International Journal of Pharmaceutics 479 (2015) 41–51
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Improved delivery of the natural anticancer drug tetrandrine Chen Shi a,b , Saeed Ahmad Khan a , Kaiping Wang b , Marc Schneider a, * a b
Pharmaceutics and Biopharmacy, Philipps University, 35037 Marburg, Germany Wuhan Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 430022 Wuhan, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 October 2014 Received in revised form 10 December 2014 Accepted 11 December 2014 Available online 13 December 2014
The study aims at designing a nanoparticle-based delivery system to improve the efficacy of the natural compound tetrandrine against lung cancer. Nanoparticles from poly(lactic-co-glycolic acid) (PLGA) were prepared by the emulsion solvent diffusion method and characterized for their physicochemical properties and drug-loading efficiency. Furthermore, the cellular uptake and the anti-cancerous activity was studied on A549 cell line. To investigate the surface properties and uptake, three different stabilizers were used to analyze the effect on size and zeta potential of nanoparticles as well as the effect on the cellular uptake. Nanoparticles in the size range of 180–200 nm with spherical shape were obtained with polyvinyl alcohol (PVA), Pluronic-F127 (PF127) and didodecyldimethylammonium bromide (DMAB), 2%, 1% and 0.1%, respectively. An entrapment efficiency of 50–60% with a loading of 1.5–2% was observed. In vitro release profile at pH 7.4 PBS solution showed a consistent release over 168 h. All particle systems showed an improved performance over the pure drug at the same drug concentration. DMAB stabilized particles demonstrated the most pronounced effect against A549 cells compared to pure drug while PVA stabilized particles were least effective in terms of antitumor activity. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Anti-cancer activity PLGA Cell uptake Surface properties Drug delivery
1. Introduction Lung cancer is the major cause of cancer-related mortality throughout the world (Edwards et al., 2014). Among all lung cancers types, over 75% belong to the non-small cell lung cancer (NSCLC). The surgery and chemotherapeutic options seem to be inadequate in curing NSCLC and the overall 5-year survival rate of all stages of NSCLC could only remain approximately 10–15% (Zochbauer-Muller et al., 2002). Unsatisfactory therapeutic effect is mainly due to the narrow therapeutic window of anticancer drugs, the occurrence of side effects (Simon, 2008), and chemotherapy agents not reaching effectively the target tissue and cells. Tetrandrine (Tet), a bisbenzylisoquinoline alkaloid is the main active ingredient of Stephania tetrandra S. Moore, extracted from the root tuber. Recent pharmacodynamics studies have shown considerable activity against lung cancer cells: for example, Cho and colleagues reported Tet could selectively inhibit the proliferation of lung cancer cells (Cho et al., 2009). Liou et al. studied the molecular mechanism of growth inhibiting and apoptosis
* Corresponding author at: Pharmaceutics and Biopharmacy, Philipps-Universität Marburg, Ketzerbach 63, D-35037 Marburg, Germany. Tel.: +49 6421 282 5885; fax: +49 6421 282 7016. E-mail address: [email protected] (M. Schneider). http://dx.doi.org/10.1016/j.ijpharm.2014.12.022 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.
inducing when Tet treated lung cancer cell line A549 and showed the anticancer activity of Tet is due to an up-regulation of the cyclin-dependent kinase inhibitor p21, an activation of the apoptosis mediator caspase-3, and a down-regulation of cyclin D1 (Liou et al., 2008). Furthermore, Liu et al. tested Tet in combination with gemcitabine treatment for 240 patients with advanced NSCLC and found Tet may improve short-term efficacy, survival, and mitigate adverse reactions to chemotherapy for patients with NSCLC (Liu et al., 2012). However, poor water-solubility (Tet csaturation = 0.015 mg/mL in pH 7.4 phosphate buffer solution (PBS)) and toxicities such as localized ulcer are some of the limitations of Tet application (Li et al., 2009). Furthermore, free agents fail to achieve long retention in tumor tissue. Therefore, alternative strategies are needed to improve Tet delivery to target the site of action effectively. Drug loaded nanoparticles (NPs) are promising for intracellular delivery and offer the possibility to be used for local pulmonary delivery of therapeutics for treating lung diseases (Hein et al., 2009; Nafee et al., 2012, 2014). Delivery of therapeutic agents to the site of action for lung diseases may allow for efficient treatment of lung cancers, lung infections and some other respiratory pathologies (Gelperina et al., 2005). Besides the common benefits obtained from the administration of drug-loaded nanoparticles like improved therapeutic efficiency, reduced side effects and toxicity (Moghimi et al., 2005; Wang et al., 2008), drug loaded nanoparticles have a greater chance to escape
42
C. Shi et al. / International Journal of Pharmaceutics 479 (2015) 41–51
from the clearance mechanisms of the lung defense systems, compared to microparticles (Chono et al., 2006; Schürch et al., 1990). Even though not approved nanocarriers from biocompatible and biodegradable materials such as poly(lactic-co-glycolic acid) (PLGA) offer good prerequisites for pulmonary application (De Souza Carvalho et al., 2014). Particle size or better mean mass aerodynamic diameter are key factors for deposition (De Souza Carvalho et al., 2014). An optimal size range is defined in the micrometer range (Chow et al., 2007) but nanoparticulate carriers are also expected to be efficiently inhaled and deposited (Henning et al., 2010) or applied as structured microparticles from nanoparticles. The size also impacts on the clearance mechanisms and the fate of the particles in the deep lung. Diameters lower than 260 nm will only be weakly taken up by macrophages (Mura et al., 2011; Shoyele and Cawthorne, 2006). Moreover, small particles are more efficiently internalized into cancer cells than larger particles (Panyam and Labhasetwar, 2003). Tahara et al. (2009) reported that A549 cellular uptake of PLGA NP increased, when the particle size was controlled to below the sub- micron region (98%) was obtained from Wuhan Dinghui Chemical Co., Ltd., (Wuhan, China). PLGA 50:50 (Resomer RG 503, MW: 24,000–38,000 Da) was purchased from Evonik Industries, Germany. Polyvinyl alcohol (PVA) (Mowiol 4-88, Kuraray Specialities Europe GmbH, Frankfurt, Germany), Pluronic1 F-127 (PF127), didodecyldimethylammonium bromide (DMAB), fluoresceinamine and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (DMAP) (Sigma–Aldrich, St. Louis, MO, USA) were used as obtained. Dimethylthiazoly-2,5-diphenyltetrazolium bromide (MTT) was obtained from (Promega, Darmstadt, Germany). Millipore water with a resistivity of 18.2 mV cm was used throughout the experiment. All other solvents and chemicals were from the highest grade and commercially available.
FA (0.0583 g) were dissolved entirely in 30 mL of acetonitrile with 0.0408 g of DMAP and incubated at room temperature for 24 h under light protection and gentle stirring. The resultant FA–PLGA was precipitated by the addition of purified water and separated by centrifugation. The polymer was rinsed from excessive reagents (dissolution in acetone and precipitation with ethanol) and then lyophilized (Alpha 2–4 LSC, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode Germany). 2.3. Preparation of the tetrandrine-loaded nanoparticles or fluorescently-labeled PLGA nanoparticles with different surfactants Tetrandrine-loaded PLGA NPs were prepared by the emulsion solvent diffusion evaporation method as previously used (Nafee et al., 2012). Briefly, 20 mg/mL PLGA and Tetrandrine (0.2, 0.3, 0.4, 0.5 mg/mL) were dissolved in ethyl acetate (organic phase). This was then added dropwise into the aqueous phase (containing PVA, PF127 or DMAB), under continuous stirring. After 1 h of stirring the crude emulsion was homogenized using ultrasonifier with 500 J for 30 s (G560E, Scientific Industries, Inc., USA) and then diluted with water to allow ethyl acetate diffusion into the aqueous phase. Subsequently, the suspension was stirred overnight to evaporate ethyl acetate. Nanoparticles were collected using a centrifuge (Rotina420, Hettich, Tuttlingen, Germany) at 14,000 g, for 40 min and redispersed in demineralized water. The fluorescently-labeled PLGA nanoparticles were prepared by the same method using FA–PLGA instead of normal PLGA. For purification of Tet-loaded PLGA NPs, centrifugation of the nanoparticles suspension in Vivaspin-20 tubes (Sartorius tubes with a MWCO of 100,000 Da, Goettingen, Germany) at 14,000 g, for 40 min was performed. The NPs on the filter were collected and washed with demineralized water. Then, nanoparticle suspensions were freeze dried and stored until use at room temperature. 2.4. Characterization of nanoparticles 2.4.1. Physicochemical characterization 2.4.1.1. Size, polydispersity and zeta potential determination. The colloidal properties size, size distribution (polydispersity, PDI), and the zeta potential of the NP was measured using a Malvern Zetasizer Nano (Malvern Instruments, Worcestershire, UK) The mean values were calculated from the measurements performed at least in triplicate. 2.4.1.2. Stability evaluation of colloidal properties. Tetrandrineloaded PLGA NPs were resuspended after centrifugation in pH 7.4 PBS solution and kept at room temperature. Particle sizes were then determined by dynamic light scattering (DLS) every 3 days for 12 days to evaluate colloidal stability. 2.4.2. Morphological characterization The morphology of NPs was visualized by SEM (SEM EVO HD series, Carl Zeiss, Jena, Germany) using high vacuum, applying an acceleration voltage of 1.5 or 4.0 kV. For SEM measurement, a drop of the nanoparticles suspension was placed on a silica wafer. The sample was dried under ambient conditions and coated with gold using a Quorum Q 150 ES sputter coater (Quorum Technologies Ltd., Laughton, UK) to render the sample conductive.
2.2. Preparation of the fluorescently-labeled PLGA polymer
2.5. Measurement of drug loading and in vitro release
Fluoresceinamine (FA) bound PLGA (FA–PLGA) was prepared based upon the method introduced by Horisawa et al. (2002) and modified by Weiss et al. (2007). Briefly, PLGA (3.07 g) and
2.5.1. HPLC analysis for tetrandrine Drug concentrations were analyzed using a HPLC system (Dionex Summit System with P680 gradient pump) with a
C. Shi et al. / International Journal of Pharmaceutics 479 (2015) 41–51
UV detector (Dionex Ultimate 3000 UV/vis detector). A RP18-select B (125 nm 4 mm) column (VWR Corporate, PA, U.S) was used with a column temperature of 35 C. 20 mL of sample was injected into the mobile phase composed of water-acetonitrile-triethylamine (700:300:1) and the pH was adjusted to pH 7.4 with phosphoric acid. Flow rate was 0.7 mL min1 and the retention time was 2.5 min. Detection of tetrandrine was done at a wavelength of l = 280 nm. 2.5.2. Drug encapsulation Encapsulation of the drug into the nanoparticles was evaluated in terms of the encapsulation efficiency (% EE) with respect to the overall used drug amount and the percentage of drug with respect to the overall particle mass (% drug loading). The lyophilized tetrandrine-loaded PLGA NPs were dissolved in acetonitrile, filtered with a chromatic filter 0.2 mm and then distributed into HPLC vials. Tetrandrine content in the samples was determined using HPLC. Encapsulation efficiency and % drug loading were calculated by the Eqs. (1) and (2): % EE ¼
amount of Tet in nanoparticles 100% total amount of Tet
%Drugloading ¼
(1)
ODðsample wellÞ ODðpositive controlÞ ODðnegative wellÞ ODðpositive controlÞ 100%
Cell viabilityð%Þ ¼
2.6. In vitro cell activity studies and particle cellular uptake studies 2.6.1. Dose dependent and time dependent cell activity assay The in vitro dose dependent activity of the nanoparticles was determined by standard MTT assays using A549 human lung carcinoma cells. Briefly, cells were seeded in a 96-well plate (CytoOne STARLAB GmbH, Hamburg, Germany) at a density of 1 104 cells per well 24 h prior to the assay. Then a series of doses of free tetrandrine, tetrandrine-loaded PLGA nanoparticles and blank PLGA NPs modified with different surfactants were incubated with the cells. The dose of tetrandrine was consistent in all nanoparticle formulations compared to free tetrandrine. Meanwhile, as positive control 1% TritonX-100 solution and as negative control only medium was used. After 4 h incubation, 200 mL of 5 mg/mL MTT solution was added to each well and the plate was incubated for another 4 h. Then 100 mL of dimethyl sulphoxide (DMSO) were used to dissolve the dark-blue formazan crystals. The optical density (OD) of each well was measured by an Infinite M200 plate reader (Tecan group Ltd., Mannedorf, Switzerland) at lmax = 570 nm. Cell viability was determined by the following formula (3):
(3)
The concentration which was chosen for the time dependent cell activity assay based on the result of dose dependent cell activity studies. The free Tet, Tet-loaded PLGA NPs, and blank PLGA NPs were applied with the same procedure as for the dose dependent assay but with different incubation times (1 h, 4 h, 8 h and 24 h). Cells viability was then calculated by the same formula. 2.6.2. Cellular uptake study To investigate the particles cellular uptake, A549 cells were seeded in a 96-well plate (Opaque, BRAND GMBH + CO KG, Wertheim, Germany) at a density of 1 104 cells per well. The medium was then replaced with 100 mL fluorescent nanoparticles (0.225 mg/mL, 0.45 mg/mL in medium, pH 7.4) using fluoresceinamine-labeled PLGA (Weiss et al., 2007). The cells were incubated for 1, 4 and 8 h, and then the suspension was removed and the cells were washed three times with PBS. For the cell uptake experiment, cell lysis solution (150 mM NaCl, 1% Triton X-100, 0.1% SDS, 50 mM tris pH 7.4) was used. The uptake efficiency of nanoparticles was determined by the fluoresce signal from the
amount of Tet in nanoparticles 100% amount of Tet in nanoparticlesþamount of the polymeric material used
2.5.3. In vitro release The lyophilized tetrandrine-loaded PLGA NPs with different stabilizers were suspended in 6 mL, 0.01 M phosphate buffered saline (PBS) at pH 7.4. The suspension was then placed in a pre-swelled dialysis bag (3.5 kDa MWCO, Fisher Scientific Inc., Rockford, IL, USA) and immersed into 94 mL, 0.01 M PBS of pH7.4 at constant temperature (37 C) in a shaker. Samples were withdrawn periodically and replaced with the same amount of fresh release medium. For each group, at each time point, three samples were taken (n = 3) for measurements. The amount of released tetrandrine was determined by HPLC analysis.
43
(2)
particles using the Infinite M200 plate reader. The excitation wavelength and emission wavelength was 489 and 518 nm for fluoresceinamine, respectively. The concentration of particles was linearly proportional to the fluorescence intensities in the cell lysis solution, hence cellular uptake efficiency was calculated by the ratio between the fluorescence intensities of particles taken up in cells and the fluorescence intensities of the control (Inegative). The cell uptake efficiency was calculated based on Eq. (4): Uptake efficiencyð%Þ ¼
Isample Inegative 100% Ipositive Inegative
(4)
where Isample, Ipositive and Inegative are the fluorescence intensities of the sample, positive control and negative control, respectively. In these tests, particle suspension was added to five wells without cells as the positive control. In contrast, the negative control should be the case where nanoparticles without fluorescent label were added to the cells. Beside the quantitative analysis of fluoresceinamine-labeled PLGA NPs, confocal laser scanning microscopy (CLSM) was applied to visualize the particles’ uptake behavior. A549 cells were seeded in 12-well plates with a density of 3.0 105 cells/well. Two days after seeding, the growth medium was replaced with fluorescent PLGA NPs in PBS buffer and the uptake was observed after incubation for 1 h, 4 h and 8 h at 37 C by CLSM (ZEISS LSM 510 META NLO, Jena, Germany). The objective used was a C-Apochromat 63/1.2w. The excitation of the NP was performed using lex = 488 nm. The green fluorescent particles were located with respect to the DAPI-stained cell nucleus. Fluorescence excitation of DAPI was performed based on two photon absorption with a pulsed Ti:Sapphire laser (Chameleon XR, Coherent, Germany) operated at lex = 720 nm (150 fs pulses, 80 MHz repetition rate). The fluorescence signal of the particles was detected with a band pass (BP 505–550) (Nafee et al., 2012).
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C. Shi et al. / International Journal of Pharmaceutics 479 (2015) 41–51
2.7. Statistics analysis All data were expressed as mean standard deviation. Statistical analysis were performed using an unpaired, two tailed Student t-test. The result of P < 0.05 was considered to be significant. 3. Results and discussion Nanoparticles should ideally be designed to deliver maximum amount of drug using as few carriers as possible. This can be achieved if the nanoparticles show good entrapment efficiency. Several factors can govern the encapsulation of drugs in nanoparticles, such as the interaction with polymer matrix, the solubility of the drug in the solvent and polymer etc. The choice of polymer and preparation method for entrapment of a certain drug depends on the solubility of the drug. As a universal rule hydrophobic drugs are well encapsulated in hydrophobic polymers and vice versa. PLGA was chosen providing a hydrophobic matrix for Tet and due to its biocompatibility and -degradability. Therefore, our goal was to produce drug loaded PLGA NPs with uniform particle size but different surface. In this attempt stabilizer concentration was primarily studied. 3.1. Effect of stabilizers on physicochemical properties The purpose of stabilizer was not only to prevent coalescence and hence prevent agglomeration (Vandervoort and Ludwig, 2002), but also to vary particles surface properties. This is an important factor for cellular uptake and anti-cancer efficiency of the nanoparticles but also for drug encapsulation and release behavior (Shakweh et al., 2005). Concentration of the three different stabilizers was varied in order to see the effect on size and size distribution (polydispersity index, PDI). Table 1 shows that increasing the stabilizer concentration decreases the size of nanoparticles as expected (Bhardwaj et al., 2009). For instance, the size decreased from 260 to around 200 nm when PVA concentration was increased from 1% to 3%. Similarly, an increase of the PF127 concentration from 1% to 4% reduced the size from around 187 nm to around 145 nm. Whereas in case of DMAB, the size dropped from 205 nm to 164 nm when concentration was increased from 0.1 to 1%. After a certain stabilizer concentration no substantial decrease in size was observed due to full saturation of created surface by the stabilizer, the rest was just excess material. For example, from 1% until 2% of DMAB only around 20 nm
Table 2 Particle size, polydispersity and zeta potential data for Tet-loaded PLGA NPs, blank PLGA NPs and FA-labled PLGA NPs. *SD denotes the standard deviation of (n = 3).
2% PVA–Tet–PLGA 2% PVA–PLGA 2% PVA–FA–PLGA 1% PF127–Tet–PLGA 1% PF127–PLGA 1% PF–FA–PLGA 0.1% DMAB–Tet–PLGA 0.1% DMAB–PLGA 0.1% DMAB–FA–PLGA
Diameter (nm *SD)
Polydispersity (PDI *SD)
Zeta potential (mV *SD)
211.1 2.3 206.1 1.3 207.5 3.1 189.4 3.2 187.4 2.2 192.3 4.2 209.4 2.7 205.4 2.7 203.4 3.5
0.055 0.01 0.075 0.01 0.048 0.021 0.073 0.031 0.065 0.021 0.077 0.024 0.088 0.041 0.059 0.044 0.098 0.037
23.3 3.2 26.0 3.2 25.7 2.9 12.7 1.2 11.6 2.2 13.4 2.1 44.8 3.2 46.8 2.2 42.6 4.4
decrease was seen, and only a few nanometers decrease for PVA and PF127 from 2.5 to 3% and 4 to 5%, respectively. All nanoparticle formulations independent of the stabilizer concentration had a narrow size distribution (PDI
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Improved delivery of the natural anticancer drug tetrandrine Chen Shi a,b , Saeed Ahmad Khan a , Kaiping Wang b , Marc Schneider a, * a b
Pharmaceutics and Biopharmacy, Philipps University, 35037 Marburg, Germany Wuhan Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 430022 Wuhan, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 October 2014 Received in revised form 10 December 2014 Accepted 11 December 2014 Available online 13 December 2014
The study aims at designing a nanoparticle-based delivery system to improve the efficacy of the natural compound tetrandrine against lung cancer. Nanoparticles from poly(lactic-co-glycolic acid) (PLGA) were prepared by the emulsion solvent diffusion method and characterized for their physicochemical properties and drug-loading efficiency. Furthermore, the cellular uptake and the anti-cancerous activity was studied on A549 cell line. To investigate the surface properties and uptake, three different stabilizers were used to analyze the effect on size and zeta potential of nanoparticles as well as the effect on the cellular uptake. Nanoparticles in the size range of 180–200 nm with spherical shape were obtained with polyvinyl alcohol (PVA), Pluronic-F127 (PF127) and didodecyldimethylammonium bromide (DMAB), 2%, 1% and 0.1%, respectively. An entrapment efficiency of 50–60% with a loading of 1.5–2% was observed. In vitro release profile at pH 7.4 PBS solution showed a consistent release over 168 h. All particle systems showed an improved performance over the pure drug at the same drug concentration. DMAB stabilized particles demonstrated the most pronounced effect against A549 cells compared to pure drug while PVA stabilized particles were least effective in terms of antitumor activity. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Anti-cancer activity PLGA Cell uptake Surface properties Drug delivery
1. Introduction Lung cancer is the major cause of cancer-related mortality throughout the world (Edwards et al., 2014). Among all lung cancers types, over 75% belong to the non-small cell lung cancer (NSCLC). The surgery and chemotherapeutic options seem to be inadequate in curing NSCLC and the overall 5-year survival rate of all stages of NSCLC could only remain approximately 10–15% (Zochbauer-Muller et al., 2002). Unsatisfactory therapeutic effect is mainly due to the narrow therapeutic window of anticancer drugs, the occurrence of side effects (Simon, 2008), and chemotherapy agents not reaching effectively the target tissue and cells. Tetrandrine (Tet), a bisbenzylisoquinoline alkaloid is the main active ingredient of Stephania tetrandra S. Moore, extracted from the root tuber. Recent pharmacodynamics studies have shown considerable activity against lung cancer cells: for example, Cho and colleagues reported Tet could selectively inhibit the proliferation of lung cancer cells (Cho et al., 2009). Liou et al. studied the molecular mechanism of growth inhibiting and apoptosis
* Corresponding author at: Pharmaceutics and Biopharmacy, Philipps-Universität Marburg, Ketzerbach 63, D-35037 Marburg, Germany. Tel.: +49 6421 282 5885; fax: +49 6421 282 7016. E-mail address: [email protected] (M. Schneider). http://dx.doi.org/10.1016/j.ijpharm.2014.12.022 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.
inducing when Tet treated lung cancer cell line A549 and showed the anticancer activity of Tet is due to an up-regulation of the cyclin-dependent kinase inhibitor p21, an activation of the apoptosis mediator caspase-3, and a down-regulation of cyclin D1 (Liou et al., 2008). Furthermore, Liu et al. tested Tet in combination with gemcitabine treatment for 240 patients with advanced NSCLC and found Tet may improve short-term efficacy, survival, and mitigate adverse reactions to chemotherapy for patients with NSCLC (Liu et al., 2012). However, poor water-solubility (Tet csaturation = 0.015 mg/mL in pH 7.4 phosphate buffer solution (PBS)) and toxicities such as localized ulcer are some of the limitations of Tet application (Li et al., 2009). Furthermore, free agents fail to achieve long retention in tumor tissue. Therefore, alternative strategies are needed to improve Tet delivery to target the site of action effectively. Drug loaded nanoparticles (NPs) are promising for intracellular delivery and offer the possibility to be used for local pulmonary delivery of therapeutics for treating lung diseases (Hein et al., 2009; Nafee et al., 2012, 2014). Delivery of therapeutic agents to the site of action for lung diseases may allow for efficient treatment of lung cancers, lung infections and some other respiratory pathologies (Gelperina et al., 2005). Besides the common benefits obtained from the administration of drug-loaded nanoparticles like improved therapeutic efficiency, reduced side effects and toxicity (Moghimi et al., 2005; Wang et al., 2008), drug loaded nanoparticles have a greater chance to escape
42
C. Shi et al. / International Journal of Pharmaceutics 479 (2015) 41–51
from the clearance mechanisms of the lung defense systems, compared to microparticles (Chono et al., 2006; Schürch et al., 1990). Even though not approved nanocarriers from biocompatible and biodegradable materials such as poly(lactic-co-glycolic acid) (PLGA) offer good prerequisites for pulmonary application (De Souza Carvalho et al., 2014). Particle size or better mean mass aerodynamic diameter are key factors for deposition (De Souza Carvalho et al., 2014). An optimal size range is defined in the micrometer range (Chow et al., 2007) but nanoparticulate carriers are also expected to be efficiently inhaled and deposited (Henning et al., 2010) or applied as structured microparticles from nanoparticles. The size also impacts on the clearance mechanisms and the fate of the particles in the deep lung. Diameters lower than 260 nm will only be weakly taken up by macrophages (Mura et al., 2011; Shoyele and Cawthorne, 2006). Moreover, small particles are more efficiently internalized into cancer cells than larger particles (Panyam and Labhasetwar, 2003). Tahara et al. (2009) reported that A549 cellular uptake of PLGA NP increased, when the particle size was controlled to below the sub- micron region (98%) was obtained from Wuhan Dinghui Chemical Co., Ltd., (Wuhan, China). PLGA 50:50 (Resomer RG 503, MW: 24,000–38,000 Da) was purchased from Evonik Industries, Germany. Polyvinyl alcohol (PVA) (Mowiol 4-88, Kuraray Specialities Europe GmbH, Frankfurt, Germany), Pluronic1 F-127 (PF127), didodecyldimethylammonium bromide (DMAB), fluoresceinamine and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (DMAP) (Sigma–Aldrich, St. Louis, MO, USA) were used as obtained. Dimethylthiazoly-2,5-diphenyltetrazolium bromide (MTT) was obtained from (Promega, Darmstadt, Germany). Millipore water with a resistivity of 18.2 mV cm was used throughout the experiment. All other solvents and chemicals were from the highest grade and commercially available.
FA (0.0583 g) were dissolved entirely in 30 mL of acetonitrile with 0.0408 g of DMAP and incubated at room temperature for 24 h under light protection and gentle stirring. The resultant FA–PLGA was precipitated by the addition of purified water and separated by centrifugation. The polymer was rinsed from excessive reagents (dissolution in acetone and precipitation with ethanol) and then lyophilized (Alpha 2–4 LSC, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode Germany). 2.3. Preparation of the tetrandrine-loaded nanoparticles or fluorescently-labeled PLGA nanoparticles with different surfactants Tetrandrine-loaded PLGA NPs were prepared by the emulsion solvent diffusion evaporation method as previously used (Nafee et al., 2012). Briefly, 20 mg/mL PLGA and Tetrandrine (0.2, 0.3, 0.4, 0.5 mg/mL) were dissolved in ethyl acetate (organic phase). This was then added dropwise into the aqueous phase (containing PVA, PF127 or DMAB), under continuous stirring. After 1 h of stirring the crude emulsion was homogenized using ultrasonifier with 500 J for 30 s (G560E, Scientific Industries, Inc., USA) and then diluted with water to allow ethyl acetate diffusion into the aqueous phase. Subsequently, the suspension was stirred overnight to evaporate ethyl acetate. Nanoparticles were collected using a centrifuge (Rotina420, Hettich, Tuttlingen, Germany) at 14,000 g, for 40 min and redispersed in demineralized water. The fluorescently-labeled PLGA nanoparticles were prepared by the same method using FA–PLGA instead of normal PLGA. For purification of Tet-loaded PLGA NPs, centrifugation of the nanoparticles suspension in Vivaspin-20 tubes (Sartorius tubes with a MWCO of 100,000 Da, Goettingen, Germany) at 14,000 g, for 40 min was performed. The NPs on the filter were collected and washed with demineralized water. Then, nanoparticle suspensions were freeze dried and stored until use at room temperature. 2.4. Characterization of nanoparticles 2.4.1. Physicochemical characterization 2.4.1.1. Size, polydispersity and zeta potential determination. The colloidal properties size, size distribution (polydispersity, PDI), and the zeta potential of the NP was measured using a Malvern Zetasizer Nano (Malvern Instruments, Worcestershire, UK) The mean values were calculated from the measurements performed at least in triplicate. 2.4.1.2. Stability evaluation of colloidal properties. Tetrandrineloaded PLGA NPs were resuspended after centrifugation in pH 7.4 PBS solution and kept at room temperature. Particle sizes were then determined by dynamic light scattering (DLS) every 3 days for 12 days to evaluate colloidal stability. 2.4.2. Morphological characterization The morphology of NPs was visualized by SEM (SEM EVO HD series, Carl Zeiss, Jena, Germany) using high vacuum, applying an acceleration voltage of 1.5 or 4.0 kV. For SEM measurement, a drop of the nanoparticles suspension was placed on a silica wafer. The sample was dried under ambient conditions and coated with gold using a Quorum Q 150 ES sputter coater (Quorum Technologies Ltd., Laughton, UK) to render the sample conductive.
2.2. Preparation of the fluorescently-labeled PLGA polymer
2.5. Measurement of drug loading and in vitro release
Fluoresceinamine (FA) bound PLGA (FA–PLGA) was prepared based upon the method introduced by Horisawa et al. (2002) and modified by Weiss et al. (2007). Briefly, PLGA (3.07 g) and
2.5.1. HPLC analysis for tetrandrine Drug concentrations were analyzed using a HPLC system (Dionex Summit System with P680 gradient pump) with a
C. Shi et al. / International Journal of Pharmaceutics 479 (2015) 41–51
UV detector (Dionex Ultimate 3000 UV/vis detector). A RP18-select B (125 nm 4 mm) column (VWR Corporate, PA, U.S) was used with a column temperature of 35 C. 20 mL of sample was injected into the mobile phase composed of water-acetonitrile-triethylamine (700:300:1) and the pH was adjusted to pH 7.4 with phosphoric acid. Flow rate was 0.7 mL min1 and the retention time was 2.5 min. Detection of tetrandrine was done at a wavelength of l = 280 nm. 2.5.2. Drug encapsulation Encapsulation of the drug into the nanoparticles was evaluated in terms of the encapsulation efficiency (% EE) with respect to the overall used drug amount and the percentage of drug with respect to the overall particle mass (% drug loading). The lyophilized tetrandrine-loaded PLGA NPs were dissolved in acetonitrile, filtered with a chromatic filter 0.2 mm and then distributed into HPLC vials. Tetrandrine content in the samples was determined using HPLC. Encapsulation efficiency and % drug loading were calculated by the Eqs. (1) and (2): % EE ¼
amount of Tet in nanoparticles 100% total amount of Tet
%Drugloading ¼
(1)
ODðsample wellÞ ODðpositive controlÞ ODðnegative wellÞ ODðpositive controlÞ 100%
Cell viabilityð%Þ ¼
2.6. In vitro cell activity studies and particle cellular uptake studies 2.6.1. Dose dependent and time dependent cell activity assay The in vitro dose dependent activity of the nanoparticles was determined by standard MTT assays using A549 human lung carcinoma cells. Briefly, cells were seeded in a 96-well plate (CytoOne STARLAB GmbH, Hamburg, Germany) at a density of 1 104 cells per well 24 h prior to the assay. Then a series of doses of free tetrandrine, tetrandrine-loaded PLGA nanoparticles and blank PLGA NPs modified with different surfactants were incubated with the cells. The dose of tetrandrine was consistent in all nanoparticle formulations compared to free tetrandrine. Meanwhile, as positive control 1% TritonX-100 solution and as negative control only medium was used. After 4 h incubation, 200 mL of 5 mg/mL MTT solution was added to each well and the plate was incubated for another 4 h. Then 100 mL of dimethyl sulphoxide (DMSO) were used to dissolve the dark-blue formazan crystals. The optical density (OD) of each well was measured by an Infinite M200 plate reader (Tecan group Ltd., Mannedorf, Switzerland) at lmax = 570 nm. Cell viability was determined by the following formula (3):
(3)
The concentration which was chosen for the time dependent cell activity assay based on the result of dose dependent cell activity studies. The free Tet, Tet-loaded PLGA NPs, and blank PLGA NPs were applied with the same procedure as for the dose dependent assay but with different incubation times (1 h, 4 h, 8 h and 24 h). Cells viability was then calculated by the same formula. 2.6.2. Cellular uptake study To investigate the particles cellular uptake, A549 cells were seeded in a 96-well plate (Opaque, BRAND GMBH + CO KG, Wertheim, Germany) at a density of 1 104 cells per well. The medium was then replaced with 100 mL fluorescent nanoparticles (0.225 mg/mL, 0.45 mg/mL in medium, pH 7.4) using fluoresceinamine-labeled PLGA (Weiss et al., 2007). The cells were incubated for 1, 4 and 8 h, and then the suspension was removed and the cells were washed three times with PBS. For the cell uptake experiment, cell lysis solution (150 mM NaCl, 1% Triton X-100, 0.1% SDS, 50 mM tris pH 7.4) was used. The uptake efficiency of nanoparticles was determined by the fluoresce signal from the
amount of Tet in nanoparticles 100% amount of Tet in nanoparticlesþamount of the polymeric material used
2.5.3. In vitro release The lyophilized tetrandrine-loaded PLGA NPs with different stabilizers were suspended in 6 mL, 0.01 M phosphate buffered saline (PBS) at pH 7.4. The suspension was then placed in a pre-swelled dialysis bag (3.5 kDa MWCO, Fisher Scientific Inc., Rockford, IL, USA) and immersed into 94 mL, 0.01 M PBS of pH7.4 at constant temperature (37 C) in a shaker. Samples were withdrawn periodically and replaced with the same amount of fresh release medium. For each group, at each time point, three samples were taken (n = 3) for measurements. The amount of released tetrandrine was determined by HPLC analysis.
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(2)
particles using the Infinite M200 plate reader. The excitation wavelength and emission wavelength was 489 and 518 nm for fluoresceinamine, respectively. The concentration of particles was linearly proportional to the fluorescence intensities in the cell lysis solution, hence cellular uptake efficiency was calculated by the ratio between the fluorescence intensities of particles taken up in cells and the fluorescence intensities of the control (Inegative). The cell uptake efficiency was calculated based on Eq. (4): Uptake efficiencyð%Þ ¼
Isample Inegative 100% Ipositive Inegative
(4)
where Isample, Ipositive and Inegative are the fluorescence intensities of the sample, positive control and negative control, respectively. In these tests, particle suspension was added to five wells without cells as the positive control. In contrast, the negative control should be the case where nanoparticles without fluorescent label were added to the cells. Beside the quantitative analysis of fluoresceinamine-labeled PLGA NPs, confocal laser scanning microscopy (CLSM) was applied to visualize the particles’ uptake behavior. A549 cells were seeded in 12-well plates with a density of 3.0 105 cells/well. Two days after seeding, the growth medium was replaced with fluorescent PLGA NPs in PBS buffer and the uptake was observed after incubation for 1 h, 4 h and 8 h at 37 C by CLSM (ZEISS LSM 510 META NLO, Jena, Germany). The objective used was a C-Apochromat 63/1.2w. The excitation of the NP was performed using lex = 488 nm. The green fluorescent particles were located with respect to the DAPI-stained cell nucleus. Fluorescence excitation of DAPI was performed based on two photon absorption with a pulsed Ti:Sapphire laser (Chameleon XR, Coherent, Germany) operated at lex = 720 nm (150 fs pulses, 80 MHz repetition rate). The fluorescence signal of the particles was detected with a band pass (BP 505–550) (Nafee et al., 2012).
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2.7. Statistics analysis All data were expressed as mean standard deviation. Statistical analysis were performed using an unpaired, two tailed Student t-test. The result of P < 0.05 was considered to be significant. 3. Results and discussion Nanoparticles should ideally be designed to deliver maximum amount of drug using as few carriers as possible. This can be achieved if the nanoparticles show good entrapment efficiency. Several factors can govern the encapsulation of drugs in nanoparticles, such as the interaction with polymer matrix, the solubility of the drug in the solvent and polymer etc. The choice of polymer and preparation method for entrapment of a certain drug depends on the solubility of the drug. As a universal rule hydrophobic drugs are well encapsulated in hydrophobic polymers and vice versa. PLGA was chosen providing a hydrophobic matrix for Tet and due to its biocompatibility and -degradability. Therefore, our goal was to produce drug loaded PLGA NPs with uniform particle size but different surface. In this attempt stabilizer concentration was primarily studied. 3.1. Effect of stabilizers on physicochemical properties The purpose of stabilizer was not only to prevent coalescence and hence prevent agglomeration (Vandervoort and Ludwig, 2002), but also to vary particles surface properties. This is an important factor for cellular uptake and anti-cancer efficiency of the nanoparticles but also for drug encapsulation and release behavior (Shakweh et al., 2005). Concentration of the three different stabilizers was varied in order to see the effect on size and size distribution (polydispersity index, PDI). Table 1 shows that increasing the stabilizer concentration decreases the size of nanoparticles as expected (Bhardwaj et al., 2009). For instance, the size decreased from 260 to around 200 nm when PVA concentration was increased from 1% to 3%. Similarly, an increase of the PF127 concentration from 1% to 4% reduced the size from around 187 nm to around 145 nm. Whereas in case of DMAB, the size dropped from 205 nm to 164 nm when concentration was increased from 0.1 to 1%. After a certain stabilizer concentration no substantial decrease in size was observed due to full saturation of created surface by the stabilizer, the rest was just excess material. For example, from 1% until 2% of DMAB only around 20 nm
Table 2 Particle size, polydispersity and zeta potential data for Tet-loaded PLGA NPs, blank PLGA NPs and FA-labled PLGA NPs. *SD denotes the standard deviation of (n = 3).
2% PVA–Tet–PLGA 2% PVA–PLGA 2% PVA–FA–PLGA 1% PF127–Tet–PLGA 1% PF127–PLGA 1% PF–FA–PLGA 0.1% DMAB–Tet–PLGA 0.1% DMAB–PLGA 0.1% DMAB–FA–PLGA
Diameter (nm *SD)
Polydispersity (PDI *SD)
Zeta potential (mV *SD)
211.1 2.3 206.1 1.3 207.5 3.1 189.4 3.2 187.4 2.2 192.3 4.2 209.4 2.7 205.4 2.7 203.4 3.5
0.055 0.01 0.075 0.01 0.048 0.021 0.073 0.031 0.065 0.021 0.077 0.024 0.088 0.041 0.059 0.044 0.098 0.037
23.3 3.2 26.0 3.2 25.7 2.9 12.7 1.2 11.6 2.2 13.4 2.1 44.8 3.2 46.8 2.2 42.6 4.4
decrease was seen, and only a few nanometers decrease for PVA and PF127 from 2.5 to 3% and 4 to 5%, respectively. All nanoparticle formulations independent of the stabilizer concentration had a narrow size distribution (PDI
