Accepted Manuscript Title: Formulation optimization, characterization, and evaluation of in vitro cytotoxic potential of Curcumin loaded solid lipid nanoparticles for improved anticancer activity Authors: Sri Vishnu Kiran, Bhatt Himanshu, Aashma Shah, Neeraja Komanduri, Dhanya Vijayasarathy, Balaram Ghosh, Swati Biswas PII: DOI: Reference:
S0009-3084(17)30152-4 http://dx.doi.org/10.1016/j.chemphyslip.2017.08.009 CPL 4585
To appear in:
Chemistry and Physics of Lipids
Received date: Revised date: Accepted date:
16-6-2017 21-7-2017 17-8-2017
Please cite this article as: Kiran, Sri Vishnu, Himanshu, Bhatt, Shah, Aashma, Komanduri, Neeraja, Vijayasarathy, Dhanya, Ghosh, Balaram, Biswas, Swati, Formulation optimization, characterization, and evaluation of in vitro cytotoxic potential of Curcumin loaded solid lipid nanoparticles for improved anticancer activity.Chemistry and Physics of Lipids http://dx.doi.org/10.1016/j.chemphyslip.2017.08.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Manuscript type: Research Article Title of the article: “Formulation optimization, characterization, and evaluation of in vitro cytotoxic potential of Curcumin loaded solid lipid nanoparticles for improved anticancer activity”. Name of authors: Sri Vishnu Kiran Rompicharla1, Bhatt Himanshu1, Aashma Shah1, Neeraja Komanduri1, Dhanya Vijayasarathy1, Balaram Ghosh1, Swati Biswas1* Affiliations 1
Department of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad
Campus, Shameerpet, Hyderabad, Telangana 500078, India.
Name, Address, Phone number and E-mail address of the corresponding author: *Dr. Swati Biswas, Assistant professor, Department of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad Campus, Shameerpet, Hyderabad, Telangana 500078, India. Phone: +91-40-66303630 Email ID:
[email protected]
Graphical abstract
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Highlights
Curcumin loaded SLN using cholesterol (Chol CUR SLN) were prepared by High shear homogenization.
Box-Behnken Design was applied to study the effect of variables on formulation parameters.
DSC, XRD, FTIR and drug release studies were performed to characterize the formulated SLN.
Chol CUR SLN exhibited superior cell uptake and higher cytotoxicity in MDA-MB231 cell line compared to free drug.
Greater percentage of apoptosis was induced by Chol CUR SLN as studied by Annexin V/PI binding assay.
Abstract The aim of the present research was to develop a novel, biocompatible, amenable to industrial scale up and affordable solid lipid nanoparticles (SLN) preparation of curcumin and evaluate the therapeutic efficacy in vitro using cancer cells. We have incorporated cholesterol as the lipid to prepare SLN along with the Poloxamer-188 as stabilizer. High shear homogenization was used to prepare the SLN and formulation was optimized using Quality by Design. The optimized Chol CUR SLN exhibited a narrow size distribution with a particle size of 166.4±3.5 nm. Percentage encapsulation (%EE) was found to be 76.9±1.9%. The SLN were further characterized by DSC, FTIR, XRD and drug release. In vitro cell studies in MDAMB-231 (Human Breast cancer) cell line revealed that the Chol CUR SLN showed superior cytotoxicity and uptake in comparison to the free curcumin. Furthermore, Chol CUR SLN induced a significantly higher apoptosis compared to free CUR treatment. These results indicated that the curcumin encapsulated in Chol SLN was able to significantly improve the cytotoxic potential and induction of apoptosis in MDA-MB-231 cells. The promising result from our study could lead a further exploration of this nanoparticle formulation to be utilized clinically for cancer treatment. Keywords: Solid lipid nanoparticles; Curcumin; Cholesterol; Box-Behnken design; anticancer; cell uptake
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1. Introduction Curcumin, a naturally occurring bioflavonoid, is extracted from the rhizomes of a traditional herb, turmeric, scientifically known as Curcuma longa Linn (Mulik et al., 2010). The uses of this phyto- extract dates back to ancient times as a medicine to treat wide range of ailments due to its varied pharmacological properties. It has been explored and known to possess antiinflammatory, antioxidant, wound healing, anti-microbial and anti-cancer activities (Jeengar et al., 2016; Ji et al., 2016; Sun et al., 2013; Wang et al., 2013). The strong anti-cancer potential of curcumin has been investigated against various cancers, including breast, colon, brain, blood, prostrate and skin (Kumari et al., 2017). The studies demonstrated multiple mechanisms of action exhibited by curcumin against the cancer cells. Anti-cancer activity of curcumin involves interference with various signal transduction pathways, cell cycle arrest or apoptosis. In addition, it inhibits cancer cell proliferation and carcinogenesis by blocking NFkB activity and anti-apoptotic proteins, including Bcl-2 (Jeengar et al., 2016; Kumari et al., 2017; Mulik et al., 2010; Wang et al., 2013). Curcumin has also been investigated in combination with other chemotherapeutic agents to overcome the multiple drug resistance (Pawar et al., 2016). However, the delivery of curcumin has always been challenging due to its poor pharmacokinetic properties. It suffers the problem of bioavailability owing to the poor solubility and rapid metabolism of curcumin which limits its clinical application. In order to address these concerns, several attempts have been made to formulate an effective delivery system such as encapsulating curcumin into polymeric nanoparticles, solid dispersions, liposomes and inclusion complexes which experienced many drawbacks (Ji et al., 2016; Wang et al., 2013). Solid Lipid Nanoparticles (SLN) have attracted great consideration as an alternative system to traditional carriers due to the numerous advantages it offers in delivering the poorly soluble drugs (Harde et al., 2011). Moreover, SLN were proved to have superior stability and biocompatibility over other delivery systems without posing problems of toxicity and drug leakage (Mulik et al., 2010; Nooli et al., 2017; Radhakrishnan et al., 2016). SLN can be given by various administration routes such as oral, parenteral as well as transdermal/topical (Harde et al., 2011). Compared to other nanoparticles system known to load drugs efficiently, SLN have several other benefits such as their facile preparation routes, need of few formulation excipients including lipid and surfactant, excellent stability, high drug loading, and affordability (Rostami et al., 2014). Cholesterol is an example of animal steroid and is a building block of cell membrane (Varshosaz et al., 2014). It is also plays a vital role in lipid 3
organization, signal transduction, cell attachment, and cell migration (Belletti et al., 2016). Cholesterol has been used extensively with other lipids to improve the biocompatibility, cell permeability and intracellular transport of the nanocarriers (Kumari et al., 2017). These nanocarriers also possess the advantages of accumulating in the tumor by a passively targeting phenomenon called enhanced permeation and retention effect (Harde et al., 2011; Kiran Rompicharla et al., 2017). In recent years, Quality by Design approach has often been applied for the optimization of nanoparticles, considering a comprehensive understanding of the connection between inprocess parameters for better product quality (Kaithwas et al., 2017; Shah et al., 2015). Hence, various response surface methodologies can be applied like Box–Behnken design, which helps in optimization of formulation variables and responses with relatively few experimental runs (Baig et al., 2016; Ji et al., 2016). In the present study, we have prepared solid lipid nanoparticles of curcumin using cholesterol as the single lipid to form the nanoparticles' core compartment. High shear homogenizer was used to prepare SLN and optimized using Box-Behnken experiment design. The optimized formulation was characterized and evaluated in vitro using a human breast cancer cell line. The treatment of cells with Chol CUR SLN was compared with the treatment with free curcumin for all the performed studies. 2. Materials and methods 2.1 Materials Curcumin (CUR) was purchased from Sigma-Aldrich (Bangalore, India). Nuclear stain Diamidino-phenylindole dihydrochloride (DAPI), Cholesterol and Poloxamer 188 (Pol-188) were also obtained from Sigma-Aldrich (Bangalore, India). Trypan blue solution, phosphate buffered saline (PBS), Growth medium RPMI 1640, antibiotic solution 100× liquid with 10,000 units Penicillin and 10 mg streptomycin per milliliter, fetal bovine serum, trypsin, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were procured from Himedia Laboratories Pvt. Ltd. (Mumbai, India). Dialysis membrane made of regenerated cellulose with a molecular weight cut-off (MWCO) of 3,500 Da and 12,000–14,000 Da was obtained from Spectrum Laboratories Inc., (CA, USA). AnnexinV-FITC/PI apoptosis kit was procured from Molecular Probes, ThermoFisher Scientific, USA. All other chemicals and reagents were of analytical quality. 2.2 Formulation of Chol CUR SLN
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High Shear Homogenization (HSH) method was employed to prepare SLN following a previously reported procedure (Mehnert and Mäder, 2001). Briefly, CUR and Cholesterol were solubilized in an organic solvent (chloroform-methanol 1:1), and was added drop-wise to the aqueous solution of Poloxamer-188 under homogenization condition (Polytron PT 3100D, Kinematica, Switzerland). After 15 minutes of homogenization, the dispersion was further put under magnetic stirring to remove traces of the solvent (Das et al., 2011; Shah et al., 2015). The nanoparticles were collected by centrifugation (10,000 RPM for 20 min) and washed twice with millipore water to remove un-entrapped curcumin. 2.3 Box-Behnken Design (BBD) BBD with 3 independent factors (X1= drug to lipid ratio, X2= concentration of surfactant and X3= HSH speed) and 3 level viz., low level (-1), mid-level (0), high level (+1) was employed for analysis using Design-Expert Software (Design-Expert®10). BBD explores quadratic response and constructs polynomial models in-turn giving fewer experimental runs. BBD encloses points at the center of cube as well as the points lying at the middle of each edge. (Shah et al., 2016). The polynomial equation for BBD is as follows: Yi = b0 + b1 X1 + b2 X2 + b3 X3 + b12 X1 X2 + b23 X2 X3 + b13 X1 X3 + b11 X21 + b22 X22 + b33 X23 Where, Yi is the dependent variable; b0 is the arithmetic mean response of the seventeen runs; b1, b2, b3 are linear coefficients for corresponding factors X1, X2, X3 respectively; b12, b23, b13 are interaction coefficients for corresponding factors X12, X23, X13 respectively; b11, b22 and b33 are quadratic coefficients for corresponding factors X11, X22 and X33 respectively. All selected variables with their high (+1), medium (0) and low levels (-1) are represented in Table 1. Seventeen trials of Chol CUR SLN were formulated in accordance with BBD. The influence of independent variables on responses is represented in Table 2. In the polynomial equations, the linear coefficients for corresponding factors (X1, X2, X3) have positive or negative sign, describing synergistic or antagonistic effect respectively. The best suitable model was selected by ANOVA graphically by three dimensional surface plot derived from Design-Expert software. The significance level was determined at p value < 0.05. 2.4 Data optimization and validation of experimental model To ensure the desired quality of CUR SLN, the design space was established to evaluate the effect of each independent variable using BBD. The optimization was carried out with the 5
aim to achieve low particle size, narrow PDI, and high %EE. Graphical optimization was done using overlay plot while numerical optimization was done using desirability criteria. Check-point analysis was carried out to verify the reliability of designed model by executing two validation batches viz., B1 and B2 (Table 2). The extent of error was evaluated between observed and predicted values.
2.5 Characterization of Chol CUR SLN 2.5.1 Photon correlation spectroscopy Particle size, polydispersity index (PDI) and zeta potential of the Chol CUR SLN and blank SLN were determined by Zetasizer (Nano ZS, Malvern Instruments, UK) at a laser backscattering angle of 173°. Analysis was performed after samples were suitably diluted. 2.5.2 % Entrapment efficiency (%EE) The Chol CUR SLN were centrifuged at 10,000 rpm for 20 min to get a solid pellet of nanoparticles. The upper layer was collected, diluted and analyzed for free CUR content by UV-Vis spectroscopy. The %EE was determined from the below equation: (Rahman et al., 2010) %encapsulation efficiency =
Initial amount of CUR − Amount of free CUR Initial amount of CUR
2.5.3 Thermal analysis by Differential Scanning Calorimetry (DSC) DSC analysis of CUR, Cholesterol and Chol CUR SLN was performed using DSC-60 (Shimadzu, Japan). Chol CUR SLN were lyophilized prior to DSC measurement. For each sample, 3 to 4 mg dried sample was filled in different aluminum pans and sealed. The measurement was executed under inert atmosphere at a nitrogen flow rate of 50 ml/min. The sample pans were heated at a rate of 10°C/min from room temperature to 300°C. An empty standard aluminum pan was taken as reference. 2.5.4 FTIR studies FTIR analysis was performed by making a pellet of each sample along with KBr. CUR, Chol and lyophilized Chol CUR SLN were mixed individually with KBr approximately at a ratio of 1:100. Standard pellet press was used to make the pellet. Spectra were recorded in the range of 4000-400 cm–1 using FTIR spectrometer (FT/IR-4200, Jasco, USA). The resultant spectrum of the Chol CUR SLN was compared with CUR and Chol to observe the spectral changes in wave number. 6
2.5.5 Powder X-ray diffraction (XRD) The XRD analysis of CUR, Chol and lyophilized Chol CUR SLN was performed using XRay diffractometer (Rigaku Ultima IV, Japan) having Cu anode (1.54 Å) at 60 mA, 60 kV. Samples were scanned at a step size 0.017° and 2θ angle of 5–50° with scintillation counter detector. Any change in intensity or shift of characteristic XRD peak corresponding to sample was observed using standard PDXL software. 2.5.6 In-vitro drug release study The pattern of drug release was studied by dialysis bag method using a cellulose ester membrane of MWCO 3,500 Da. Chol CUR SLN and CUR solution equivalent to 5 mg of drug were filled in the dialysis bag and dropped into release medium. PBS pH 7.4 added with 30% methanol was used as the dissolution medium. Addition of alcohols such as ethanol, isopropyl alcohol or methanol, and surfactants like Tween 80 avoids saturation of dissolution medium due to poor aqueous solubility of curcumin. (Jeengar et al., 2016; Shaikh et al., 2009; Suwantong et al., 2007; Yallapu et al., 2010). The bags were placed in different beakers and stirred at 150 rpm at 37°C. Samples were taken at selected time points and same volume of fresh dissolution medium was replaced to maintain the sink conditions. The samples were diluted, filtered through 0.22 µm membrane filter if necessary and were analyzed by UV-Vis spectroscopy to determine the amount of drug released. A graph of cumulative drug release percentage versus time was plotted. 2.6 Cell culture studies 2.6.1 Cell culture The MDA-MB-231 (Human breast cancer) cells were procured from National Centre For Cell Science (NCCS; Pune, India). RPMI 1640 medium was used to culture the cells added with 1% antibiotic solution and 10% fetal bovine serum. An incubator equipped with 5% CO2 and maintained at 37°C was used to incubate the cells under humidified environment. 2.6.2 Cellular uptake study by Fluorescence microscopy and Flow cytometry The cellular uptake of free CUR and Chol CUR SLN was investigated in MDA-MB-231 (Human Breast Cancer) cell line. Cells were counted (50,000 cells/well) and plated in 12-well microplates containing surface treated coverslips to observe under fluorescence microscope (Leica DMi8, Leica microsystems, Germany). CUR and Chol CUR SLN were pipetted out into the wells at a concentration of 12.5 µg/ml and placed in incubator for 1 h and 4 h. After the incubation time, nuclei were stained with DAPI and cells were washed twice with PBS. 7
Slides were prepared after fixing the cells with 4% paraformaldehyde. Cells were visualized and captured in DAPI and FITC channels. Quantitative determination of cell uptake was performed using flow cytometer. MDA-MB231 cells were plated in 6-well microplates and left in incubator to attach overnight. Following day, cells were added with CUR and Chol CUR SLN formulations (12.5 µg/ml) and incubated. Following 1 h and 4 h of incubation, cells were collected by trypsinization and processed. After washing twice, cells were suspended in sterile PBS and analyzed using flow cytometer (Amnis Corporation, EMD Millipore, USA). For each sample, a minimum of 10,000 events were collected. 2.6.3 Cytotoxicity MTT colorimetric assay was carried out to study the cytotoxic potential of free CUR, Chol CUR SLN, and blank Chol SLN against MDA-MB-231 cells. Briefly, 8000 cells/well were seeded in 96-well plates and were grown in an incubator maintained at 37°C with 5% CO2. A 100 µl solution of different formulations were added to the cells at a curcumin concentration range of 0-50 µg/ml and placed in incubator for 6 h and 24 h. Blank Chol SLN were tested to evaluate carrier mediated toxicity, if any. The formulations were removed from the wells after 6 h and replaced with RPMI 1640 complete medium and further incubated till 24 h. MTT solution (50 μL; 5 mg/mL) was pipetted out into the wells and left in incubator for generation of formazan crystals. After 4 h, dimethylsulfoxide (150 μL) was added into each well to solubilize the purple colored formazan crystals. The color intensity was quantified by measuring absorbance using a 96-well plate reader (Spectramax™, Molecular Devices, USA) at 590 nm with a reference wavelength of 620 nm. RPMI 1640 medium treated cells were used as control. Percentage cell viability was calculated based on the absorbance of sample measured relative to control cells. 2.6.4 Apoptosis Induction of apoptosis by CUR and Chol CUR SLN was assessed by Annexin V/PI cell binding assay (Kiran Rompicharla et al., 2017). In brief, formulations with a curcumin concentration of 50 µg/ml were added to MDA-MB-231 cells cultured in 6-well plates at a population of 0.8 million cells/well and incubated for 24 h at 37°C and 5% CO2. Cells without any treatment were used as control. After the treatment time, cells were washed twice with cold PBS and harvested. Annexin V, PI were added to the cells suspended in annexin V binding buffer as per manufacturer’s protocol, and incubated for 15 minutes in dark. The 8
samples were analyzed using flow cytometer (Amnis Corporation, EMD Millipore, USA). The percentage of live (Annexin V-/PI-), early apoptotic (Annexin V+/PI-), late apoptotic (Annexin V+/PI+), and necrotic (Annexin V-/PI+) cells was determined from the scatter plot of FITC channel and PI channel, using Ideas Software Version 6.0. 2.7 Statistical Analysis All the experiments in the present study were performed at least three times and presented as mean ± standard deviation. To determine the statistical significance, unpaired Student’s t test was applied. The differences among the groups were considered significant for p values less than 0.05. 3. Results and discussion 3.1 Optimization of Chol CUR SLN by Box-Behnken design (BBD) From the experimental design, it was observed that CUR to Chol ratio, Poloxamer 188 concentration and HSH speed significantly influenced the size, PDI and %EE of Chol CUR SLN. The main effect (X1, X2, X3), interaction effect (X12, X23, X13) and quadratic effect (X11, X22, X33) were analyzed using BBD. Seventeen trials were executed and their outcomes were shown in Table 2. Model F-value for dependent factors Y1, Y2 and Y3 were 52.35, 442.79 and 38.16 respectively. The value of “Prob > F” was
