Enhanced Apoptotic Effect of Curcumin Loaded Solid Lipid

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Enhanced Apoptotic Effect of Curcumin Loaded Solid Lipid Nanoparticles Kakkar Vandita,†,§ Bhushan Shashi,‡,§ Kumar Guru Santosh,‡ and Kaur Indu Pal*,† †

Department of Pharmaceutics, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160014, India Division of Cancer Pharmacology, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu-180001, India

‡

ABSTRACT: Curcumin is reported to show potent in vitro anticancer effects in a surfeit of human cancer cell lines and majorly in the carcinogenesis of GIT, in animals. Its poor pharmacokinetics and stability limit its vivo clinical efficacy for the other systemic cancers. We recently reported on a 32−155 times enhancement in bioavailability of curcumin when incorporated into solid lipid nanoparticles (C-SLNs). Presently we report on a 54−85% reduction in IC 50 values with developed C-SLNs in comparison to free curcumin against a panel of human cancer cell lines (HL-60, A549, and PC3). Results demonstrate mechanisms similar to those claimed for free curcumin, including induction of cellular apoptosis by activation of caspases, release of cyctochrome c, loss of membrane potential, blockade of nuclear factor kappa B (NF-κB) activation, and upregulation of TNF-R for C-SLNs. However, the extent of cell death provided by C-SLNs in all these tests was significantly higher (p < 0.001). This may be attributed to the presentation of curcumin in a dispersible/soluble form which enhanced permeability across the cell surface. The display of significantly better in vitro anticancer effect coupled with high in vivo bioavailability points toward a great potential of using CSLNs for cancer therapeutics. KEYWORDS: apoptosis, bioavailability, caspases, curcumin, solid lipid nanoparticles

1.0. INTRODUCTION

apoptotic and other mechanistic studies with C-SLNs. Results were compared with those obtained for free curcumin. Cell cytotoxicity was evaluated after treatment with curcumin and its developed solid lipid nanoparticles (C-SLNs) (2.7−54 μM) in A549, PC3, and human leukemia HL-60 cell lines. An insight into the molecular events leading to C-SLNs induced apoptosis in HL 60 cells were also demonstrated. The observed apoptotic activity of this bioavailable form of curcumin was also evaluated in terms of loss of mitochondrial membrane potential, release of pro-apoptotic factors, and activation of caspases, which together account for apoptotic cell death. The latter were supplemented with fluorescence microscopy, flowcytometry, and fluorescence intensity, to establish the mechanism of enhanced efficacy of developed C-SLNs over free curcumin.

The role of curcumin as an anticancer agent is well established in the literature.1,2 It has been shown to protect against different cancers, including leukemia and lymphoma, gastrointestinal cancers, genitourinary cancers, breast cancer, ovarian cancer, head and neck squamous cell carcinoma, lung cancer, melanoma, neurological cancers, and sarcoma, in in vitro studies including cancerous cell lines such as HL-60, K562, MCF-7, and HeLa.3−6 The multitargeting ability of curcumin may be the key to its therapeutic anticancer potential, which helps modulate as many as 33 different proteins, including thioredoxin reductase, cyclooxygenase-2 (COX-2), protein kinase C (PKC), 5-lipoxygenase, and tubulin.6 Other molecular targets modulated by this agent include transcription factors, growth factors and their receptors, cytokines, enzymes, and genes regulating cell proliferation and apoptosis.6−8 Studies by various researchers working in the area of nanotechnology have elaborated on the preparation9,10 of nanoparticles and their evaluation in term of in vitro anticancer efficacy.11−13 We have previously established a significantly enhanced bioavailability (32−155 times) of curcumin achieved with solid lipid nanoparticles (C-SLNs) using a highly sensitive and validated LC/MS/MS method.14,15 In a view to observe the effect of prepared lipidic nanoparticles on improvising and maintaining the anticancer potential of curcumin, we conducted © 2012 American Chemical Society

2.0. MATERIALS AND METHODS 2.1. Chemicals and Antibodies. RPMI-1640 medium, rhodamine-123 (Rh-123), propidium iodide (PI), DNase-free RNase, proteinase K, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT), eukaryotic protease inhibitor Received: Revised: Accepted: Published: 3411

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treated with C-SLNs or curcumin dissolved in dimethyl sulfoxide (DMSO) while the untreated control cultures received only the vehicle (DMSO, ≤0.2%). 2.6. Cell Proliferation Assay using MTT. This assay is a quantitative colorimetric method for determination of cell survival and proliferation. The assessed parameter is the metabolic activity of viable cells. Metabolically active cells reduce pale yellow tetrazolium salt (MTT) to a dark blue water-insoluble formazan which can be, after solubilization with DMSO, directly quantified. The absorbance of the formazan directly correlates with the number of viable cells.17 The cells were plated in 96-well plates at a density of 2.0 × 104 in 200 μL of medium per well. Cultures were incubated with different concentrations of test material and incubated for 48 h. A longer incubation of 48 h (instead of 24 h) was used to ensure a high extent (almost 100%) of kill, taking HL-60 as the reference. The medium was replaced with fresh medium containing 100 μg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for 4 h. The supernatant was aspirated, and MTT-formazan crystals were dissolved in 100 μL of DMSO, and the OD of the resulting solution was measured at λ540 nm (reference wavelength, λ620 nm) on an ELISA reader (Thermo Laboratories, USA). Cell growth was calculated by comparing the absorbance of treated versus untreated cells. 2.7. DNA Agarose Gel Electrophoresis. Apoptosis was also assessed by electrophoresis of extracted genomic DNA from cells. Briefly, 2 × 106 cells after various 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, 5% Triton X-100) containing 400 μg/mL DNase-free RNase and incubated at 37 °C for 90 min followed by 1 h incubation with proteinase-K (200 μg/mL) at 50 °C for 1 h. The DNA was extracted with 150 μL of phenol for 1 min and centrifuged at 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 by incubating overnight with 3 volumes of chilled alcohol and 0.3 M sodium acetate at 20 °C. 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 a mixture of tris base, acetic acid, and EDTA (TAE) buffer. 2.8. DNA Content and Cell Cycle Phase Distribution. DNA fragmentation constitutes one biochemical hallmark of apoptosis. Thus, 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 relative nuclear DNA content, the fluorescent dye propidium iodide (PI), which becomes highly fluorescent after binding to DNA, is most commonly used. After permeabilization, PI binds to DNA in cells at all stages of the cell cycle, and the intensity with which a cell nucleus emits fluorescent light is directly proportional to its DNA content. The results of the measurement are illustrated in a histogram, where the number of cells (counts) is plotted against the relative fluorescence intensity of PI (FL-2; λem: 585 nm). The histogram reflects the cell cycle distribution of the cell population. Staining normal untreated cells with PI, most of the cells are in the G0/G1 phase (DNA content: 2n) and emit light at a uniform frequency, depicted in the prominent G0/G1 peak of the histogram. Cells treated with the test material were collected, washed in PBS, fixed in 70% cold ethanol, and placed

cocktail, penicillin, and streptomycin were purchased from Sigma Chemical Co. Fetal bovine serum was obtained from GIBCO Invitrogen Corporation USA. Caspase-8 and -9 inhibitors and ApoAlert caspases assay kits were from B.D. Clontech, USA. The mouse antihuman antibodies B-Actin and TNF-R1 were from Santa Cruz Biotechnology, USA. Electrophoresis reagents, the protein estimation kit, and protein markers were purchased from Bio-Rad Laboratories, USA. The hyper film and ECL Plus Western blotting detection kit was obtained from Amersham Biosciences, U.K. 2.2. Preparation of C-SLNs. Polysorbate 80 (45.45% v/v), soy lecithin (0.58% w/v), and water were placed together in a beaker and heated to the lipid melt temperature. Compritol 888 ATO i.e. glyceryl behenate (lipid; 7.27% w/v) was melted separately at 82−85 °C. Curcumin was added to the aqueous phase, following which the hot aqueous emulsifier mix was dropped at once into the lipid melt, under magnetic stirring to obtain a clear microemulsion. The hot microemulsion thus formed was transferred into an equivalent amount of cold water (∼2 °C) under continuous mechanical stirring (5000 rpm) for 1.5 h. In the aqueous medium, SLNs are formed by crystallization of the oil droplets present in the microemulsion.14,16 The prepared C-SLNs were evaluated for percent entrapment efficiency (EE) and total drug content and stored in a refrigerator until further analysis. A similar set of SLNs, but without the addition of curcumin, were prepared for references as plain SLNs. 2.3. Total Drug Content (TDC). TDC was estimated spectrophotometrically at λmax of 425 nm by disrupting 1 mL of the SLN dispersion using an appropriate volume of chloroform/methanol (1:1). 2.4. Entrapment Efficiency (EE). For determining the entrapment efficiency, the SLN dispersion was ultracentrifuged at 90 000 rpm for 2 h at 4 °C. The clear supernatant was decanted. The pellet of C-SLNs was then washed with methanol to remove any unentrapped drug (with curcumin being insoluble, the unentrapped free drug will also settle down along with the SLNs) and recentrifuged. The two supernatants were combined, and the absorbance of the resulting mixture was recorded. The pellet obtained from the above was dissolved in a suitable quantity of methanol/chloroform (1:1), and the absorbance of the clear solution was recorded. The amount of drug in the pellet gave a direct measure of the extent of drug entrapped, while the concentration of drug in the supernatant indicates unentrapped free drug. The latter was used to obtain the mass balance of the total drug content. The absorbance value obtained for plain SLNs treated in a similar manner was used as the control value to compensate for any interference of the ingredients. EE =

total drug entrapped × 100 total drug content

2.5. Cell Culture, Growth Conditions, and Treatment. Human promyelocytic leukemia cell line HL-60 was procured from the National Centre for Cell Sciences (NCCS), Pune, India. Human lung carcinoma cell line (A549) and prostrate cancer cell lines (PC3) were procured from the National Cancer Institute, Frederick, U.S.A. Cells were grown in RPMI1640/MEM medium containing 10% FCS, 100 unit penicillin/ 100 g streptomycin per milliliter of medium in a CO2 incubator (Thermo-con Electron Corporation, USA) at 37 °C with 98% humidity and 5% CO2 gas environment. They were further 3412

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overnight at −20 °C. Cells were washed with PBS and were subjected to proteinase-K and RNase digestion followed by staining of clean nuclear materials (nuclei) with PI using procedures and reagents as described in the instruction manual of the Cycle Test plus -DNA reagent kit (Becton Dickinson, USA). The preparations were analyzed for DNA content using a BD-LSR flow cytometer. Data were collected in list mode on 10 000 events for FL2-A versus FL2-W. Apoptotic nuclei appear as a broad hypodiploid DNA peak at lower fluorescence intensity compared to nuclei in the G0/G1 phase.17 2.9. Measurement of Mitochondrial Membrane Potential. Mitochondrial dysfunction within the apoptotic process is often associated with loss of the mitochondrial inner transmembrane potential. One possibility to visualize alterations in the mitochondrial membrane potential is staining with potentiometric fluorescent dyes. Changes in mitochondrial transmembrane potential (Ψmt) as a result of mitochondrial perturbation were measured after staining with Rhodamine123.17 Cells after various treatments in a 12-well plate were incubated with medium containing Rhodamine-123 (5 μg/mL; stock, 1 mg/mL PBS) for 1 h. Cells were washed in PBS and centrifuged at 100g for 5 min and suspended in sheath fluid. Immediately before analysis, PI (5 μg/mL; stock 1 mg/mL PBS) was added to the samples. The intensity of fluorescence from 10 000 events was analyzed in the FL-1 channel of the flow cytometer.

electrotransferred to polyvinylidene difluoride (PVDF) membranes (Bio-RAD) into Western blotting transfer frames in the following manner: sponge-blotting paper-gel-PVDF membraneWestern blotting paper-sponge and transfer overnight at 4 °C at 30 V in transfer buffer. Nonspecific bindings of the membrane are blocked by incubation with 5% nonfat milk in Tris-buffered saline (10 mM Tris-HCl, 150 mM NaCl) containing 0.1% Tween-20 (TBST) for 1 h at room temperature. The blots are probed with respective primary antihuman antibodies for 2 h (1:1000 dilutions) and washed three times with TBST. The blots are then incubated with horseradish peroxidase conjugated respective secondary antibodies for 1 h (1:1000 dilution) and washed again three times with TBST. PVDF membrane was incubated into ECL Pus Western blot detection reagent (ECL kit, Amersham Biosciences) for 5 min on a transparency sheet, in the dark. PVDF membrane was placed into the Hyper Cassette and superimposed with a high performance chemiluminescence film in a dark room for 2 min, and the protein signal was then developed onto the high performance chemiluminescence Xray film by using developer, and the signal was fixed with processing chemical fixer. The film was gently washed with tap water and dried. The density of the bands was arbitrarily quantified using Quantity One software of the Bio-RAD gel documentation system.17 3.2. Curcumin Cellular Uptake Studies by Fluorescence Method. The cellular uptake of free curcumin and CSLNs in HL-60 cells was analyzed by the fluorescence method. In brief, HL-60 cells were incubated with plain-SLN, curcumin, and C-SLNs for 6 h and then washed two times with PBS. The washed cells were resuspended in phosphate buffer saline (PBS) and divided into three parts for fluorescence microscopy, flow-cytometry, and fluorescence intensity measurement. One part of the cell suspension was then examined under an inverted fluorescence microscope (Olympus 1 × 70, magnification 20×) for curcumin autofluorescence using a green filter. The second part of the cell suspension was analyzed for curcumin intracellular uptake using a BD-LSR flow cytometer. Data were collected in list mode on 10 000 events for count versus FL1-H. The last and third part of the cell suspension was centrifuged and suspended in 200 μL of methanol (to lyse the cells) and vortexed gently. The fluorescence intensity of curcumin dissolved in methanol was measured at excitation λ 420 nm and emission λ 498 nm using methanol as a reference.

3.0. CASPASE ACTIVITY ASSAYS Most of the proteolytic cleavages during apoptosis result from the activation of caspases, a family of cysteine-dependent proteases. These enzymes recognize specific tetra- or pentapeptide motifs in their substrates and cleave exclusively on the carboxyl side of aspartate residues. Caspase activation can be measured by applying a synthetic peptide substrate which is coupled to a fluorophore. Cleavage of the fluorogenic substrate by the activated enzyme leads to increased fluorescence. The generated fluorophore is proportional to the concentration of activated caspase. The liberation of 7amino-4-trifluoromethyl coumarin (AFC) shows a blue to green shift in fluorescence at an excitation wavelength of 400 nm and an emission wavelength of 505 nm while the liberation of 7-amino-4-methylcoumarin (AMC) shows a blue to green shift at an excitation wavelength of 380 nm and an emission wavelength of 440 nm respectively. Cells (2 × 106) were incubated with test material for the indicated concentrations and time periods.17 At the end of treatment, cells were washed in PBS and pellet lysed in cell lysis buffer. The activities of caspase-3, -8, and -9 in the cell lysates were determined fluorometrically using BD Apoalert caspase fluorescent assay kits. Caspase-3 and -8 employed fluorochrome conjugated peptides DEVD-AFC and IETD-AFC as substrates, respectively, while caspase-9 employed LEHD-AMC. Release of AFC and AMC were assayed according to the instructions provided in the manual by the supplier. Specific inhibitors were used as negative control to determine whether fluorescence intensity changes were specific for the activity of caspases. The peptide based inhibitors used were DEVD-CHO for caspase-3, IETDfmk for caspase-8, and LEHD-CHO for caspase-9. 3.1. Immunoblot Analysis. The protein lysates along with a standard protein marker were subjected to discontinuous SDS-PAGE analysis. Protein aliquots (50 μg) were resolved on SDS-PAGE, run at 60 V (PowerPac HC high current power supply, BioRad), for 3 h. The resolved proteins were

4.0. STATISTICAL ANALYSIS Data is expressed as mean ± SD, unless otherwise indicated. Comparisons were made between control and treated groups unless otherwise indicated using one way ANOVA by Sigmastat Software, and P-values ≤ 0.001 were considered significant. 5.0. RESULTS 5.1. Inhibition of Cell Proliferation by C-SLNs. Using a conventional tetrazolium-based colorimetric cell proliferation assay, we evaluated the cytotoxicity18 of C-SLNs in different cancer cell lines. Treatment of cancerous cells with curcumin and C-SLNs for 48 h produced concentration dependent inhibition of cell proliferation of HL-60, A549, and PC3. CSLNs showed concentration dependent inhibition of cell growth with IC50 of 1 μg/mL, which is 85.7% less than that of free curcumin (IC50 = 7 μg/mL). Similar reductions in IC50 were observed in other cell lines also. While the IC50 was 57% 3413

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Figure 1. Influence of treatment with different concentrations of C-SLNs and curcumin on proliferation of various human cancer cell lines. The left side of the figure represents the treatment of cells with C-SLNs while the right side of the figure represents treatment with free curcumin. Data are mean ± SD (n = 8 wells) and representative of two similar experiments.

lower than that of free curcumin in the case of A549 cells, 76% higher reduction was obtained in the case of PC3 cell lines (Figure 1). 5.2. C-SLNs Induce DNA Fragmentation Typical of Apoptosis. To investigate whether treatment with C-SLNs induced faster or higher DNA fragmentation in comparison to free curcumin at similar concentration, genomic DNA was isolated from treated HL-60 cells. Apoptosis typically involves intranucleosomal chromatin cleavage by endonucleases in multiples of 180 bp leading to DNA fragmentation resulting in a typical DNA laddering. Cells treated with C-SLNs for 24 h exhibited a typical DNA ladder at both the selected concentrations (10 and 20 μg). However, with free curcumin,

at similar concentrations, the DNA ladder was not visible (Figure 2). 5.3. C-SLNs Increase Sub-G0 DNA Fraction of Cell Cycle Phase Distribution. HL-60 cells treated with C-SLNs for 24 h exhibited a concentration dependent increase in subG0 DNA fraction (≤2n DNA; Figure 3). The sub-G0 fraction was 2% in plain-SLNs treated cells, which increased to ∼96% after treatment with 10 μg/mL of C-SLNs, while the sub-G0 fraction for free curcumin was only ∼54%, at 10 μg/mL (Pvalues: ≤0.001 compared to untreated control). The sub-G0 fraction comprises both the apoptotic and debris fractions, implying the extent of death which has occurred. Results clearly indicated the potential of C-SLNs to increase the sub-G0 fraction, as compared to free drug. 3414

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fluorescence (Figure 4). C-SLNs at 10 μg caused mitochondrial damage, hence the decrease of mitochondrial membrane potential by about 44% in comparison to free curcumin, which showed only a 9% decrease at the same concentration (10 μg/mL). The loss of Δψm is largely due to the opening of mitochondrial permeability transition pores (PTP), which conduit the leakage of cytochrome c and pro-apoptotic proteins from mitochondria to the cytosol. Significant loss in membrane potential, post treatment with SLNs, is a proof of its effectiveness to cause cell death. 5.5. C-SLNs Stimulate Caspase Activities in HL-60 Cells. C-SLNs produced a dose dependent increase in the activities of caspase-8 and -9 in HL-60 cells (Figure 5). After incubation for 6 h with C-SLNs, the effect was 3 times that achieved with free curcumin at the same dose (10 μg/mL). Furthermore, there was an even greater increase (4.4 times; p ≤ 0.001) of caspase 3 activity, post treatment with curcumin SLNs at 10 μg/mL in comparison to free curcumin. 5.6. Effect of C-SLNs on Other Apoptotic Related Genes Expression. Treatment with C-SLNs at 10 and 20 μg/ mL concentration also increased the release of cytochrome c into the cytoplasm of HL-60 cells (Figure 6). It is known that cytochrome c releases from mitochondria into the cytosol and binds to the apoptotic protease activating factor (Apaf) complex and triggers the activation of procaspase-9 to the active caspase-9.19 As shown in Figure 6, we observed a significant increase (p ≤ 0.001) in the marked fraction of the cytochrome c released from the mitochondria, of C-SLNs treated cells as compared to treatment with free curcumin at 6 h. Further, treatment with curcumin and C-SLNs resulted in suppression of NF-kB, with results being significantly better (p ≤ 0.001) post treatment with curcumin SLNs. NF-kB is a transcription factor present in the cytoplasm and regulates the expression of genes involved in antiapoptosis (e.g., bcl-2 and bcl-XL); proliferation (COX-2 and cyclin D1); and metastasis (e.g., MMP-9). Among the cytokines, TNF-R1 is the most

Figure 2. C-SLNs and free curcumin induced DNA fragmentation in HL-60 cell lines. Genomic DNA was extracted from cells treated with different concentrations of C-SLNs and free curcumin for 24 h. Cell culture controls without any treatment (not shown) exhibited a pattern similar to that obtained for plain SLNs.

5.4. Loss of Mitochondrial Membrane Potential by CSLNs. HL-60 cells exposed to C-SLNs and free curcumin (1 and 10 μg/mL for 1 h) were analyzed for mitochondrial membrane potential (Δψm) loss employing Rh-123 uptake by flow cytometery. In the untreated plain SLNs treated cells, almost all cells were functionally active with high Rh-123

Figure 3. DNA cell cycle analyses of HL-60 cells treated with C-SLNs and free curcumin at 1 and 10 μg concentration for 24 h. Fraction of cells for hypodiploid (sub-G0, ≤2n DNA) population analyzed from FL2-A vs cell counts is shown (%). Data are representative of one of two similar experiments. Values for plain-SLNs were similar to those obtained for cell culture controls (no treatment). 3415

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Figure 4. C-SLNs induced loss of mitochondrial membrane potential (Δψm). HL-60 cells (1.2 mL/12 well plates) were incubated with plain SLNs, 1 and 10 μg/mL of C-SLNs (SLN), and free curcumin (Cur) for 6 h. Thereafter, cells were stained with Rhodamine-123 (5 μg/mL) for 1 h and PI (5 μg/mL) and analyzed in FL-1 vs FL-2 channels of the flow cytometer. Results for cell culture controls are not shown (plain-SLNs showed similar effects).

2.95 mg/mL) was entrapped within the SLNs and even the unentrapped curcumin was in solubilized form. For all practical purposes, the prepared C-SLN dispersion was suitably diluted with water to result in various stocks, suitable volumes of which were used for different experiments. Each milliliter of the prepared SLN dispersion contained 3600 μg/mL of curcumin; 40 mg/mL of glyceryl behenate; 275 mg/mL of polysorbate 80; and 3.2 mg/mL of soya lecithin. The in vitro release was predominantly by diffusion phenomenon and was prolonged up to 7 days. In vivo pharmacokinetics performed after oral administration of C-SLNs (50, 25, 12.5, and 1 mg/kg dose) and free curcumin (50 mg/kg), using a validated LC-MS/MS method in rat plasma, revealed significant improvement (at p < 0.05) in bioavailability (BA) (39 times at 50 mg/kg; 155 times at 1 mg/kg; and 59 and 32 times at 12.5 and 25 mg/kg, respectively) after administration of C-SLNs at all the doses with respect to free curcumin.14,15,20 In view of the enhanced BA achieved with the developed CSLNs,14 we intended to confirm whether the developed system retains, improves, or decreases the anticancer potential of free curcumin. For this purpose, the in vitro anticancer effect of free curcumin and C-SLNs was evaluated in a variety of cancer cell lines. Hundreds of papers elaborate on the anticancer effects of curcumin at varying dose ranges,21−24 but a relatively poor stability and poor BA have been highlighted as one of the major problems in its therapeutic applications. The stability of curcumin in a cell culture medium containing 10% FBS or in human blood is greater than that in phosphate buffer, but 50% of the molecule still decomposes within 8 h of incubation.25 Even though curcumin holds a multitargeting potential, it cannot be translated into an efficient clinical accomplishment due to its compromised stability and BA, with the latter being assigned to its poor aqueous solubility, permeation, and absorption across the gut, alongwith a high rate of metabolic transformation.26,27

potent activator of NF-kB. Whether constitutive activation of NF-kB in HL-60 cells is due to autocrine expression of TNF-R1 was examined. An efficient up regulation of TNF-R1 post treatment with C-SLNs was observed. Thus, an upregulation of TNF-R1 resulted in efficient downregulation of NF-κB, suggesting that TNF-R1 plays a major role in activation of NF-kB. 5.7. Curcumin Loaded Solid Lipid Nanoparticles Exhibit Enhanced Cellular Uptake. We investigated the cellular uptake of curcumin and C-SLNs by incubating HL-60 cells with curcumin or C-SLNs at 1 and 10 μg/mL concentration and with plain-SLNs for 6 h. The results showed that the cellular uptake of curcumin in vitro was almost two times higher in cells treated with C-SLNs in comparison to curcumin alone; however, as expected plain-SLNs did not show any fluorescence (Figure 7A and B). It was observed that curcumin as C-SLNs rapidly penetrated the cells and a significant amount was accumulated in the cytoplasmic granula located close to the nucleus (Figure 7C). Intracellular curcumin kept increasing even up to 6 h at 10 μg/mL of curcumin concentration. Cellular uptake studies were performed up to 6 h instead of 24 h and 48 h, as uptake of drug or its SLNs into the cells is an early event which ideally starts just after the treatment.

6.0. DISCUSSION Curcumin-loaded solid lipid nanoparticles (C-SLNs) with an average particle size of 134.6 nm and a total drug content of 92.33 ± 1.63% were produced using a microemulsification technique. The particles were spherical in shape, with a high drug entrapment of 81.92 ± 2.91% at 10% drug loading (proportion of curcumin to the amount of lipid used in the preparation of SLNs). It may be noted that C-SLNs initially contained 3.6 mg/mL of curcumin, out of which 81.92% (i.e. 3416

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Figure 5. Free curcumin and C-SLNs induced differential activation of various caspases: (a) caspase 3; (b) caspase 8; (c) caspase 9 in HL-60 cells. The cells in culture were exposed to 1 and 10 μg/mL of C-SLNs and free curcumin for indicated time periods. Data are mean ± SD from three similar experiments. Values at P ≤ 0.001 were considered to be significantly different as compared to untreated control or plain-SLNs treated cells (showed similar results as untreated control) and curcumin treated cells.

followed by pouring the hot microemulsion into cold water. Microemulsions are clear, thermodynamically stable systems. Hence, it may be said that once the microemulsion is formed, the drug is transformed into a solubilized form. Pouring of drug loaded microemulsion into cold water results in the formation of SLNs. The latter incorporates the drug in its amorphous state (evidenced by XRD and DSC studies14) and, thus, in a more soluble form. Additionally, the enhanced permeation is also attributed to the small size of the SLNs (134.6 nm), which perhaps leads to an enhanced efficacy. Unpublished work on the stability and permeability of C-SLNs from our lab clearly ̈ curcumin, which otherwise degrades up established that naive to 90% in 1 h, at physiological pH was protected after packaging into lipidic particles (85−89% of curcumin was remaining after 24 h). Similarly, in situ intestinal permeation studies clearly indicated a 3−4 times enhanced permeation across the ileum wall of the rats. Altogether, the protection against degradation under physiological conditions and in the culture media and enhanced permeation across cellular

The results of the present study clearly indicate the advantage of incorporation of curcumin into SLNs. The extent of cell death induced by C-SLNs was significantly higher (p ≤ 0.001) at all the tested doses and in all the cell lines. Possible reasons for the better effect could be the following: (i) presentation of curcumin in a soluble form for interaction with the cancerous cells, and (ii) better permeability of C-SLNs into the various cancerous cells, either because of a nanoparticle size which allows easy transport across the cellular membranes or because of their composition. SLNs constituted a lipid (glyceryl behenate in the present study), and surfactants included phospholipids. Small lipidic particles with a coating of surfactant/phospholipid have an intrinsic nature to intermingle with cellular membranes at the molecular level. Further, the presence of surfactants can also enhance the permeability characteristics of SLNs. (iii) Another reason for the better effect is that the stability of curcumin is ensured when incorporated into SLNs. The SLNs were prepared by a hot microemulsification (using a high melting point lipid) method, 3417

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since higher incubation periods (48 h) have shown significant differences between free curcumin and curcumin loaded SLNs, and the intent of the present study was also to compare the activity of free curcumin with C-SLNs. In lieu of the latter fact, a shorter incubation of 24 h was considered sufficient. Furthermore, the cellular uptake studies (Figure 7) show a significantly higher (2 times) uptake of curcumin when present as C-SLNs even at 6 h. A later time was chosen in view of the fact that the cellular uptake is an early event which starts soon after treatment of cells with the drug/SLNs. Most of its part was found to be concentrated in cytoplasmic granules close to the nucleus. Hence, a closer or faster interaction and permeation into the nucleus at subsequent times cannot be ruled out. We have earlier shown the presence of these SLNs within the neuronal cells using confocal microscopy.31 Inhibition of dysregulated cell cycle progression in cancer cells is an effective strategy to halt tumor growth. Our results demonstrate that treatment of HL-60 cells with C-SLNs induces sub-G0 phase arrest of cell cycle progression, indicating it to be one of the mechanisms by which they inhibit proliferation. Additionally, C-SLNs remarkably increased caspase-9 activity, with activation of the latter being solely regulated by mitochondrial release of apoptotic protease activating factor-1 (Apaf-1) and cytochrome c, which is a part of the mitochondrial dependent apoptosis pathway.32 Further, caspase-8 activation in HL-60 cells was also induced, probably because of the upstream overexpression of cell surface receptors such as TNF-R1 and suppression of NF-κB.33 Various study reports enumerate the effect of curcumin induced apoptosis in human melanoma cells (30−60 μM for 24 h)34 and human leukemia HL-60 cells (10−40 μM for 16−24 h)35,36 apart from AK-5 tumor cells and MCF-7 breast cancer cells,37−39 but no such report exists wherein the effect of the curcumin loaded drug delivery system has been established to have a prospective apoptotic effect as compared to the case of free drug. Additionally, all the above attained effects can be attributed to the enhanced penetration of curcumin after incorporation into the solid lipid nanoparticles, as was depicted in the cellular fluorescent uptake studies. The latter clearly established the internalization mechanism of the lipidic nanoparticles, indicating their cellular uptake instead of adhesion to the surface, thus proving to be a preferentially targeted system for the cancer cells. Similar results have being reported by another group of researchers with the prepared polymeric nanoparticles of curcumin.40 From the above it can be said that there are varying mechanisms responsible for apoptosis induced by curcumin. The latter include Akt dephosphorylation; Bcl-2, Bcl-XL, and inhibitor of apoptosis protein inhibition; cytochrome c release; and caspase-3 activation.37 To verify the hypothesis that the mitochondrial DNA damage induced by curcumin in HL-60 cells may be the event triggering apoptosis by C-SLNs, we analyzed the Δψm during the time-course experiments by using rhodamine 123. Interestingly, after an incubation of 6 h, hyperpolarization of mitochondrial membrane was observed with C-SLNs treatment, which was significantly more (p ≤ 0.001) than that of free curcumin. Hyperpolarization seems to represent a prerequisite for rapid mitochondrial-mediated apoptotic cell death, which eventually leads to the loss of mitochondrial membrane potential.41 Cytochrome c seems to be a critical factor in apoptotic processes.19 Cytochrome c released from the mitochondria into

Figure 6. Influence of curcumin, C-SLNs, and plain-SLNs on the expression of important proteins involved in the initiation of apoptosis. HL-60 cells were treated with 10 and 20 μg/mL of C-SLNs and free curcumin for 6 h. B-Actin was used as internal control to represent the same amount of proteins applied for SDS-PAGE. Specific antibodies were used for detection of cytochrome c, NF-κB, and TNF-R1. Data are representative of one of two similar experiments. P values ≤ 0.001 were considered to be significantly different as compared to untreated control or plain-SLNs treated cells (showed similar results as untreated control) and free curcumin treated cells.

membranes have attributed to the attained anticancer effects in all the studies being conducted. A study report on polymeric nanoparticles of curcumin demonstrated that nanocurcumin formulation has comparable efficacy to free curcumin against pancreatic cancer cell lines in vitro.28 Further, they confirmed that nanocurcumin retains the mechanistic specificity of free curcumin, inhibiting the activation of the seminal transcription factor NF-κB and reducing the steady state levels of pro-inflammatory cytokines such as interleukins and TNF-α.28 However, the authors do not report on the molecular cascade of events associated with the induction of apoptotis in these cancer cell lines. In another study, a liposomal curcumin formulation also demonstrated a comparable potency to free curcumin.29 Even as further studies with this liposomal formulation are awaited, it is emphasized that liposomes, which are metastable aggregates of lipids, tend to be more heterogeneous and larger in size (typically 100−200 nm) than most nanoparticles. The results of the present study describes the significantly higher apoptotic activity of developed C-SLNs on human leukemia HL-60 cells, for the first time. This is evidenced from the low IC50 values achieved with C-SLNs as compared to free curcumin in the MTT test in all the cell lines and especially in HL-60 cells. Such effects can be assigned to higher permeability (Figure 7) due to small particle size, the lipidic nature of CSLNs, and presentation of curcumin in a soluble and probably more bioavailable form. Furthermore, protection of curcumin against degradation in a physiological environment could be another important factor. Further, measurement of DNA fragmentation indicated that the cancer cell death is due to the induction of apoptosis by C-SLNs on HL-60 cells while no such fragmentation was observed with free curcumin at the same concentration. It may be highlighted here that previous studies28,30 report on only a similar effect being achieved with a modified/novel delivery system of curcumin while we report on much faster onset of an apoptotic effect with C-SLNs, which is not at all observed with free curcumin at 24 h along with a 7 times lower IC50 value confirming its enhanced apoptotic potential. It may be noted that even though a significant effect was observed with free curcumin in the MTT assay, reducing the incubation time to 24 h in DNA fragmentation studies showed a complete absence of an effect with free curcumin, 3418

dx.doi.org/10.1021/mp300209k | Mol. Pharmaceutics 2012, 9, 3411−3421

Molecular Pharmaceutics

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Figure 7. Cellular uptake of free curcumin and C-SLNs in HL-60 cells after 6 h of incubation. HL-60 cells were incubated for 6 h with 1 and 10 μg/ mL concentrations of free curcumin and C-SLNs. (A) Cellular uptake (green fluorescence) monitored by fluorescence microscopy. (B) Intracellular uptake of curcumin measured with a flow-cytometer on the FL1 channel. (C) Cells lysed with methanol and fluorescence intensity of free curcumin measured at excitation λ 420 nm and emission λ 498 nm using methanol as a reference. Data are mean ± SD from three similar experiments. Significance, P value: *