Cancer J. Clin. 61 (2011) 69–90.
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