Article pubs.acs.org/molecularpharmaceutics
Enhanced Anticancer Potential of Encapsulated Solid Lipid Nanoparticles of TPD: A Novel Triterpenediol from Boswellia serrata Shashi Bhushan,*,† Vandita Kakkar,‡ Harish Chandra Pal,§ Santosh Kumar Guru,† Ajay Kumar,† D. M. Mondhe,† P. R. Sharma,† Subhash Chandra Taneja,† Indu Pal Kaur,‡ Jaswant Singh,† and A. K. Saxena† †
Indian Institute of Integrative Medicine, CSIR, Canal Road, Jammu 180001, India University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160014, India § Department of Dermatology, University of Alabama at Birmingham, Alabama 35205, United States ‡
S Supporting Information *
ABSTRACT: A pentacyclic triterpenediol (TPD) from Boswellia serrata has significant cytotoxic and apoptotic potential in a large number of human cancer cell lines. To enhance its anticancer potential, it was successfully formulated into solid lipid nanoparticles (SLNs) by the microemulsion method with 75% drug entrapment efficiency. SEM and TEM studies indicated that TPD-SLNs were regular, solid, and spherical particles in the range of 100−200 nm, and the system indicated that they were more or less stable upon storing up to six months. TPD loaded SLNs showed significantly higher cytotoxic/antitumor potential than the parent drug. TPD-SLNs have 40−60% higher cytotoxic and apoptotic potential than the parent drug in terms of IC50, extent of apoptosis, DNA damage, and expression of pro-apoptotic proteins like TNF-R1, cytochrome-c, and PARP cleavage in HL-60 cells. Moreover, blank SLNs did not have any cytotoxic effect on the cancer as well as in normal mouse peritoneal macrophages. The in vivo antitumor potential of TPDSLNs was significantly higher than that of TPD alone in Sarcoma-180 solid tumor bearing mice. Therefore, SLNs of TPD successfully increased the apoptotic and anticancer potential of TPD at comparable doses (both in vitro and in vivo). This work provides new insight into improvising the therapeutic efficacy of TPD by adopting novel delivery strategies such as solid lipid nanoparticles. KEYWORDS: TPD, Boswellia serrata, SLNs, apoptosis, Sarcoma-180 tumor
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INTRODUCTION Plants have been utilized as medicines for thousands of years, and their dominant role is evident from the fact that approximately 60% of anticancer compounds are either natural products or natural product derivatives.1 Many compounds derived from nature or other sources may be cytotoxic, but the selection of only those compounds, which kill the cancer cells by apoptosis rather than necrosis is desirable. Evasion of apoptosis is the hallmark of all cancers, so targeting it provides a bull’s-eye-oriented approach in novel anticancer drug development. Apoptosis has been accepted as the predominant mechanism of drug-induced cell death in preclinical experimental models and in clinically sensitive tumors.2 On the basis of folkloric knowledge, a pentacyclic triterpenediol (TPD) was isolated from gum resin extracts of Boswellia serrata. Some of our preliminary studies have indicated that TPD is able to inhibit proliferation of a large number of human cancer cell lines and strongly induce apoptosis in human leukemia HL-60 cells.3 TPD seems to be a potential plant derived anticancer drug, and several attempts will be required to further increase its anticancer efficacy. Physico-chemicals characteristics of TPD, such as the partition coefficient (log P) and water solubility, were not excellent. Therefore, low solubility, faster metabolic rate, and not very high log P of TPD indicate the © XXXX American Chemical Society
need for appropriate formulation and development so as to achieve improved and sustained bioavailability. In recent years, it has become more and more evident that the development of new drugs alone is not sufficient to ensure progress in drug therapy. Exciting experimental data obtained in vitro are very often followed by disappointing results in vivo. Several problems were encountered with anticancer compounds, such as poor bioavailability, normal tissue toxicity, poor specificity, stability, and high incidence of drug-resistance to tumor cells. Conventionally administered cytotoxic agents often extensively and indiscriminately bind to body tissues and serum protein in a highly unpredictable manner. Only a small fraction of the drugs reaches the tumor site.4 Therefore, the most important goal of anticancer drug delivery is to achieve high intratumor drug concentration and minimize the exposure of drugs to normal tissues. Cancer cells have a defense mechanism categorized as a “cellular” drug resistance or multidrug resistance (MDR) phenotype, which involves active efflux of a broad range of cytotoxic drug from the cytoplasm by Received: July 16, 2012 Revised: November 24, 2012 Accepted: December 3, 2012
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dx.doi.org/10.1021/mp300385m | Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Molecular Pharmaceutics
Article
membrane-bound transporters.5,6 In addition, cancer cells in solid tumors tend to be more resistant to chemotherapy due to various drug permeation barriers, which makes it difficult to achieve high intratumoral drug concentration in solid tumors.5 This type of drug resistance, sometimes referred as noncellular drug resistance, may further lead to compromised clinical outcomes even though an anticancer drug has strong in vitro efficacy. These problems related to cancer chemotherapy can be partially overcome by delivering them using nanoparticles including solid lipid nanoparticles (SLNs).7 SLNs, also referred to as lipospheres or solid lipid nanospheres, are particles of submicrometer size (50 to 1000 nm) made from lipids that remain in a solid state at room and body temperature. They are relatively a new class of drug delivery system and an excellent future prospect to improved cancer chemotherapy. In this study, we report for the first time, the anticancer potential of TPD loaded solid lipid nanoparticles, which were formulated through a microemulsion method with 75% drug entrapment efficiency.8,9 High entrapment efficiency confirms the suitability of the formula and method of preparation. Particle size of TPD loaded SLNs was in the range of 100−200 nm, and it is stable in storage up to 6 months. SEM and TEM studies indicated SLNs to be regular, solid, spherical particles. TPD-SLNs have higher in vitro and in vivo cytotoxic potential than the parent drug. TPD-SLNs, TPD, and blank SLNs (SLNs without TPD) did not have any cytotoxic effect on the cancer as well as normal cells. The present formulation of TPD successfully passed all desired points established before designing the experiments. Therefore, the method and composition of the formulation were accurate and successfully increased the apoptotic and antitumor potential of TPD at comparative doses. This work provides a platform to improvise the therapeutic efficacy of TPD by adopting a novel drug delivery strategy, such as solid lipid nanoparticles (SLNs).
tion technique by dispersing the o/w warm microemulsion in a cold aqueous medium under mechanical stirring.10 Briefly, 4% solution of soy lecithin, Tween-80 and water (1:3:8) were placed together in a beaker and heated to the melting temperature of lipids. However, 400 mg of lipid (Compritol 888 ATO) was also melted (82−85 °C) and the drug (80−100 mg) added to it. The hot aqueous emulsifier mix was dropped into the lipid melt followed by stirring on a magnetic stirrer. The stirring was continued until a clear microemulsion was formed. The hot microemulsion thus formed was transferred, with the help of a hot syringe (to avoid the clogging of the syringe by solidification of the microemulsion) fitted with a 18G needle into cold distilled water (∼2 °C) under stirring at 5000 rpm for 1.5 h. Total Drug Content. One milliliter of the SLN dispersion was taken and disrupted using an appropriate volume of chloroform/methanol (1:1) to get a clear solution. Total drug content of the prepared dispersion was calculated. Determination of Drug Entrapment. SLN dispersion was ultracentrifuged at 90,000 rpm for 2 h at 4 °C. The clear supernatant was decanted, and the drug content in both the supernatant and the pellet was calculated. The SLN pellet was washed with methanol to remove the unentrapped drug (the unentrapped drug will also settle down along with the SLNs) and recentrifuged. Blank SLNs treated in a similar manner were used as the control value to compensate for any interference of the ingredients. The amount of drug in the pellet gave a direct measure of the extent of drug entrapped. Particle Size Analysis. The mean particle size of the drug loaded SLNs was determined by photon correlation spectroscopy (based on the dynamic light scattering method) using a particle size analyzer (Mastersizer 2000−1.00, Malvern Instruments). Appropriate dilutions of the dispersion were made for particle size determination.11−13 Zeta Potential. The surface charge of SLNs was determined by measurement of the zeta potential of the particles extracted from their electrophoretic mobility. The zeta potentials of the formulated SLNs were determined using a Zetasizer (Malvern Instruments, England). Each sample was diluted with water (pH 7.0) and the electrophoretic mobility determined at 25 °C. Scanning Electron Microscopy (SEM) Analysis. The surface morphology of SLNs was examined by SEM.14 The samples were diluted with ultra pure water. The diluted sample was spread over a coverslip and allowed to dry at room temperature. The coverslips were mounted over the stubs. The samples were coated with gold using a Sputter coater (Polaron) and examined with an electron microscope. Transmission Electron Microscopy (TEM) Analysis. The morphology of SLNs was examined by transmission electron microscopy (TEM). The samples were diluted 1:25 with ultra pure water. The specimens were made by placing a small drop of 2% (w/v) phosphotungstic acid (PTA) on a carbon coated, 400-mesh specimen grid and adding an approximately equal quantity of diluted material. The material was sucked up into a drawn out capillary tube, and a small quantity was returned to the grid. Most of this liquid was then removed after a few seconds by touching momentarily the edge of the grid with a filter paper. The grids were then examined with a JEOL 100CXII electron microscope at 60 kV.15 In Vitro Drug Release. The in vitro drug release studies of TPD-SLNs were carried out by the dialysis membrane method.16 The receptor media comprising of phosphate buffer
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MATERIALS AND METHODS Chemicals and Antibodies. RPMI-1640 medium, propidium iodide (PI), DNase-free RNase, proteinaseK, 3-(4,5dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT), phenol/chloroform/iso-amylalcohol, penicillin, streptomycin, L-glutamine, pyruvic acid, ethidium bromide, EDTA, bromophenol blue, agarose, phosphate buffer saline (PBS), triton-X, Hoechst 33258, camptothecin, soy lecithin, and Tween-80 were purchased from Sigma chemical Co. Fetal bovine serum (FBS) was obtained from the GIBCO Invitrogen Corporation U.S.A. AnnexinV-FITC apoptosis detection kit was from B.D. Clontech, U.S.A. Compritol 888 ATO (medium chain triglycerides) was a gift sample from Colorcon Asia Pacific Pvt. Ltd., Singapore. Mouse antihuman antibodies to PARP-1 (#SC8007), TNF-R1 (#SC8436), β-actin (#SC47778), and goat antimouse IgG-HRP (#SC2031) were from Santa Cruz Biotechnology, U.S.A. A mouse antihuman antibody to cytochrome-c (#556433, clone 7H8.2C12) was from BD Biosciences, Pharmingen, U.S.A. Electrophoresis reagents, protein estimation kit, PVDF membrane, and protein markers were from Bio-Rad Laboratories, U.S.A. Hyper film and ECL Plus Western blotting detection kit were from Amersham Biosciences, UK. All other chemicals used were of analytical grade and available locally. Synthesis of TPD. The source of TPD was the same as that described earlier.3 Preparation of Solid Lipid Nanoparticles. Solid lipid nanoparticles (SLNs) were prepared by the micro-emulsificaB
dx.doi.org/10.1021/mp300385m | Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Molecular Pharmaceutics
Article
from HL-60 cells. Briefly, 2 × 106 cells after treatments were centrifuged at 100g for 10 min and washed in PBS containing 20 mM EDTA. The pellet was lysed in 250 μL of lysis buffer (100 mM NaCl, 5 mM EDTA, 10 mM Tris-HCl, pH 8.0, and 5% Triton X-100) containing 400 μg/mL DNase-free RNase and incubated at 37 °C for 90 min followed by 1 h of incubation with proteinase-K (200 μg/mL) at 50 °C. The DNA was extracted with 150 μL of phenol for 1 min and centrifuged 13,000g for 2 min. The aqueous phase was further extracted with phenol/chloroform/isoamylalcohol (25:24:1) and centrifuged. DNA was precipitated from the aqueous phase with 3 volumes of chilled alcohol and 0.3 M sodium acetate at 20 °C overnight. The precipitate was centrifuged at 13,000g for 10 min. The DNA pellet was washed in 80% alcohol, dried, dissolved in 50 μL of TE buffer, mixed in loading buffer, and electrophoresed in 1.8% agarose gel at 50 V for 1.5 h in TAE buffer.3 Hoechst 33258 Staining of Cells for Nuclear Morphology. HL-60 cells were grown in T-25 flasks (2 × 106 cells/4 mL) and treated with TPD and TPD-SLN at 20 μg/ mL for 24 h. Cells were centrifuged at 100g for 5 min and washed twice with PBS. Cells were gently suspended in 100 μL of PBS and fixed in 400 μL of cold acetic acid/methanol (1 + 3, v/v) overnight at 4 °C. Cells were washed again in 1 mL of fixing solution, suspended in the residual volume of about 50 μL, spread on a clean slide, and dried overnight at room temperature. One milliliter of staining solution (Hoechst 33258, 10 μg/mL 0.01 M citric acid, and 0.45 M disodium phosphate containing 0.05% Tween-20) was poured onto each slide and stained for 30 min under subdued light at room temperature. Slides were washed under the gentle flow of tap water, rinsed in distilled water, followed by PBS. While wet, 50 μL of mounting fluid (PBS/glycerol, 1:1) was poured over the center of a slide and covered with a glass coverslip. The slides were sealed with nail polish and observed for any nuclear morphological alterations and apoptotic bodies under inverted fluorescence microscope (Olympus 1X70, magnification 30×) using UV excitation.3 Flow Cytometric Analysis of Apoptosis and Necrosis Using Annexin V/PI Dual Staining. An early event in apoptosis involves the loss of asymmetry in cell membrane phospholipids, altering both the hydrophobicity and charge of the membrane surface. Upon induction of apoptosis, the amount of phosphatidyl serine (PS) on the outer surface of the membrane increases. Annexin V, a calcium-dependent phospholipid-binding protein, has a high affinity for PS.21 Hence, FITC-labeled Annexin V can be used to identify apoptotic cells by flow cytometry. Additional incubation with propidium iodide (PI) is used to distinguish between viable, early apoptotic, necrotic, or late apoptotic cells. Detection of apoptosis by Annexin V-FITC was performed using a Annexin V-FITC Apoptosis Detection Kit from BD Pharmingin (Sandigo U.S.A) according to the manufacturer’s instruction. Cells treated with test material were washed twice with PBS and then resuspended in 100 μL of the annexin binding buffer. The fraction of cell populations in different quadrants was analyzed using quadrant statistics. Cells in the lower right quadrant represented apoptosis and in the upper right quadrant represented postapoptotic necrosis.22 DNA Content and Cell Cycle Phase Distribution. Measurement of DNA content makes it possible to identify apoptotic cells, to recognize the cell cycle phase specificity, and to quantitate apoptosis. For flow cytometry analysis of the
at pH 7.4 pre-equilibrated at 37 °C was used for the studies. Aqueous SLN dispersion was placed in the dialysis tubing, sealed from both the ends, dipped into the receptor media, and stirred continuously at 200−300 rpm. Samples were withdrawn from the receptor medium with replacement at indicated time intervals up to 48 h, and the amount of free or released TPD was calculated. Stability Studies. The SLN dispersion was filled into glass vials and stored at varying conditions. Storage was performed at refrigerated temperature (4 °C) and ambient room temperature (25 °C). For each storage condition, 20 mL of SLN dispersion was packed in 20 mL glass vials. After 6 months, dispersions were evaluated for the change in particle size, total drug content, drug entrapment, and zeta potential.17 Cell Lines, Medium, and Growth Conditions. Human leukemia HL-60 cells were procured from the National Cancer Institute, Frederick, Maryland, U.S.A. Mouse peritoneal macrophages were isolated and cultured as described earlier.18 The cells were grown in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 units/mL), streptomycin (100 μg/mL), L-glutamine (0.3 mg/ mL), pyruvic acid (0.11 mg/mL), 0.37% NaHCO3, and 50 μM of 2-mercaptoethanol at 37 °C in an atmosphere of 95% air and 5% CO2 with 98% humidity. Cells grown in logarithmic phase were treated with tested materials dissolved in a suitable vehicle (DMSO/water), while the untreated control cultures received only the vehicle (
