Colloids and Surfaces B: Biointerfaces 117 (2014) 21–28
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Formulation, characterization and cytotoxicity studies of alendronate sodium-loaded solid lipid nanoparticles Jafar Ezzati Nazhad Dolatabadi a,b , Hamed Hamishehkar c , Morteza Eskandani a,b , Hadi Valizadeh c,d,∗ a
Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran Student research committee, Tabriz University of Medical Sciences, Tabriz, Iran c Pharmaceutical Technology Laboratory, Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran d Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran b
a r t i c l e
i n f o
Article history: Received 15 August 2013 Received in revised form 4 January 2014 Accepted 24 January 2014 Available online 9 February 2014 Keywords: Solid lipid nanoparticle SLN Alendronate sodium Compritol Cytotoxicity
a b s t r a c t Aim: Solid lipid nanoparticles (SLNs) are novel drug delivery system for drug targeting in various routs of administration such as parenteral, oral, ophthalmic and topical. These carriers have some advantages such as high drug payload, increased drug stability, the possibility of incorporation of lipophilic and hydrophilic drugs, and low biotoxicity. In this study, alendronate sodium was used as a hydrophilic model drug and was incorporated into SLNs. Methods: Hot homogenization method was used for preparation of alendronate sodium-loaded SLN formulations and the encapsulation efficiency of drug in SLNs was determined by ultrafiltration method using centrifugal devices. Scanning electron microscopy (SEM) was carried out to study the morphological behaviors of prepared SLNs like sphericity. Several cytotoxicity studies including MTT, DAPI staining and DNA fragmentation assays were used for biocompatibility assays. Results: High drug encapsulation efficiency (70–85%) was achieved by drug determination through derivatization with o-phthalaldehyde. The physical stability of drug-loaded SLNs in aqueous dispersions was assessed in terms of size and drug leakage during two weeks. Scanning electron microscopy images showed spherical particles in the nanometer range confirming the obtained data from size analyzer. Several cytotoxicity studies including MTT, DAPI staining and DNA fragmentation assays as well as flow cytometry analysis confirmed the low toxicity of alendronate-loaded SLNs. Conclusion: The cost-efficient procedure, the avoidance of organic solvents application, acceptable reproducibility, ease of manufacturing under mild preparation conditions, high level of drug encapsulation, desirable physical stability and biocompatibility are the advantages of the proposed SLN formulations. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Solid lipid nanoparticles (SLNs) are novel drug carrier system with submicron size particles (50–1000 nm), which consists of a solid lipid matrix at both room and body temperatures, stabilized by surfactant. SLNs are considered as substitute carriers to traditional colloidal systems, for controlled and targeted delivery. SLNs possess high biocompatibility and biodegradability which
∗ Corresponding author at: Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. Tel.: +98 411 3392649; fax: +98 411 3344790. E-mail addresses: [email protected], [email protected] (H. Valizadeh). http://dx.doi.org/10.1016/j.colsurfb.2014.01.055 0927-7765/© 2014 Elsevier B.V. All rights reserved.
are capable of incorporating both lipophilic and hydrophilic compounds [1,2]. In addition, enhanced cell uptake of various drugs has also been described [3–5]. Their production involves a very simple emulsification/solidification process which does not require any organic solvents. This would enable successful scale up for industry. Besides, compared with nano-emulsions, which are prepared with liquid lipids, SLNs have more potential for controlled release, owing to their solid matrix [6–8]. Alendronate sodium is a hydrophillic, amphiprotic drug, which is administered orally for treatment of bone Pagets disease, postmenopausal osteoporosis, primary hyperparathyroidism, malignant hypercalcemia and metastatic bone diseases [9–12]. It increases bone formation and enhances osteoblast proliferation and maturation and leads to inhibition of osteoblast apoptosis [11].
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Fig. 1. The differences in formulated SLNs size between the initial (A) and 4 weeks (B) timepoint were not found to be statistically significant.
However, oral administration of alendronate sodium is associated with gastrointestinal intolerance. Gastric and esophageal lesions like erosions and ulcers have been reported due to a local irritant effect of the drug [13–15]. This medication in patients with gastrointestinal problems as well as esophageal abnormalities such as achalasia is contraindicated [15]. On the other hand, oral bioavailability of alendronate sodium is very low. Intake together with meals and beverages other than water reduces its bioavailability even more [16,17]. Poor absorption is attributed to its high hydrophilicity and complexation with divalent cations, like Ca2+ [16]. To overcome the poor bioavailability of alendronate sodium and the adverse gastrointestinal effects, investigation on alternative administration strategies have been performed [18]. Intravenous administration of alendronate sodium may raise the risk of nephrotoxicity. Subcutaneous and intramuscular administrations may result in local soft tissue damage and irritation at the site of injection [16,19]. Sutton and co-workers demonstrated the nasal delivery of alendronate sodium in dogs and rats as an alternative to per oral and parenteral administrations. Pulmonary delivery may help to avoid gastrointestinal tract problems such as, low bioavailability, gut irritability, unwanted metabolites, and food effects [20]. To the best of our knowledge no inhalable alendronate SLN formulations have been introduced in the literature [21]. The aim of this study was to design and optimize a novel alendronate sodiumloaded SLNs carrier system composed of a Compritol 888 ATO as lipid matrix for the pulmonary delivery of alendronate sodium. Laser light scattering and zeta potential were performed to characterize the properties of SLNs. All excipients evaluated in this study showed low toxicity on A549 cell line.
2. Materials and methods 2.1. Materials Alendronate monosodium trihydrate was obtained by Modava Company (Iran). Poloxamer 407 and Trypsin-EDTA (0.02–0.05%) were purchased from Sigma Aldrich Co. (Poole, UK). Tween 20 was purchased from Oleon (Olegem, Belgium). RPMI1640 medium and fetal bovine serum (FBS) was supplied by Gibco, Invitrogen (Paisley, UK). Annexin V-FITC apoptosis detection kit was purchased from Oncogene Research Products (San Diego, CA, USA). Precirol® ATO 5 and Compritol 888 ATO were obtained from Gattefosse (France). A549 lung carcinoma cell line, cell culture plates and flasks were obtained from the national cell bank of Iran (Pasteur institute, Iran) and IWAKI, Japan, respectively. 2.2. Preparation of SLNs Alendronate sodium-loaded SLN formulations were prepared by hot homogenization according to published methods [22]. Solid lipid (Precirol® ATO 5 or Compritol 888 ATO® ) with 100 l Tween 20 as a surfactant was heated at 80 ◦ C in a boiling water bath to be melt under continuous stirring (oil phase). In a separate container, surfactant (Poloxamer 407) was dissolved in ultra-pure water and heated to the same temperature of the oil phase (aqueous phase). The drug was dissolved in 2 ml of aqueous phase and added into the oil phase under homogenization at 20,000 rpm (Heidolph, Germany) to form the initial water-in-oil emulsion. Then the hot aqueous phase was added dropwise into the oil phase under the homogenization at 20,000 rpm while maintaining the temperature
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at 80 ◦ C. In order to obtain a nanoparticle suspension, the produced nano-emulsion was cooled down to room temperature [6,22].
on a glass lamella, air-dried, gold coated under vacuum, and then analyzed.
2.3. Particle size, polydispersity index and zeta potential measurement
2.7. Cell culture and viability assessment
Particle size of the SLN was measured by laser light scattering technique using particle size analyzer. Polydispersity index (PI) and zeta potential (ZP) of the nanoparticle were measured by dynamic light scattering technique using Malvern Zetasizer Nano ZS (Malvern Instruments, UK). For zeta potential measurement, disposable folded capillary cuvette was used. Air bubbles were removed from the capillary before measurement. All measurements were carried out in triplicate. 2.4. Drug encapsulation efficiency (EE) and drug loading (DL) The encapsulation efficiency of alendronate sodium in SLNs was determined by ultrafiltration method using centrifugal devices (Amicon® Ultra-4 100k, Millipore, USA) with a 100 kDa molecular weight cut-off membrane. Briefly, filter device was filled with SLNs nano-suspension and centrifuged at 5000 rpm for 10 min. The ultra-filtrate solution was analyzed for determination of unloaded drug concentration. Since alendronate has no UV–visible absorbing molecular functional group, samples were analyzed through derivatization reaction with o-phthalaldehyde (OPA) followed by UV–vis spectrophotometry (UV-mini 1240; Shimadzu, Japan) at 333 nm [12]. The method was fully validated for linearity, accuracy and precision. The samples of blank SLNs exhibited no UV interfering substances. To evaluate the total concentration of alendronate sodium in SLNs, an aliquot of SLN dispersion was dissolved in a mixture of tetrahydrofuran (THF)/methanol (MeOH) 40:60 (v/v). This mixture, which keeps alendronate sodium in solution but causes lipid precipitation, was centrifuged for 10 min at 5000 rpm. The supernatant was analyzed for drug content. To assess the non-encapsulated alendronate sodium, an aliquot (2 ml) of the SLNs nano-suspension was submitted to ultrafiltration (10 min at 5000 rpm) to separate the aqueous phase from SLNs. The aqueous ultrafilterate was analyzed by aforementioned method. The encapsulation efficiency (EE) and drug loading (DL) were calculated using the following equations [7,23]: EE (%) =
(CT − CAP ) × 100 CT
DL (mg/g) =
WDL WNP
where CT is total added drug concentration, CAP drug concentration in aqueous phase, WDL weight of drug loaded in nanoparticles and WNP is the weight of nanoparticles solid mass.
To evaluate the influence of alendronate sodium, alendronate sodium loaded SLNs and blank SLNs on the cellular viability; A549 cells (adenocarcinomic human alveolar basal epithelial cells) were seeded and cultured up to 40–50% confluency in the 96-well plates prior to treatment. The cells were exposed to a range of sample concentrations. Following incubation for 24 h in a humidified incubator (95% air and 5% CO2 ) at 37 ◦ C, the cells were washed once with phosphate buffered saline (PBS). The culture medium was replaced with 150 l of fresh medium. Fifty microliters of MTT reagent (2 mg/ml in PBS) was added to each well. After 4 h incubation at 37 ◦ C, medium was removed and cells were exposed to 200 l of DMSO and 25 l of Sorenson buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5). The cells were incubated for 30 min at 37 ◦ C to ensure complete dissolution of formazan crystals formed by metabolically active cells. absorbance was measured at 570 nm using a spectrophotometric plate reader, ELx 800 (Biotek, CA, USA) [24]. 2.8. DAPI staining assay Chromatin fragmentation can be determined using DAPI staining assay. A549 cells were seeded in six-well plates (5 × 104 cells/well) containing 12 mm cover-slips and subsequently treated for various times with alendronate sodium, blank SLNs, drug-loaded SLNs and with DMSO (200 l) as positive control. Cells then were fixed with 4% paraformaldehyde, permeabilized in 0.1% (w/v) Triton X-100 for 5 min, washed in PBS and stained with DAPI and washed again with PBS. 2.9. FITC-labeled annexin V apoptosis assay To detect the induction of early and late apoptosis, FITC-labeled annexin V assay was carried out. In brief, treated cells were washed three times in PBS buffer gently and detached by tripsinization and washed three times with 500 l 1× binding buffer. Then 1–4 × 106 of single unfixed cell suspensions were re-suspended in 100 l 1× binding buffer containing 5 l FITC-labeled Annexin V in the dark for 14 min at 37 ◦ C. Cells were washed again with 500 l 1× binding buffer and were exposed to 100 l 1× binding buffer plus 5 l propidium iodide (PI). Cells were analyzed by Becton Dickinson FACS Calibur System (San Jose, USA) with an emission filters of 515–545 nm for FITC (green) and 600 nm for PI (red) [25]. 2.10. DNA fragmentation assay
Physical stability of the samples was determined by particle size measurements and was evaluated at 1, 2 and 4 weeks at 4–9 ◦ C. All reported particle size data is the mean of three separate measurements. The DL and drug leakage were also analyzed on the same time intervals.
The probable DNA fragmentation was analyzed by agarose gel electrophoresis. In brief, treated cells were incubated in the lysis buffer (pH 7.4) containing 50 mM Tris base, 10 mM EDTA, 0.5% sodium dodecyl sulfate (SDS) and 5 units RNase for 5 min at 37 ◦ C. After denaturation of proteins with 500 l of chloroform/isoamyl alcohol (24:1), total DNA was separated by centrifugation at 12,000 rpm. The total DNA was precipitated with isopropranol and electrophoresed in 1.2% agarose gel [26,27].
2.6. Particle morphological examination
2.11. Statistical analysis
Scanning electron microscopy (SEM, Vega Tescan., Czech Republic) of the prepared SLNs was performed to study the morphological behaviors like sphericity and aggregation. Briefly, SLN formulations were diluted 2-fold with ultrapure water, mounted
All particle size, drug encapsulation efficiency and in vitro leakage rate measurements were performed in triplicate. Means and standard deviations were calculated using Microsoft® Excel 2010. Mean values were compared using Student’s t-test.
2.5. Physical stability
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9.25 ± 0.330 16.26 ± 0.53 16.22 ± 0.74 15.44 ± 0.67 21.44 ± 0.89 21.39 ± 0.92 20.61 ± 0.89 20.88 ± 0.79 21.10 ± 0.94 23.39 ± 1.02 24.53 ± 1.09 45.46 ± 2.02 46.51 ± 2.11 85.10 ± 4.36 92.82 ± 4.65 51.58 ± 3.12 80.06 ± 4.23 82.67 ± 4.01 55.41 ± 3.23 85.62 ± 4.27 78.93 ± 3.97 82.43 ± 3.89 73.67 ± 3.25 76.05 ± 3.45 78.82 ± 4.01 75.95 ± 3.64 79.63 ± 4.12 80.45 ± 4.24 84.23 ± 4.26 85.36 ± 4.31 0.623 ± 0.031 0.645 ± 0.045 0.676 ± 0.049 0.246 ± 0.013 0.256 ± 0.012 0.253 ± 0.014 0.162 ± 0.009 0.171 ± 0.010 0.262 ± 0.014 0.259 ± 0.012 0.264 ± 0.015 0.223 ± 0.011 0.234 ± 0.015 0.261 ± 0.017 0.227 ± 0.012 Poloxamer 407 was used as aqueous phase surfactant in all formulations.
Particle size (nm) Aqueous phase surfactant concentration* (g) Oil phase surfactant type and concentration (g)
316 ± 9.23 402 ± 9.62 684 ± 9.54 98 ± 4.12 105 ± 4.45 105 ± 4.32 77 ± 3.56 76 ± 3.72 95 ± 4.57 80 ± 4.04 85 ± 4.12 95 ± 4.36 96 ± 4.47 88 ± 3.89 86 ± 3.92 *
MTT assay as cell viability study was conducted to estimate the toxicity of alendronate sodium and alendronate sodium-loaded SLNs (Fig. 3). The influence of drug-loaded SLNs on the viability of A549 cells was tiny and even lower than that of pure intact drug. As
Precirol (9.00) Precirol (5.44) Precirol (5.40) Compritol (6.00) Compritol (4.00) Compritol (4.00) Compritol (4.20) Compritol (4.00) Compritol (4.00) Compritol (4.00) Compritol (4.00) Compritol (2.00) Compritol (2.00) Compritol (1.10) Compritol (1.10)
3.5. Effects of drug-loaded SLNs on cell growth
F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15
Morphology of SLNs was observed using scanning electron microscopy (SEM). The SEM images of drug-loaded SLNs showed spherical particles in shape in the nanometer range (Fig. 2). The mean particle size obtained from the SEM measurements was around 100 nm, confirming results obtained from laser particle size analyzer.
Drug concentration (g)
3.4. Morphology of selected SLN formulation
Lipid type and concentration (g)
Formulations composed of 10% (w/v) alendronate, a surfactant system consisting of 4% Tween 80 and 42% ploxamer 407 in a solid lipid matrix of 100% Compritol was placed on long term stability at 4–9 ◦ C for up 4 weeks. At pre-determined time intervals, samples were withdrawn and the nano-suspension was checked for any potential aggregation. The polydispersity index values of 0.227, 0.254 and 0.262 were obtained for the initial, 2 weeks, and 4 weeks of storage respectively. There was no significant change in mean particle size upon aging (Fig. 1; p > 0.05). Similar observations have been reported by other researchers [2,7,8]. The DL of the SLNs on the production day was 92.82 mg/g, indicating the suitability of the formulation for alendronate incorporation. Moreover, after 4 weeks of storage at 4 ◦ C DL remained high; 91.04 mg/g. The small decrease in DL is indicative of a negligible drug expulsion from the SLNs. This is a typical feature of SLN formulations, and has been illustrated previously [28,29]. These values were in accordance with the results obtained for particle sizes and PI. Storage at a low temperature provided better stability with regards to both particle size and DL.
Formulation code
3.3. Physical stability
Table 1 Composition, particle size and encapsulation efficiency (EE) of Alendronate loaded SLNs formulation. Data is expressed as mean ± SD (n = 3).
Table 1 shows the influence of fatty acid and surfactant type on EE. There is an evident improvement of EE with application of Compritol. The EE was 51.58 for SLNs prepared with Precirol without any surfactant. When 4% Tween 20 was used, EE was increased to 82.67 and 85.62 in SLNs prepared with Precirol and Compritol, respectively. The EE for the SLNs prepared with Compritol was greater than that of SLNs prepared with Precirol. The particle size of SLNs prepared with Compritol was smaller than SLNs prepared with Precirol. SLNs prepared with Compritol containing 2% of Tween 80 exhibited the highest DL values (92.82 mg/g) (Table 1). Because of favorable results in terms of encapsulation efficiency, particle size and zeta potential, the next steps were focused on formulations composed of Compritol.
Poly-dispersity
3.2. Drug loading
8.14 4.00 4.00 4.20 3.00 3.00 3.10 3.20 4.00 4.00 4.00 2.00 2.00 1.00 1.00
Encapsulation efficiency (EE %)
SLNs were prepared using Precirol® ATO 5 and Compritol 888 ATO® , stabilized using a surfactant, i.e. Tween 80 or Tween 20 and a co-surfactant (Poloxamer 407). The size of particles was generally smaller in the case of SLNs prepared with Compritol when compared to SLNs prepared with Precirol (Table 1). Formulations composed of Compritol exhibited small particle size of below 95 nm and zeta potential of −1.74. The polydispersity index (PDI) was well below 0.27, indicating a narrow particle size distribution (Table 1).
Tween 20 (0.00) Tween 20 (0.24) Tween 20 (0.30) Tween 20 (0.00) Tween 20 (0.30) Tween 20 (0.30) Tween 20 (0.30) Tween 20 (0.30) Tween 20 (0.35) Tween 20 (0.35) Tween 80 (0.35) Tween 80 (0.20) Tween 80 (0.10) Tween 80 (0.05) Tween 80 (0.05)
3.1. Preparation and characterization SLNs
0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.18 0.20 0.21 0.20 0.20 0.20 0.22
Drug loading (DL, mg/g)
3. Results
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the nucleus of cell’s treated with alendronate sodium and alendronate sodium-loaded SLN and their morphology was the same as untreated normal cells. 3.7. FITC-labeled annexin V apoptosis assay To further clarify the nature of the chromatin fragmentation and to quantify early and late apoptosis in the treated cells, annexinV assay was carried out. The obtained results confirmed the result of DAPI staining assay. As shown in Fig. 5, drug-loaded SLNs does not induce considerable apoptosis or necrosis of A549 cells. The mortality/apoptosis of A549 cells treated with drug-loaded SLNs was approximately the same as that of the control cells. In contrast, the positive control cells treated with 5% DMSO for 24 h, showed increased apoptosis (44%). 3.8. DNA fragmentation assay DNA fragmentation assay has long been applied to determine the side effects of various materials and discriminate apoptosis from necrosis, and it is among the most reliable methods for detection of apoptotic cells [26]. In the current study, we examined DNA fragmentation assay to confirm whether SLNs exhibit side effect on DNA of A549 cells. According to the results, drug-loaded SLNs neither induced apoptosis nor necrosis on A549 cells. Fig. 6 clearly indicates that alendronate-loaded SLNs did not result in the formation of DNA-ladder (trailing) in treated cells. 4. Discussion
Fig. 2. SEM images of SLN formulations: (A) blank SLNs and (B) alendronate-loaded SLNs.
it is obvious in Fig. 3, at the highest concentration more than 90% of the cell viability remains. 3.6. DAPI staining assay Fig. 4 represents DAPI staining for nucleus shrinkage assessment in A549 cells treated with alendronate sodium and alendronate sodium-loaded SLNs. Morphology of DAPI stained cells showed no obvious fragmentation in the chromatin and DNA rings within
Alendronate is currently one of the major classes of drugs for the treatment of osteoporosis. von Konch et al. indicated that alendronate take a promoting effect on the osteogenic differentiation of mesenchymal stem cells. However, a severe gastrointestinal irritation has been reported for this drug which may even lead to discontinue the treatment and prevention of osteoporosis [30,31]. Drug encapsulation offers a way to evade tissue toxicity caused by its direct drug exposure [32,33]. SLNs have attracted major attention as particulate systems to develop the delivery of lipophilic drugs due their affinity to the lipid matrix. However, the encapsulation of hydrophilic alendronate sodium might be a challenging issue which has not been reported yet. We hypothesized that lipids, such as the Precirol and Compritol, could be an interesting alternative to improve alendronate sodium encapsulation into SLNs, while reducing alendronate sodium cytotoxicity [34–36]. It has been reported that the preparation process and the composition of SLNs have significant impact on physicochemical characteristics such as size and drug loading efficiency [9,37]. The application of Tween 20 and Tween 80 as oil phase surfactant lead to smaller size and steady-state of alendronate-loaded SLNs [38]. But we preferred to replace Tween 80 with Tween 20 after finding some evidences about cytotoxicity effect of Tween 20 on A549 and HUVEC cell lines [25]. The best EE and DL of obtained SLNs was around 85.62% and 92.82 mg/g, respectively. The data clearly showed a significant increase of the alendronate sodium encapsulation when the Compritol was used. Besides, the size of the prepared SLNs was considerably smaller than that of SLNs prepared by Precirol. Therefore, we can suggest that Compritol is a suitable lipid for the formulation of alendronate sodium-loaded SLNs. SEM images of drug-loaded SLNs showed that the particles are spherical with uniform shapes. The round shape of the particles supported the hypothesis of partial transformation of glyceryl behenate (Compritol) from the metastable  to the more stable i configuration [7,8,33]. For biocompatibility assessment, the prepared formulations were evaluated for cyto/genotoxicity with MTT, DAPI staining and
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Fig. 3. Dose- and time- dependent inhibition properties of alendronate and alendronate-loaded SLNs on A549 cells. The figure illustrates that alendronate-loaded SLNs have not shown any significant cytotoxicity on A549 cells compared to alendronate. Error bars represent SD (n = 3).
DNA fragmentation assays and flow cytometry analysis. The cytocompatibility of the drug-loaded SLNs was compared with that of pure intact drug. Viability of cells treated with alendronate-loaded SLNs was considerably higher than that of alendronate [39,40]. The lack of toxicity of both intact drug and SLNs on nucleus of cells was further confirmed by DAPI staining and DNA fragmentation
assessments. DNA cleavage and chromatin fragmentation is seen when cell apoptosis occurs [24,25,31,41]. No morphological changes were observed in the nucleus of treated cells. Moreover, cell cycle analysis of treated A549 cells, determined by FITC-labeled annexin V flow cytometry, indicated that alendronate-loaded SLNs did not promote significant apoptosis and even necrosis. Overall,
Fig. 4. Fluorescent and light microscopy images of treated and untreated A549 cells, which have been stained with DAPI. The figure shows no chromatin fragmentation occurrence in the nucleus of treated cells with alendronate and alendronate-loaded SLNs.
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Fig. 6. DNA ladder assay. Genomic DNA of (1) alendronate, (2) blank SLNs, (3) alendronate sodium loaded SLN, (4) untreated and (5) DMSO (5%) treated A549 cells.
5. Conclusions Alendronate sodium-loaded SLN suspensions were successfully formulated by simple hot homogenization technique with high encapsulation efficiency (85%) and nano ranged particle size with narrow distribution. The safety and lack of toxicity of alendronate sodium-loaded SLN to A549 cells was confirmed by various cytotoxicity assay procedures. The obtained findings suggest that the proposed SLN formulation could be employed as a surrogate for the traditionally available products used in the treatment of different diseases such as bone Pagets disease, postmenopausal osteoporosis, primary hyperparathyroidism, malignant hypercalcemia and metastatic bone diseases. Acknowledgments Authors would like to thank Research Center for Pharmaceutical Nanotechnology (RCPN), Tabriz University of Medical Sciences for supporting this project (grant No: 5/87/260, which was a part of PhD thesis No: 90/014/104/2). References
Fig. 5. FITC-labeled annexin V flow cytometric detection of apoptosis in A549 cells. (A) Untreated control cells, (B) alendronate-loaded SLNs-treated cells (2 mg/ml) and (C) positive control (DMSO-treated cells). As it is clear in the figure, alendronate sodium loaded SLN does not induce considerable apoptosis or necrosis of A549 cells and the mortality/apoptosis of alendronate sodium loaded SLN-treated A549 cells is approximately the same as that of the control cells.
it can be deduced that alendronate-loaded SLNs did not inhibit the growth of A549 cell line and may have no significant cyotoxicity. Therefore, it might be a suitable candidate for delivery of alendronate with reduced side effects.
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Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Formulation, characterization and cytotoxicity studies of alendronate sodium-loaded solid lipid nanoparticles Jafar Ezzati Nazhad Dolatabadi a,b , Hamed Hamishehkar c , Morteza Eskandani a,b , Hadi Valizadeh c,d,∗ a
Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran Student research committee, Tabriz University of Medical Sciences, Tabriz, Iran c Pharmaceutical Technology Laboratory, Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran d Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran b
a r t i c l e
i n f o
Article history: Received 15 August 2013 Received in revised form 4 January 2014 Accepted 24 January 2014 Available online 9 February 2014 Keywords: Solid lipid nanoparticle SLN Alendronate sodium Compritol Cytotoxicity
a b s t r a c t Aim: Solid lipid nanoparticles (SLNs) are novel drug delivery system for drug targeting in various routs of administration such as parenteral, oral, ophthalmic and topical. These carriers have some advantages such as high drug payload, increased drug stability, the possibility of incorporation of lipophilic and hydrophilic drugs, and low biotoxicity. In this study, alendronate sodium was used as a hydrophilic model drug and was incorporated into SLNs. Methods: Hot homogenization method was used for preparation of alendronate sodium-loaded SLN formulations and the encapsulation efficiency of drug in SLNs was determined by ultrafiltration method using centrifugal devices. Scanning electron microscopy (SEM) was carried out to study the morphological behaviors of prepared SLNs like sphericity. Several cytotoxicity studies including MTT, DAPI staining and DNA fragmentation assays were used for biocompatibility assays. Results: High drug encapsulation efficiency (70–85%) was achieved by drug determination through derivatization with o-phthalaldehyde. The physical stability of drug-loaded SLNs in aqueous dispersions was assessed in terms of size and drug leakage during two weeks. Scanning electron microscopy images showed spherical particles in the nanometer range confirming the obtained data from size analyzer. Several cytotoxicity studies including MTT, DAPI staining and DNA fragmentation assays as well as flow cytometry analysis confirmed the low toxicity of alendronate-loaded SLNs. Conclusion: The cost-efficient procedure, the avoidance of organic solvents application, acceptable reproducibility, ease of manufacturing under mild preparation conditions, high level of drug encapsulation, desirable physical stability and biocompatibility are the advantages of the proposed SLN formulations. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Solid lipid nanoparticles (SLNs) are novel drug carrier system with submicron size particles (50–1000 nm), which consists of a solid lipid matrix at both room and body temperatures, stabilized by surfactant. SLNs are considered as substitute carriers to traditional colloidal systems, for controlled and targeted delivery. SLNs possess high biocompatibility and biodegradability which
∗ Corresponding author at: Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. Tel.: +98 411 3392649; fax: +98 411 3344790. E-mail addresses: [email protected], [email protected] (H. Valizadeh). http://dx.doi.org/10.1016/j.colsurfb.2014.01.055 0927-7765/© 2014 Elsevier B.V. All rights reserved.
are capable of incorporating both lipophilic and hydrophilic compounds [1,2]. In addition, enhanced cell uptake of various drugs has also been described [3–5]. Their production involves a very simple emulsification/solidification process which does not require any organic solvents. This would enable successful scale up for industry. Besides, compared with nano-emulsions, which are prepared with liquid lipids, SLNs have more potential for controlled release, owing to their solid matrix [6–8]. Alendronate sodium is a hydrophillic, amphiprotic drug, which is administered orally for treatment of bone Pagets disease, postmenopausal osteoporosis, primary hyperparathyroidism, malignant hypercalcemia and metastatic bone diseases [9–12]. It increases bone formation and enhances osteoblast proliferation and maturation and leads to inhibition of osteoblast apoptosis [11].
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Fig. 1. The differences in formulated SLNs size between the initial (A) and 4 weeks (B) timepoint were not found to be statistically significant.
However, oral administration of alendronate sodium is associated with gastrointestinal intolerance. Gastric and esophageal lesions like erosions and ulcers have been reported due to a local irritant effect of the drug [13–15]. This medication in patients with gastrointestinal problems as well as esophageal abnormalities such as achalasia is contraindicated [15]. On the other hand, oral bioavailability of alendronate sodium is very low. Intake together with meals and beverages other than water reduces its bioavailability even more [16,17]. Poor absorption is attributed to its high hydrophilicity and complexation with divalent cations, like Ca2+ [16]. To overcome the poor bioavailability of alendronate sodium and the adverse gastrointestinal effects, investigation on alternative administration strategies have been performed [18]. Intravenous administration of alendronate sodium may raise the risk of nephrotoxicity. Subcutaneous and intramuscular administrations may result in local soft tissue damage and irritation at the site of injection [16,19]. Sutton and co-workers demonstrated the nasal delivery of alendronate sodium in dogs and rats as an alternative to per oral and parenteral administrations. Pulmonary delivery may help to avoid gastrointestinal tract problems such as, low bioavailability, gut irritability, unwanted metabolites, and food effects [20]. To the best of our knowledge no inhalable alendronate SLN formulations have been introduced in the literature [21]. The aim of this study was to design and optimize a novel alendronate sodiumloaded SLNs carrier system composed of a Compritol 888 ATO as lipid matrix for the pulmonary delivery of alendronate sodium. Laser light scattering and zeta potential were performed to characterize the properties of SLNs. All excipients evaluated in this study showed low toxicity on A549 cell line.
2. Materials and methods 2.1. Materials Alendronate monosodium trihydrate was obtained by Modava Company (Iran). Poloxamer 407 and Trypsin-EDTA (0.02–0.05%) were purchased from Sigma Aldrich Co. (Poole, UK). Tween 20 was purchased from Oleon (Olegem, Belgium). RPMI1640 medium and fetal bovine serum (FBS) was supplied by Gibco, Invitrogen (Paisley, UK). Annexin V-FITC apoptosis detection kit was purchased from Oncogene Research Products (San Diego, CA, USA). Precirol® ATO 5 and Compritol 888 ATO were obtained from Gattefosse (France). A549 lung carcinoma cell line, cell culture plates and flasks were obtained from the national cell bank of Iran (Pasteur institute, Iran) and IWAKI, Japan, respectively. 2.2. Preparation of SLNs Alendronate sodium-loaded SLN formulations were prepared by hot homogenization according to published methods [22]. Solid lipid (Precirol® ATO 5 or Compritol 888 ATO® ) with 100 l Tween 20 as a surfactant was heated at 80 ◦ C in a boiling water bath to be melt under continuous stirring (oil phase). In a separate container, surfactant (Poloxamer 407) was dissolved in ultra-pure water and heated to the same temperature of the oil phase (aqueous phase). The drug was dissolved in 2 ml of aqueous phase and added into the oil phase under homogenization at 20,000 rpm (Heidolph, Germany) to form the initial water-in-oil emulsion. Then the hot aqueous phase was added dropwise into the oil phase under the homogenization at 20,000 rpm while maintaining the temperature
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at 80 ◦ C. In order to obtain a nanoparticle suspension, the produced nano-emulsion was cooled down to room temperature [6,22].
on a glass lamella, air-dried, gold coated under vacuum, and then analyzed.
2.3. Particle size, polydispersity index and zeta potential measurement
2.7. Cell culture and viability assessment
Particle size of the SLN was measured by laser light scattering technique using particle size analyzer. Polydispersity index (PI) and zeta potential (ZP) of the nanoparticle were measured by dynamic light scattering technique using Malvern Zetasizer Nano ZS (Malvern Instruments, UK). For zeta potential measurement, disposable folded capillary cuvette was used. Air bubbles were removed from the capillary before measurement. All measurements were carried out in triplicate. 2.4. Drug encapsulation efficiency (EE) and drug loading (DL) The encapsulation efficiency of alendronate sodium in SLNs was determined by ultrafiltration method using centrifugal devices (Amicon® Ultra-4 100k, Millipore, USA) with a 100 kDa molecular weight cut-off membrane. Briefly, filter device was filled with SLNs nano-suspension and centrifuged at 5000 rpm for 10 min. The ultra-filtrate solution was analyzed for determination of unloaded drug concentration. Since alendronate has no UV–visible absorbing molecular functional group, samples were analyzed through derivatization reaction with o-phthalaldehyde (OPA) followed by UV–vis spectrophotometry (UV-mini 1240; Shimadzu, Japan) at 333 nm [12]. The method was fully validated for linearity, accuracy and precision. The samples of blank SLNs exhibited no UV interfering substances. To evaluate the total concentration of alendronate sodium in SLNs, an aliquot of SLN dispersion was dissolved in a mixture of tetrahydrofuran (THF)/methanol (MeOH) 40:60 (v/v). This mixture, which keeps alendronate sodium in solution but causes lipid precipitation, was centrifuged for 10 min at 5000 rpm. The supernatant was analyzed for drug content. To assess the non-encapsulated alendronate sodium, an aliquot (2 ml) of the SLNs nano-suspension was submitted to ultrafiltration (10 min at 5000 rpm) to separate the aqueous phase from SLNs. The aqueous ultrafilterate was analyzed by aforementioned method. The encapsulation efficiency (EE) and drug loading (DL) were calculated using the following equations [7,23]: EE (%) =
(CT − CAP ) × 100 CT
DL (mg/g) =
WDL WNP
where CT is total added drug concentration, CAP drug concentration in aqueous phase, WDL weight of drug loaded in nanoparticles and WNP is the weight of nanoparticles solid mass.
To evaluate the influence of alendronate sodium, alendronate sodium loaded SLNs and blank SLNs on the cellular viability; A549 cells (adenocarcinomic human alveolar basal epithelial cells) were seeded and cultured up to 40–50% confluency in the 96-well plates prior to treatment. The cells were exposed to a range of sample concentrations. Following incubation for 24 h in a humidified incubator (95% air and 5% CO2 ) at 37 ◦ C, the cells were washed once with phosphate buffered saline (PBS). The culture medium was replaced with 150 l of fresh medium. Fifty microliters of MTT reagent (2 mg/ml in PBS) was added to each well. After 4 h incubation at 37 ◦ C, medium was removed and cells were exposed to 200 l of DMSO and 25 l of Sorenson buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5). The cells were incubated for 30 min at 37 ◦ C to ensure complete dissolution of formazan crystals formed by metabolically active cells. absorbance was measured at 570 nm using a spectrophotometric plate reader, ELx 800 (Biotek, CA, USA) [24]. 2.8. DAPI staining assay Chromatin fragmentation can be determined using DAPI staining assay. A549 cells were seeded in six-well plates (5 × 104 cells/well) containing 12 mm cover-slips and subsequently treated for various times with alendronate sodium, blank SLNs, drug-loaded SLNs and with DMSO (200 l) as positive control. Cells then were fixed with 4% paraformaldehyde, permeabilized in 0.1% (w/v) Triton X-100 for 5 min, washed in PBS and stained with DAPI and washed again with PBS. 2.9. FITC-labeled annexin V apoptosis assay To detect the induction of early and late apoptosis, FITC-labeled annexin V assay was carried out. In brief, treated cells were washed three times in PBS buffer gently and detached by tripsinization and washed three times with 500 l 1× binding buffer. Then 1–4 × 106 of single unfixed cell suspensions were re-suspended in 100 l 1× binding buffer containing 5 l FITC-labeled Annexin V in the dark for 14 min at 37 ◦ C. Cells were washed again with 500 l 1× binding buffer and were exposed to 100 l 1× binding buffer plus 5 l propidium iodide (PI). Cells were analyzed by Becton Dickinson FACS Calibur System (San Jose, USA) with an emission filters of 515–545 nm for FITC (green) and 600 nm for PI (red) [25]. 2.10. DNA fragmentation assay
Physical stability of the samples was determined by particle size measurements and was evaluated at 1, 2 and 4 weeks at 4–9 ◦ C. All reported particle size data is the mean of three separate measurements. The DL and drug leakage were also analyzed on the same time intervals.
The probable DNA fragmentation was analyzed by agarose gel electrophoresis. In brief, treated cells were incubated in the lysis buffer (pH 7.4) containing 50 mM Tris base, 10 mM EDTA, 0.5% sodium dodecyl sulfate (SDS) and 5 units RNase for 5 min at 37 ◦ C. After denaturation of proteins with 500 l of chloroform/isoamyl alcohol (24:1), total DNA was separated by centrifugation at 12,000 rpm. The total DNA was precipitated with isopropranol and electrophoresed in 1.2% agarose gel [26,27].
2.6. Particle morphological examination
2.11. Statistical analysis
Scanning electron microscopy (SEM, Vega Tescan., Czech Republic) of the prepared SLNs was performed to study the morphological behaviors like sphericity and aggregation. Briefly, SLN formulations were diluted 2-fold with ultrapure water, mounted
All particle size, drug encapsulation efficiency and in vitro leakage rate measurements were performed in triplicate. Means and standard deviations were calculated using Microsoft® Excel 2010. Mean values were compared using Student’s t-test.
2.5. Physical stability
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9.25 ± 0.330 16.26 ± 0.53 16.22 ± 0.74 15.44 ± 0.67 21.44 ± 0.89 21.39 ± 0.92 20.61 ± 0.89 20.88 ± 0.79 21.10 ± 0.94 23.39 ± 1.02 24.53 ± 1.09 45.46 ± 2.02 46.51 ± 2.11 85.10 ± 4.36 92.82 ± 4.65 51.58 ± 3.12 80.06 ± 4.23 82.67 ± 4.01 55.41 ± 3.23 85.62 ± 4.27 78.93 ± 3.97 82.43 ± 3.89 73.67 ± 3.25 76.05 ± 3.45 78.82 ± 4.01 75.95 ± 3.64 79.63 ± 4.12 80.45 ± 4.24 84.23 ± 4.26 85.36 ± 4.31 0.623 ± 0.031 0.645 ± 0.045 0.676 ± 0.049 0.246 ± 0.013 0.256 ± 0.012 0.253 ± 0.014 0.162 ± 0.009 0.171 ± 0.010 0.262 ± 0.014 0.259 ± 0.012 0.264 ± 0.015 0.223 ± 0.011 0.234 ± 0.015 0.261 ± 0.017 0.227 ± 0.012 Poloxamer 407 was used as aqueous phase surfactant in all formulations.
Particle size (nm) Aqueous phase surfactant concentration* (g) Oil phase surfactant type and concentration (g)
316 ± 9.23 402 ± 9.62 684 ± 9.54 98 ± 4.12 105 ± 4.45 105 ± 4.32 77 ± 3.56 76 ± 3.72 95 ± 4.57 80 ± 4.04 85 ± 4.12 95 ± 4.36 96 ± 4.47 88 ± 3.89 86 ± 3.92 *
MTT assay as cell viability study was conducted to estimate the toxicity of alendronate sodium and alendronate sodium-loaded SLNs (Fig. 3). The influence of drug-loaded SLNs on the viability of A549 cells was tiny and even lower than that of pure intact drug. As
Precirol (9.00) Precirol (5.44) Precirol (5.40) Compritol (6.00) Compritol (4.00) Compritol (4.00) Compritol (4.20) Compritol (4.00) Compritol (4.00) Compritol (4.00) Compritol (4.00) Compritol (2.00) Compritol (2.00) Compritol (1.10) Compritol (1.10)
3.5. Effects of drug-loaded SLNs on cell growth
F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15
Morphology of SLNs was observed using scanning electron microscopy (SEM). The SEM images of drug-loaded SLNs showed spherical particles in shape in the nanometer range (Fig. 2). The mean particle size obtained from the SEM measurements was around 100 nm, confirming results obtained from laser particle size analyzer.
Drug concentration (g)
3.4. Morphology of selected SLN formulation
Lipid type and concentration (g)
Formulations composed of 10% (w/v) alendronate, a surfactant system consisting of 4% Tween 80 and 42% ploxamer 407 in a solid lipid matrix of 100% Compritol was placed on long term stability at 4–9 ◦ C for up 4 weeks. At pre-determined time intervals, samples were withdrawn and the nano-suspension was checked for any potential aggregation. The polydispersity index values of 0.227, 0.254 and 0.262 were obtained for the initial, 2 weeks, and 4 weeks of storage respectively. There was no significant change in mean particle size upon aging (Fig. 1; p > 0.05). Similar observations have been reported by other researchers [2,7,8]. The DL of the SLNs on the production day was 92.82 mg/g, indicating the suitability of the formulation for alendronate incorporation. Moreover, after 4 weeks of storage at 4 ◦ C DL remained high; 91.04 mg/g. The small decrease in DL is indicative of a negligible drug expulsion from the SLNs. This is a typical feature of SLN formulations, and has been illustrated previously [28,29]. These values were in accordance with the results obtained for particle sizes and PI. Storage at a low temperature provided better stability with regards to both particle size and DL.
Formulation code
3.3. Physical stability
Table 1 Composition, particle size and encapsulation efficiency (EE) of Alendronate loaded SLNs formulation. Data is expressed as mean ± SD (n = 3).
Table 1 shows the influence of fatty acid and surfactant type on EE. There is an evident improvement of EE with application of Compritol. The EE was 51.58 for SLNs prepared with Precirol without any surfactant. When 4% Tween 20 was used, EE was increased to 82.67 and 85.62 in SLNs prepared with Precirol and Compritol, respectively. The EE for the SLNs prepared with Compritol was greater than that of SLNs prepared with Precirol. The particle size of SLNs prepared with Compritol was smaller than SLNs prepared with Precirol. SLNs prepared with Compritol containing 2% of Tween 80 exhibited the highest DL values (92.82 mg/g) (Table 1). Because of favorable results in terms of encapsulation efficiency, particle size and zeta potential, the next steps were focused on formulations composed of Compritol.
Poly-dispersity
3.2. Drug loading
8.14 4.00 4.00 4.20 3.00 3.00 3.10 3.20 4.00 4.00 4.00 2.00 2.00 1.00 1.00
Encapsulation efficiency (EE %)
SLNs were prepared using Precirol® ATO 5 and Compritol 888 ATO® , stabilized using a surfactant, i.e. Tween 80 or Tween 20 and a co-surfactant (Poloxamer 407). The size of particles was generally smaller in the case of SLNs prepared with Compritol when compared to SLNs prepared with Precirol (Table 1). Formulations composed of Compritol exhibited small particle size of below 95 nm and zeta potential of −1.74. The polydispersity index (PDI) was well below 0.27, indicating a narrow particle size distribution (Table 1).
Tween 20 (0.00) Tween 20 (0.24) Tween 20 (0.30) Tween 20 (0.00) Tween 20 (0.30) Tween 20 (0.30) Tween 20 (0.30) Tween 20 (0.30) Tween 20 (0.35) Tween 20 (0.35) Tween 80 (0.35) Tween 80 (0.20) Tween 80 (0.10) Tween 80 (0.05) Tween 80 (0.05)
3.1. Preparation and characterization SLNs
0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.18 0.20 0.21 0.20 0.20 0.20 0.22
Drug loading (DL, mg/g)
3. Results
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the nucleus of cell’s treated with alendronate sodium and alendronate sodium-loaded SLN and their morphology was the same as untreated normal cells. 3.7. FITC-labeled annexin V apoptosis assay To further clarify the nature of the chromatin fragmentation and to quantify early and late apoptosis in the treated cells, annexinV assay was carried out. The obtained results confirmed the result of DAPI staining assay. As shown in Fig. 5, drug-loaded SLNs does not induce considerable apoptosis or necrosis of A549 cells. The mortality/apoptosis of A549 cells treated with drug-loaded SLNs was approximately the same as that of the control cells. In contrast, the positive control cells treated with 5% DMSO for 24 h, showed increased apoptosis (44%). 3.8. DNA fragmentation assay DNA fragmentation assay has long been applied to determine the side effects of various materials and discriminate apoptosis from necrosis, and it is among the most reliable methods for detection of apoptotic cells [26]. In the current study, we examined DNA fragmentation assay to confirm whether SLNs exhibit side effect on DNA of A549 cells. According to the results, drug-loaded SLNs neither induced apoptosis nor necrosis on A549 cells. Fig. 6 clearly indicates that alendronate-loaded SLNs did not result in the formation of DNA-ladder (trailing) in treated cells. 4. Discussion
Fig. 2. SEM images of SLN formulations: (A) blank SLNs and (B) alendronate-loaded SLNs.
it is obvious in Fig. 3, at the highest concentration more than 90% of the cell viability remains. 3.6. DAPI staining assay Fig. 4 represents DAPI staining for nucleus shrinkage assessment in A549 cells treated with alendronate sodium and alendronate sodium-loaded SLNs. Morphology of DAPI stained cells showed no obvious fragmentation in the chromatin and DNA rings within
Alendronate is currently one of the major classes of drugs for the treatment of osteoporosis. von Konch et al. indicated that alendronate take a promoting effect on the osteogenic differentiation of mesenchymal stem cells. However, a severe gastrointestinal irritation has been reported for this drug which may even lead to discontinue the treatment and prevention of osteoporosis [30,31]. Drug encapsulation offers a way to evade tissue toxicity caused by its direct drug exposure [32,33]. SLNs have attracted major attention as particulate systems to develop the delivery of lipophilic drugs due their affinity to the lipid matrix. However, the encapsulation of hydrophilic alendronate sodium might be a challenging issue which has not been reported yet. We hypothesized that lipids, such as the Precirol and Compritol, could be an interesting alternative to improve alendronate sodium encapsulation into SLNs, while reducing alendronate sodium cytotoxicity [34–36]. It has been reported that the preparation process and the composition of SLNs have significant impact on physicochemical characteristics such as size and drug loading efficiency [9,37]. The application of Tween 20 and Tween 80 as oil phase surfactant lead to smaller size and steady-state of alendronate-loaded SLNs [38]. But we preferred to replace Tween 80 with Tween 20 after finding some evidences about cytotoxicity effect of Tween 20 on A549 and HUVEC cell lines [25]. The best EE and DL of obtained SLNs was around 85.62% and 92.82 mg/g, respectively. The data clearly showed a significant increase of the alendronate sodium encapsulation when the Compritol was used. Besides, the size of the prepared SLNs was considerably smaller than that of SLNs prepared by Precirol. Therefore, we can suggest that Compritol is a suitable lipid for the formulation of alendronate sodium-loaded SLNs. SEM images of drug-loaded SLNs showed that the particles are spherical with uniform shapes. The round shape of the particles supported the hypothesis of partial transformation of glyceryl behenate (Compritol) from the metastable  to the more stable i configuration [7,8,33]. For biocompatibility assessment, the prepared formulations were evaluated for cyto/genotoxicity with MTT, DAPI staining and
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Fig. 3. Dose- and time- dependent inhibition properties of alendronate and alendronate-loaded SLNs on A549 cells. The figure illustrates that alendronate-loaded SLNs have not shown any significant cytotoxicity on A549 cells compared to alendronate. Error bars represent SD (n = 3).
DNA fragmentation assays and flow cytometry analysis. The cytocompatibility of the drug-loaded SLNs was compared with that of pure intact drug. Viability of cells treated with alendronate-loaded SLNs was considerably higher than that of alendronate [39,40]. The lack of toxicity of both intact drug and SLNs on nucleus of cells was further confirmed by DAPI staining and DNA fragmentation
assessments. DNA cleavage and chromatin fragmentation is seen when cell apoptosis occurs [24,25,31,41]. No morphological changes were observed in the nucleus of treated cells. Moreover, cell cycle analysis of treated A549 cells, determined by FITC-labeled annexin V flow cytometry, indicated that alendronate-loaded SLNs did not promote significant apoptosis and even necrosis. Overall,
Fig. 4. Fluorescent and light microscopy images of treated and untreated A549 cells, which have been stained with DAPI. The figure shows no chromatin fragmentation occurrence in the nucleus of treated cells with alendronate and alendronate-loaded SLNs.
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Fig. 6. DNA ladder assay. Genomic DNA of (1) alendronate, (2) blank SLNs, (3) alendronate sodium loaded SLN, (4) untreated and (5) DMSO (5%) treated A549 cells.
5. Conclusions Alendronate sodium-loaded SLN suspensions were successfully formulated by simple hot homogenization technique with high encapsulation efficiency (85%) and nano ranged particle size with narrow distribution. The safety and lack of toxicity of alendronate sodium-loaded SLN to A549 cells was confirmed by various cytotoxicity assay procedures. The obtained findings suggest that the proposed SLN formulation could be employed as a surrogate for the traditionally available products used in the treatment of different diseases such as bone Pagets disease, postmenopausal osteoporosis, primary hyperparathyroidism, malignant hypercalcemia and metastatic bone diseases. Acknowledgments Authors would like to thank Research Center for Pharmaceutical Nanotechnology (RCPN), Tabriz University of Medical Sciences for supporting this project (grant No: 5/87/260, which was a part of PhD thesis No: 90/014/104/2). References
Fig. 5. FITC-labeled annexin V flow cytometric detection of apoptosis in A549 cells. (A) Untreated control cells, (B) alendronate-loaded SLNs-treated cells (2 mg/ml) and (C) positive control (DMSO-treated cells). As it is clear in the figure, alendronate sodium loaded SLN does not induce considerable apoptosis or necrosis of A549 cells and the mortality/apoptosis of alendronate sodium loaded SLN-treated A549 cells is approximately the same as that of the control cells.
it can be deduced that alendronate-loaded SLNs did not inhibit the growth of A549 cell line and may have no significant cyotoxicity. Therefore, it might be a suitable candidate for delivery of alendronate with reduced side effects.
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