International Journal of Pharmaceutics 477 (2014) 601–612

Contents lists available at ScienceDirect

International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Pharmaceutical nanotechnology

Lipid based nanocarrier system for the potential oral delivery of decitabine: Formulation design, characterization, ex vivo, and in vivo assessment Yub Raj Neupane a , Manish Srivastava a , Nafees Ahmad a , Neeraj Kumar b , Aseem Bhatnagar b , Kanchan Kohli a, * a b

Formulation Development Laboratory, Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard University, New Delhi, India Department of Nuclear Medicine, Institute of Nuclear Medicine and Allied Sciences, New Delhi, India

A R T I C L E I N F O

A B S T R A C T

Article history: Received 13 September 2014 Received in revised form 31 October 2014 Accepted 2 November 2014 Available online 6 November 2014

The aim of this study was to design and fabricate nanostructured lipid carrier (NLC) for the potential oral delivery of decitabine (DCB). NLC was prepared by cold homogenization technique and optimized by the Box–Behnken experimental design. It was further characterized by particle size, zeta potential, transmission electron microscopy (TEM), atomic force microscopy (AFM), differential scanning calorimetry (DSC), X-ray diffraction (XRD), in vitro release study, and stability study. Moreover, ex vivo and in vivo efficacy of the NLC was assessed by gut permeation study, g scintigraphy imaging, and MTT assay. NLC was found to have particle size (116.64
6.67 nm), zeta potential (31.8
0.96 mV) and sustained drug release (80.23
4.67%) up to 24 h. TEM and AFM proved that the particles were spherical in shape and smooth surface. DSC and XRD studies had demonstrated the reduced crystallinity and stability enhancing effect of the NLC. Stability studies revealed the changes in the observed parameters up to 45 days were not significantly differences (p > 0.05). Ex vivo gut permeation study showed 4-folds increment in the permeation of drug compared with the plain drug solution. g Scintigraphy imaging and MTT assay results inferred that DCB loaded NLC possesses excellent cytotoxic activity against cancer cells. Thus, NLC holds high potential for the oral delivery of DCB to treat cancer cells and future prospects for the industrial purpose. ã 2014 Elsevier B.V. All rights reserved.

Keywords: Atomic force microscopy Decitabine g Scintigraphy Lipid based nanocarrier Oral drug delivery

1. Introduction DCB, a novel drug that inhibits deoxyribonucleic acid (DNA) methylation and approved by the U.S. Food and Drug Administration (USFDA) to Myelodysplastic Syndromes (MDS) in May 2006 (Gore et al., 2006). DCB is an analogue of the natural nucleoside 20 deoxycytidine reported to have therapeutic activity against various solid tumors and MDS by inhibiting the enzyme DNA methyl transferases, which results in hypomethylation of DNA (Jones and Taylor, 1980; Issa et al., 2005). MDS is a heterogeneous group of bone marrow disorders characterized by ineffective hematopoiesis resulting in anemia, neutropenia, and thrombocytopenia with survival time range from weeks to years (Heaney and Golde, 1999; Silverman and Mufti, 2005).

* Corresponding author. Tel.: +91 11 26059688. E-mail addresses: [email protected] (Y.R. Neupane), [email protected] (K. Kohli). http://dx.doi.org/10.1016/j.ijpharm.2014.11.001 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.

The reported oral bioavailability of the drug is low (3.9–14%) (Liu, 2012) because it was sparingly soluble in water (22.67 mg/ mL), less permeable and unstable in the acidic condition (Garcia et al., 2010). It is also found that DCB gets metabolized by the enzyme cytidine deaminase which is present in the liver (Stresemann and Lyko, 2008). Reduction of the particle size to the nano scale is one of the major approach to enhance the oral bioavailability of the drug (Liversidge and Cundy, 1995). There is a need to design such nanoformulation that could protect DCB from acidic condition and release in the intestinal medium. Although some colloidal drug carriers like polymeric nanoparticles, nanosuspensions, nanoemulsions had tried to overcome the problems like solubility and bioavailability of the many drugs but they possess the disadvantages of the mammalian tissue toxicity due to use of organic solvents, limited physical stability and leakage of drug during storage (Blasi et al., 2011; Radtke et al., 2005; Muller et al., 1996). Hence, the current focus of the research based on the search of bio-compatible lipids as a carrier for low bioavailable drugs to minimize the above mentioned problems.

602

Y.R. Neupane et al. / International Journal of Pharmaceutics 477 (2014) 601–612

NLC has emerged as a potential drug carrier with lower incidence of tissue toxicity due to use of bio-compatible and biodegradable lipids (Muller et al., 1996; Narvekar et al., 2012). It has reported that lipid protects drug from acidic degradation, increases intestinal permeability, promotes oral absorption through lymphatic transport by reducing first pass metabolism and higher amount of drug loading which enhances the availabile of drug to the systemic circulation (Xie et al., 2011; Pouton, 2006; Porter et al., 2007; Charman, 2000). In addition, NLC showed sustained release of the drug from the lipid matrix which results in the prolongation of the drug concentration within therapeutic window (Xie et al., 2011). Hence, NLC is becoming one of the best selected drug delivery system among the researchers. Basically, lipid nanocarriers are composed of solid lipids combine with liquid lipids to enhance the drug loading and stabilized by using aqueous surfactant solution. These are produced using high pressure homogenization, ultrasonication, solvent injection, high shear homogenization and solvent evaporation techniques (Neupane et al., 2013; Mehnert and Mader, 2001; Uner and Yener, 2007). This study thus, aims to design and fabricate thermodynamically stable NLC of DCB by cold homogenization technique using high pressure cell homogenizer. The prepared NLC was characterized for various parameters and its efficacy against cancer cells was evaluated. 2. Materials and methods 2.1. Materials DCB was taken as the gift sample from the Dabur Research Foundation, India. Transcutol1 HP (diethylene glycol monoethyl ether), Compritol1 888 ATO (glyceryl behenate) and Precirol1 ATO 5 (glyceryl palmitostearate) were kindly donated by Gattefosse (Mumbai, India). Tween1 80 (polyoxyethylene (20) sorbitan monooleate) (SD Fine Chem), Poloxamer 188 (BASF) and Solutol1 HS 15 (polyoxyl 15 hydroxystearate) (BASF) were used as surfactant. Deionized water was obtained from interchanged columns Milli-Q (Millipore, U.S.A). Ammonium acetate (Merck, Bombay), sodium bisulfite (Merck), Methanol HPLC Grade (SRL) were used. 2.2. Selection of excipients Solid lipid and liquid lipid were selected based on saturation solubility study and miscibility study among them. For saturation solubility study, 500 mg of the solid lipid was kept in the 5 mL glass vials and heated to 5–10  C above their melting point in water bath. To this, DCB was added in small increments of 1 mg and kept for shaking up to 24 h in the water bath. Solid lipid was selected by observing the loss of transparency on addition of the drug to the melted lipid. Liquid lipid was selected from the miscibility study between solid lipid and liquid lipid. On the other hand, selection of surfactant was carried out by preparation of placebo formulations with different surface active agents. The prepared formulations were evaluated for stability upon storage, particle size, particle sedimentation (data not shown). 2.3. Preparation of DCB loaded NLC 1

2.3.1. Particle size reduction of DCB in Transcutol HP before formulating to NLC DCB was subjected to reduce the particle size prior to formulate the NLC to achieve high entrapment efficiency and drug loading capacity using high pressure cell homogenizer (FPG 12800, Stansted, UK) at room temperature (Kasongo et al., 2012). DCB

(15% w/w) was dispersed in the binary mixture of Transcutol1 HP and Tween1 80 (2% w/w), and then the mixture was vortexed to produce coarse dispersion. The coarse dispersion was homogenized using the high pressure cell homogenizer for five cycles at pressure 500 and 1000 bar (e.g., 5  500 and 5  1000 bar). Samples after each five cycles were taken for particle size monitoring using a light microscope to monitor the progress of particle size reduction during each five cycles. The product obtained after homogenization was used for the further production of NLC. 2.3.2. Production of NLC DCB loaded NLC was prepared by using DCB dispersed in Transcutol1 HP as liquid lipid and Precirol1 ATO5 as solid lipid, and cold high pressure homogenization technique (Kasongo et al., 2012) with slight modification using high pressure cell homogenizer (FPG 12800, Stansted, UK). Transcutol1 HP (4% w/v) containing DCB was added to melted Precirol1 ATO 5 (8–16% w/ v) heated at 70  C with continuous stirring. The molten lipid phase was poured immediately into dry ice to solidify it. After solidification, the dried mixture was ground by using mortar and pestle to get fine powder. The powdered mixture was then dispersed in cold aqueous solution of Tween1 80, Polaxomer 188 and Solutol1 HS 15 (1:2:3) ratio as the surfactant (4–8% w/v) by using Diax 9000 (Heidolph, Germany) at 5400 rpm for 8 min to produce coarse pre-dispersion. The resulted pre-dispersion was then homogenized using the high pressure cell homogenizer at cold condition at the pressure of 500 and 1000 bar for 8–12 cycles to produce NLC dispersion. Finally, NLC dispersion was subjected to the lyophilization to produce NLC powder using mannitol as cryoprotectant in the ratio of 2% w/v of the total formulation. Liquid sample was frozen at 20  C for 24 h and subjected to lyophilization using freeze dryer (Heto Dry Winner, Denmark) at 10  C for 24 h. The obtained NLC powder sample was subjected to further evaluation. 2.4. Design of the experiment by using response surface methodology (RSM) The experimental design was performed by using RSM in which 3-factors 3-levels study and 17 experimental runs were obtained with the help of Design Export1 Software (Version 8.0.7.1, StatEase Inc., MN, USA). This experimental design was used to investigate the effect of independent variables on various dependent variables. The independent variables were % w/v of lipid concentration (X1), % w/v of surfactant concentration (X2) and number of homogenization cycles (X3) represented by 1, 0, +1, analogous to the low, middle and high levels respectively, while the dependent variables were particle size (Y1), poly dispersity index (Y2) and %entrapment efficiency (Y3) as described in Table 1. Table 1 Variables and their coded levels in the Box–Behnken design. Factors

Coded levels

Independent variables

Low level (1)

Medium level (0)

High level (+1)

X1 = lipid concentration (%w/ v) X2 = surfactant concentration (%w/v) X3 = no. of homogenization cycle

8

12

16

4

6

8

8

10

12

Dependent variables

Constraints

Y1 = particle size (nm) Y2 = poly dispersity index (PDI) Y3 = entrapment efficiency (%w/w)

Optimum (100–200 nm) Minimum Maximum

nm = nanometer, mV = millivolt, %w/v = percentage weight/volume.

Y.R. Neupane et al. / International Journal of Pharmaceutics 477 (2014) 601–612

The polynomial equation generated from the experimental design is given below Y ¼ b0 þ b1 X 1 þ b2 X 2 þ b3 X 3 þ b12 X 1 X 2 þ b13 X 1 X 3 þ b23 X 2 X 3 þ b11 X 1 2 þ b22 X 2 2 þ b33 X 3 2 . . . where Y is the response of the dependent variables associated with each factor level combination; b0 is an intercept; b1 to b33 are regression coefficients of the respective variables and X1,X2 and X3 are the coded levels of the independent variables (Chaudhary et al., 2011; Ahmad et al., 2014b; Mujtaba et al., 2014; Srivastava et al., 2014). 2.5. Characterization of NLC 2.5.1. Particle size distribution and zeta potential measurement NLC formulation was reconstituted with Milli-Q water to get particle count between 100 and 400 kilocounts s1. Particle size distribution of the reconstituted NLC formulation was measured by dynamic light scattering (DLS) technique using Zetasizer (Nano-ZS, Malvern Instruments, UK) and analyzed by “DTS nano” software. Ten DLS measurements were performed at 25  C and at the detection angle of 90 to record the average particle size (d, nm) and poly dispersity indix (PDI). Zeta potential of the formulation was measured by using Zetasizer (Nano-ZS, Malvern Instruments, UK) and it measures the electrophoretic mobility which reflects the electric charge on the particle surface. The applied field strength was 20 V cm1 and zeta potential values were recorded automatically by Zetasizer (Neupane et al., 2013). 2.5.2. Surface morphological study 2.5.2.1. Transmission electron microscopy (TEM). TEM study was performed to observe the shape and size of the NLC nanoparticles. Negatively stained with phosphotungstic acid nanoparticle of the formulation was applied on carbon coated grid and observed under TEM (JOEL 2100F, USA) operating at 200 kV. 2.5.2.2. Atomic force microscopy (AFM). The surface morphology of the NLC was investigated by using AFM (Perk System-XE-70, Korea) equipped with non contact cantilever. The instrument was operated in the frequency of 257.903 Hz keeping on scan mode. The dried sample was analyzed in lateral force and topographic image along with 3D view was captured using XEP Software Version 1.7.6 for surface morphological analysis (Atefa et al., 2014). 2.5.3. Determination of entrapment efficiency and drug loading The entrapment efficiency and drug loading capacity of the NLC formulation was determined by measuring the amount of the unentrapped drug by using Amicon ultra centrifugal filter units (Ultra-15, MWCO 10 kDa, Sigma–Aldrich) and was quantified using HPLC method (Neupane et al., 2014). For this, 1 mL of the formulation was diluted with 4 mL of diluents to dissolve any un-loaded drug particles. The diluted sample was kept in the upper compartment of the ultra centrifuge tube and centrifuged at 12,000 rpm for 15 min. The free drug in the aqueous phase moves to the lower chamber through the semi permeable filter membrane, whereas the drug entrapped in the nanoparticle retained in the upper chamber. The collected sample in the lower chamber was measured, and entrapment efficiency and drug loading was determined by the following equations. Entrapment Efficiencyð%w=wÞ ¼

w1  w2  100% w1

Drug Loadingð%w=wÞ ¼

603

w1  w2  100% w3

where w1 = amount of drug added in the NLC, w2 = amount of unentrapped drug, w3 = amount of the lipids added. 2.5.4. In vitro drug release study The in vitro release study of the plain DCB solution and NLC was performed using dialysis bag technique in the phosphate buffer solution (PBS) pH 7.4 (Das et al., 2011; Ahmad et al., 2014a). The dialysis bag (M.W 12 kDa, Sigma–Aldrich) was activated as per the procedure and was soaked in the dissolution medium for 24 h. 6 mL of the DCB solution and NLC was placed in the pre-activated dialysis bag and both the ends were tied with thread then, kept in the beaker containing 500 mL of PBS. After this, the beaker was placed in the magnetic stirrer with continuous stirring at 100 rpm maintained at 37
0.5  C for 24 h. The samples were withdrawn at predetermined time intervals and replenished with same quantity of fresh PBS. So obtained samples were filtered and analyzed for DCB content using HPLC method (Neupane et al., 2014) and the plot between cumulative amount of drug release vs. time was plotted. 2.5.5. Differential scanning calorimetry (DSC) DSC was performed for the lyophilized NLC and DCB powder to determine the interaction between formulation excipients, stability and nature of the NLC using differential scanning calorimeter (Pyris 6 DSC, PerkinElmer, Software Pyris series). The study was conducted with the heating rate of 10  C per min over the temperature range of 35–345  C within an inert environment of nitrogen gas. The samples were kept in hermetic pan made of aluminum with keeping an empty aluminum pan as a reference. 2.5.6. X-ray diffraction (XRD) Powder XRD patterns of DCB powder and lyophilized NLC were investigated using X-ray diffractometer (PW 1830, Philips). Sample on the XRD plate was rotated during data collection to reduce orientation effects of particles. XRD patterns of all the samples were recorded at the resolution of 2u (diffraction angle) = 5 and 90 at 40 kV and 30 mA in the crystalline regions of the samples. Spectra obtained were analyzed for their physical states. 2.6. Stability studies The lyophilized NLC was subjected to stability studies for 45 days in a desiccator at room temperature. Lyophilized NLC was reconstituted with milli-Q water under gentle agitation. Reconstituted sample was analyzed for particle size, PDI, entrapment efficiency and drug loading on 1, 15, 30 and 45 days. 2.7. Ex vivo gut permeation study The gut permeation study of the NLC was conducted as per the procedure and equipment described in (Neupane et al., 2013,b; Ahmad et al., 2014a) and compared with the plain DCB solution under the same experimental conditions. The animals used for the study were taken from the animal house facility of the Jamia Hamdard with prior approval from the Institutional Animal Ethical Care Committee (IAEC) of Jamia Hamdard University, India and approved protocol number was IAEC-907. Fasted over night male albino rats weighing 200–250 g were selected for the experiments and were sacrificed using anesthetic ether. The ileum nearly 5 cm long was surgically removed and flushed with warm saline. The plain drug solution and NLC (2 mL) was filled in the mucosal side and both the ends of the sac were ligated tightly. The sac each containing drug solution and NLC was immersed in jacketed glass

604

Y.R. Neupane et al. / International Journal of Pharmaceutics 477 (2014) 601–612

assembly containing 100 mL of Krebs solution pre warmed at 37
2 C provided with continuous aeration using aerator for 2 h. The samples (2 mL) at predetermined time intervals were collected from the serosal medium and replenished with same quantity of the fresh Krebs solution. The collected samples were filtered and the amount of the drug permeated through the sac was determined using HPLC method. Then, the graph between the cumulative amount of drug permeated (mg) vs. time was plotted and slope as a permeation flux (F) of the graph was taken to calculate the apparent permeability coefficient (APC) by the following equation APC ¼

F 1 cmmin A  C0

where F is the permeation flux, A is the surface area of the barrier membrane (7.85 cm2) and C0 is the initial concentration of the drug in the mucosal medium. 2.8. In vivo study The affinity of the plain DCB and DCB loaded NLC towards the tumor cells induced in the Ehrlich Ascites Tumor (EAT) bearing mice weighing about 30 g were determined after radio-labeling with radio tracer Technetium-99m (Tc-99m) and using g scintigraphy imaging technique. 2.8.1. Radio labeling of formulations and determination of labeling efficacy Radio labeling of plain DCB solution and NLC formulation with Tc-99m was performed to produce radio-labeled complexes DCBTc-99m and NLC-Tc-99m respectively by dirct addition of stannous chloride as reducing agent as per the explained method (Ahmad et al., 2014a; Priyadarshani et al., 2010; Snehalatha et al., 2008). Briefly, 1 mL of plain DCB solution and NLC formulation was mixed individually with 50 mL of stannous chloride (2 mg/mL) and pH of this mixture was adjusted to 7.0 by addition of sodium hydrogen carbonate solution (0.5 M). This was filtered through 0.25 um

membrane filter into sterile vials. To this, approximately 50–60 MBq of Tc-99m was added, mixed and incubated for 15 min at room temperature. The percentage of radio labeling efficacy was determined by thin layer chromatography (TLC). TLC was performed using instant thin layer chromatography-silica gel (ITLC-SG) strips as stationary phase and acetone (100%) as mobile phase. The effects of stannous chloride concentration, incubation time and pH on labeling efficacy were studied to determine optimum reaction conditions.The stability of radio-labeled complex was evaluated in 0.9% (w/v) NaCl solution and in rat plasma. The stable radio-labeled complex was used for the further distribution study using g scintigraphy imaging technique. 2.8.2. g Scintigraphy imaging study g Scintigrapgy imaging study was performed on Ehrlich Ascites Tumor (EAT) bearing mice giving DCB-Tc-99m through tail vein and NLC-Tc-99m through oral route. These mice were obtained from the animal house facility at Institute of Nuclear Medicine and Allied Sciences, New Delhi, and experiments were conducted as per approved protocol. The first group was given with DCB-Tc-99m (equivalent to 160 mci) through tail vein whereas, the second group was given with NLC-Tc-99m (equivalent to 160 mci) through the oral route. At predetermined time intervals (0.25 h, 2 h, 4 h and 24 h) images were captured by Gamma camera (Spect-CT 75005 Diacam, Siemens, Germany). All the images were analyzed using Symbian Software. The percent of the radioactivity that reaches to the tumor cells were determined with respect to the total activity present in the body and compression between controlled muscle and tumor induced cells were also performed. 2.9. MTT assay The MTT assay for the NLC was executed in A549 human nonsmall cell lung cancer cells for the determination of cell cytotoxicity activity. A549 cells were seeded in 96 well plates at a density of 1 105cells per well in 120 mL Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine

Fig. 1. Microscopic images depicting DCB prior to (a) and after application of homogenization pressure (b) 5  500 and (c) 5  1000 bar at 100 magnification.

Y.R. Neupane et al. / International Journal of Pharmaceutics 477 (2014) 601–612

605

Table 2 Predicted and actual values of the optimized NLC formulation. Formulation

Optimized NLC (predicted) Optimized NLC (actual)

Composition

Characterization

X1

X2

X3

Y1

Y2

Y3

0 12

0 6

0 10

117.36 116.64
6.67

0.187 0.194
0.12

80.31 84.42
5.38

Zeta potential (mV)

Maximum release in 24 h

– 31.8
0.96

– 80.23%
4.67

X1 = lipid concentration (%w/v), X2 = surfactants concentration (%w/v), X3 = no. of homogenization cycles, Y1 = particle size (nm), Y2 = PDI, Y3 = %entrapment efficiency, n = 3.

serum and 1% penicillin/streptomycin. The cells were incubated in a humidified chamber maintained at 37  C and 100% RH supplied with 95% O2 and 5% CO2. These cells were treated with different concentrations of free DCB solution, placebo NLC and equivalent amount of DCB loaded NLC for a period of 4 h. After 4 h, cells were supplied with fresh DMEM and allowed to incubate in a humidified chamber for 24 h. Then, after 24 h the cells were treated with 250 mL of fresh MTT reagent at a concentration of 5 mg/mL in PBS and further incubated for 4 h at 37  C in dark conditions. Finally, the cells were treated with 250 mL of DMSO to dissolve the formazan crystals. Further, the plates were read on a microplate reader at 570 nm. The data were expressed as the percent cell survival of control cells receiving just the media. The %cell survival was determined by using the following equation

%Cell survival ¼

Sample absorbance at570nm  100 Control absorbance at570nm

2.10. Statistical analysis All the results obtained were expressed as mean
S.D using Graph Pad Software (Instat 3.06, USA) with more than three replicates for each experiment. Comparisons were made by using Student’s t-test and the level of significance was set p < 0.05. 3. Result and discussion 3.1. Selection of excipients From the solubility and miscibility studies, Precirol1 ATO5 as a solid lipid and Transcutol1 HP as a liquid lipid were selected to

Fig. 2. Images showing (a) particle size (b) zeta potential and (c) TEM analysis of the optimized NLC.

606

Y.R. Neupane et al. / International Journal of Pharmaceutics 477 (2014) 601–612

produce NLC (data not shown). Combination of surfactants as Tween1 80, Polaxomer 188 and Solutol1 HS 15 (1:2:3) ratio were selected to produce stable NLC system after successful trials. 3.2. Particle size reduction of DCB in Transcutol1 HP DCB was subjected to particle size reduction prior to formulate NLC to achieve high entrapment efficiency and drug loading capacity. Measurement of particle size after successive cycle of high pressure cell homogenization of the DCB in Transcutol1 HP generally required to be monitored by dynamic light scattering technique (DLS). This method required the use of water or liquids of low viscosity as a dispersion medium. Due to the hydrophilic nature of DCB and viscous nature of the Transcutol1 HP used for the process, it was not possible to monitor the size reduction of DCB using DLS technique. So, optical microscope was used to monitor the process of the size reduction after successive cycle of the homogenization. The results of the microscopic images at 100 are shown in Fig. 1. It showed the reduction in particle size after successive cycles of homogenization at different pressures with excellent dispersibility in the liquid lipid.

3.3. Optimization by RSM The independent variables were observed to be significantly influenced over the dependent variables as Y1 particle size, Y2 poly dispersity index and Y3 entrapment efficiency. The optimum value of the independent variables were determined based on the mathematical model fit analysis and polynomial equations involving the main effect and their interaction factors based on estimation of statistical parameters like sequential p-value, lack of fit and difference between adjusted and predicted R2-values (Mujtaba et al., 2014) (data and graphs not shown). The selection criterion of the NLC was set as per the desired results i.e., particle size (100–200 nm), minimum PDI and maximum entrapment efficiency. For the optimized NLC predicted values and actual values obtained from the experiments are given in the Table 2. 3.4. Particle size distribution and zeta potential DLS technique used to measure the particle size distribution of the NLC covers the measurement of particle size range from a few nanometers to about 3 mm in size (Muller et al., 2000). The particle size and PDI of the optimized NLC were found to be

Fig. 3. AFM images showing (a) topographic image at scan range 60 mm  60 mm (b) 3D image at scan range 10 mm  10 mm (c) 3D image scan range 60 mm  60 mm (d) 3D image of tip analysis.

Y.R. Neupane et al. / International Journal of Pharmaceutics 477 (2014) 601–612

607

Fig. 4. In vitro drug release studies of plain DCB solution and optimized NLC formulation.

116.64
6.67 nm and 0.194
0.12, respectively as shown in the Fig. 2(a). Particle size analysis revealed the positive effect with lipid concentration (X1), whereas the negative effect with the surfactant concentration (X2) and number of homogenization cycles (X3). This was in an agreement with the report (Schubert and MullerGoymann, 2003). Zeta potential of the optimized NLC was found to be 31.8
0.96 mV as shown in the Fig. 2(b). Zeta potential is the potential difference between the stationary layer of the dispersed particle and dispersion medium. Zeta potential indicates the stability of the dispersed particles in the dispersion medium. Higher zeta potential values indicates greater repulsion between the particles and particle aggression is less likely to occur. Negative value of the zeta potential showed that NLC had gained good stability and dispersion quality among these nanoparticles (Mehnert and Mader, 2001; Jenning et al., 2000). 3.5. Surface morphological study 3.5.1. Transmission electron microscopy (TEM) TEM images showed almost spherical surface of the optimized NLC with uniform distribution and was on corroboration with particle size measurements. This concluded that the particles were uniformLy distributed in the sample and the particles were separated from each other, which is a typical NLC system (Hatziantoniou et al., 2007; Rahman et al., 2010). TEM photomicrograph of optimized NLC is given in Fig. 2(c).

3.5.2. Atomic force microscopy (AFM) AFM investigation has been widely used to obtain the size, shape and surface morphological information of the nanoparticles (Gualbert et al., 2003; Shahgaldian et al., 2003). The surface morphology of the optimized NLC can be clearly seen in the Fig. 3 and proved smooth surface of the NLC. Spherical particles were seen in the topographic image Fig. 3(a) as well as in 3D image Fig. 3(b) and particles were separated from each other, which proved that there were no aggregation in the particles. Certain caves were observed during tip analysis Fig. 3(d) which may be due to error signals occurred during analysis. 3.6. Entrapment efficiency and drug loading The entrapment efficiency of optimized NLC was found to be 84.42
5.38% and drug loading was observed to be 8.54
2.65%. It has been observed during optimization that the increase in the lipid concentration (X1) showed a strong positive correlation with entrapment efficiency. This may be due to the dispersion of drug in Transcutol1 HP before mixing with Precirol1 ATO5. This technique resulted in the increase in the entrapment efficiency and drug loading capacity of the NLC (Kasongo et al., 2012). 3.7. In vitro drug release study The in vitro release study of the optimized NLC showed sustained release up to 24 h, whereas plain DCB solution showed

Table 3 In vitro release model fitting in terms of linear regression coefficient (R2). S. No.

Model

1. 2. 3. 4. 5.

Zero order model First order model Higuchi model Hixcon–Crowell model Korsmeyer–Peppas model

R2

Plot X-axis

Y-axis

Fraction of drug released Log %drug remaining Fraction of drug released Cube root of fraction drug remaining Log fraction of drug released

time time p time time log time

0.8669 0.9639 0.9719 0.9505 0.9281

608

Y.R. Neupane et al. / International Journal of Pharmaceutics 477 (2014) 601–612

Fig. 5. Graphs showing (a) DSC thermogram of DCB and Freeze dried NLC (b) XRD graph of DCB and Freeze dried NLC.

95.79
3.89% drug release at 4 h. NLC showed biphasic drug release behavior with initial burst release at first 2 h (29.36
2.66% cumulative %drug release) followed by a sustained release pattern for 24 h, cumulative %drug release was measured as 80.23
4.67% in PBS pH 7.4. The graph showing cumulative %drug release vs. time is shown in Fig. 4. Various factors that can influence the release of the drug from the NLC are solubility of the drug in the lipid, lipid matrix and its concentration, partition co-efficient and particle size (Muller et al., 2006). 3.8. Drug release mechanistic study The obtained in vitro release data were incorporated into various release kinetic models like Zero order model, First-order model, Higuchi model, Hixcon–Crowell model and Korsmeyer– Peppas model, and linear regression analysis were performed. The best fit model was determined on the basis of regression coefficient (R2) value. The observed best fit model was higuchi model followed by first order release model with R2 0.9719 and 0.9639, respectively. R2 values for all the models are presented in the Table 3. Korsmeyer–Peppas model suggests that for the equation Mt/ M1 = Ktn, (Mt/M1 = fraction of the drug release, K = release rate constant, t = release time and n = release exponent) for the drug release that is dependent on the shape of the matrix dosage form. We calculated the value of n by plotting a graph between the fraction of drug release and square root of time. The n-values 0–0.5, 0.5–1, 1 and >1 characterize a Fickian diffusion, non Fickian, Zero order and super diffusion respectively for spherical particles (Dash et al., 2010; Sabyasachi et al., 2014). The obtained value of n = 0.075 for NLC showed that the release behavior was fickian diffusion.

drug. Lyophilized NLC displayed delayed onset and melting temperature Precirol1 ATO 5 as compared to that of the pure lipid, whereas the peak of the DCB at around 201  C had been disappeared because drug was molecularly dispersed in the lipid matrix. The peak at 55.973  C represents the peak of lipid present in the NLC formulation indicated the stable crystalline form of the lipid and no other peaks were visible proved that the stability enhancing effect of the lipid in the formulation (Ahmad et al., 2014a; Pardeikea et al., 2011). The observed peak at about 160  C is of mannitol which was added during the lyophilization of the NLC as a cryoprotectant. 3.10. X-Ray diffraction (XRD) The XRD observations of the DCB and lyophilized NLC are shown in the Fig. 5(b). Figure showed significant differences between the diffraction patterns of the drug and lyophilized NLC. Pure DCB showed diffraction peaks at 2u values of 15 . The low intense peak of DCB in lyophilized NLC at nearly 2u = 15 showed pronounced decrease in the crystallinity which may be due to super solubility and stability of the drug loaded NLC. This proved that the drug was molecularly dispersed in the NLC matrix (Atefa et al., 2014). 3.11. Stability studies Lyohipilized DCB loaded NLC containing 2% mannitol was evaluated for stability studies at room temperature. Average particle size, PDI, entrapment efficiency and drug loading were evaluated after reconstitution of the NLC at 1, 15, 30 and, 45 days after lyophilization. Results as shown in the Fig. 6(a) and (b) proved the stability of the NLC at room temperature with no significant (p > 0.05) difference in the observations.

3.9. Differential scanning calorimetry (DSC) 3.12. Ex vivo gut permeation study DSC thermograms of DCB and optimized NLC powder is shown in the Fig. 5(a). DCB showed an exothermic sharp peak at the melting point of 201.54  C indicating the crystalline nature of the

Ex vivo gut permeation study showed nearly 4 folds increment in the permeation of the drug from NLC compared with the plain

Y.R. Neupane et al. / International Journal of Pharmaceutics 477 (2014) 601–612

609

Fig. 6. Stability studies graphs (a) particle size and PDI of reconstituted optimized NLC (b) %entrapment efficiency and %drug loading of reconstituted optimized NLC after 1, 15, 30, 45 days.

DCB solution under the same experimental conditions. The increment in the permeation of DCB was due to the reduction of particle size of the DCB in NLC along with the lipids and surfactants used for the production of NLC. This finally leads to improvement in the oral bioavailability of the drug. The APC for the plain drug solution and optimized NLC were found to be 0.356  104 cm/min and 1.64  104 cm/min, respectively. NLC composed of lipids and surfactants with simultaneous reduction in particle size had proved to increase in the permeability of the drug from the intestine. The combination of surfactants which are present in the Table 4 Stability of radio-labeled complexes DCB-Tc-99m and NLC-Tc-99m in saline and plasma at different time intervals. Time (h)

Radio-label efficacy (%)
S.D. DCB-Tc-99m Saline

0.25 2 4 24

96.34 95.83 95.08 92.62







0.34 0.89 1.08 3.56

NLC-Tc-99m Plasma

Saline

96.15
0.98 95.78
4.32 94.65
4.67 92. 81
5.43

96.98 95.21 95.42 92.73

Plasma





3.64 0.93 0.56 6.56

96.56 96.02 95.18 93.05







0.87 0.76 5.63 7.54

formulation not only provides stability to the formulation but also mitigate intestinal efflux by inhibition of P-gp efflux pumps which are present in the villus tip of enterocytes in the gastrointestinal tract (Ahmad et al., 2014a). 3.13. In vivo study 3.13.1. Radio labeling experiments Radio labeling efficacy was found to be 98.06% at 50 mL of stannous chloride (2 mg/mL) and pH 7.0 determined by ITLC technique (optimized data not shown). The concentration of stannous chloride (reducing agent) plays major role in the stability of the radio-labeled complexes at optimum pH. The stability of radio-labeled DCB solution as well as NLC formulation was determined in the normal saline and mice plasma for different time intervals as shown in Table 4. The results of stability study showed that the radio-labeled products were stable over period of 24 h and suitable for g scintigraphy imaging study. 3.13.2 g Scintigraphy imaging study g Scintigraphy imaging is one of the best non invasive imaging technique to determine the accumulation of drug and nanoparticle

610

Y.R. Neupane et al. / International Journal of Pharmaceutics 477 (2014) 601–612

Fig. 7. Whole body g scintigraphy images (a) after 4 h of oral administration of optimized NLC-Tc-99m (b) After 4 h of I.V. injection through tail vein of DCB-Tc-99m.

in the tumor cells. The affinity of these complexes (DCB-Tc-99m and NLC-Tc-99m) to the tumor cells were determined employing g scintigraphy imaging technique over time as shown in the Fig. 7. In this study, mice showed accumulation of the radio-labeled complexes in the tumor cells maximum at 4 h after I.V. administration of DCB-Tc-99m through tail vein and NLC-Tc99m after oral administration. The images were captured up to 24 h of post administration. The %activity of the radio-labeled complexes were also determined with reference to the total activity present in the body. The %activity that reaches to the tumor cells at 4 h after oral administration of NLC-Tc-99m and I.V. administration of DCB-Tc-99m through tail vein were found 9.977% and 4.938%

respectively. The amount of the activity that reaches to the normal muscles and tumor cells, and their ratio are represented in the Tables 5 and 6. 3.14. MTT assay The percent survival of the A549 human non-small cell lung cancer cells after treatment with a free DCB solution and optimized NLC were determined using MTT/cell cytotoxicity assay. Fig. 8 showed the percentage cell survival at different concentration ranges after 24 h of post treatment. This graph demonstrated that the NLC loaded with DCB had significantly improved cytotoxic

Table 5 Ratio of tumor to muscle count and %activity in tumor cell and muscle observed after oral administration of NLC-Tc-99m. Time (h)

Total count
S.D.

0.25 2 4 24

72,784 58,456 48,653 2206







6.68 8.36 6.46 8.53

Tumor cell count
S.D. 1608 4187 4854 148







8.43 8.32 7.41 6.43

Muscle count
S.D. 486 568 578 48







6.43 8.42 6.32 7.43

Tumor/muscle

%Activity in tumor

%Activity in muscle

3.31 7.37 8.40 3.08

2.209 7.163 9.977 6.709

0.668 0.972 1.188 2.176

Table 6 Ratio of tumor to muscle count and %activity in tumor cell and muscle observed after IV injection of DCB-Tc-99m solution through tail vein. Time (h)

Total count
S.D.

Tumor cell count
S.D.

Muscle count
S.D.

Tumor/muscle

%Activity in tumor

%Activity in muscle

0.25 2 4 24

55,927
4.67 48,348
8.57 40,784
6.35 4804
6.56

1123
6.46 1426
6.49 2014
8.36 210
8.21

224
6.35 237
5.32 540
8.43 68
7.23

5.01 6.02 3.72 3.09

2.008 2.949 4.938 4.371

0.401 0.490 0.441 1.415

Y.R. Neupane et al. / International Journal of Pharmaceutics 477 (2014) 601–612

611

Fig. 8. MTT assay for placebo NLC, plain DCB solution and DCB loaded NLC with A549 human non-small cell lung cancer cells.

activity of DCB as compared to plain DCB solution. Placebo formulation showed no cell cytotoxic activity. This results clearly indicated a higher cell death with the drug loaded NLC as compared to an equivalent dose of free DCB solution. This may be due to the fact that the higher amount of the DCB accumulated in the cells from the NLC. The burst release of the drug from the NLC at the initial stage made the free drug for transport into the cells through nucleoside transpoters and these nanoparticles were actively endocytosed. This biphasic release behavior of the lipid nanoparticles showed great advantages in providing both free drugs and drug loaded on the nanopaticles at the site of action. 4. Conclusions The present study conclusively illustrated the use of response surface methodology for the optimization of the NLC system. The physicochemical characterization proved that the DCB was molecularly dispersed in the lipid matrix present in the NLC system and was within the optimum size range. The result of the stability study revealed that changes in the observed parameter up to 45 days were found to be with in the statistically significant limit (p > 0.05) indicated that the optimized NLC system was stable both physically and chemically. Furthermore, g scintigraphy imaging and MTT assay proved that the DCB loaded NLC system has more affinity towards tumor cells and along with cytotoxic activity against cancer cells. Thus, DCB incorporated NLC system present an important and valuable strategy for treatment of cancer cells through oral delivery. Conflict of interest The authors report no conflicts of interest. Acknowledgments The authors are greatful to Governement of India for providing SAARC country nation scholarship to Mr. Yub Raj Neupane. The authors also wish to express their appreciation to Mr Anurag Tyagi (Gattefosse, India) for providing the lipids as a gift sample and Amit kumar from Aimil instrument for zeta potential determination in

his laboratory. Authors are also thankful to AIIMS, New Delhi and JNU, New Delhi for providing laboratory facilities during sample analysis. We greatfully acknowledge Mrs. Srijana Subedi Neupane and Mr. Nikesh Shrestha for their linguistic support during manuscript editing. References Ahmad, J., Mir, S.R., Kohli, K., Chuttani, K., Mishra, A.K., Panda, A.K., Amin, S., 2014a. Solid nanoemulsion preconcentrate for oral delivery of paclitaxel: formulation design, biodistribution, and g scintigraphy imaging. BioMed Res. Int. 984756. Ahmad, N., Amin, S., Neupane, Y.R., Kohli, K., 2014b. Anal fissure nanocarrier of lercanidipine for enhanced transdermal delivery: formulation optimization, ex vivo and in vivo assessment. Expert Opinion Drug Deliv. 11, 467–478. Atefa, M., Rezaeia, M., Behrooz, R., 2014. Preparation and characterization agarbased nanocomposite film reinforced by nanocrystalline cellulose. Int. J. Biol. Macromol. 70, 537–544. Blasi, P., Giovagnoli, S., Schoubben, S., et al., 2011. Lipid nanoparticles for brain targeting I. Formul. Optim. Int. J. Pharm. 419, 287–295. Charman, W.N., 2000. Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts. J. Pharm. Sci. 89, 967. Chaudhary, H., Kohli, K., Amin, S., Rathee, P., Kumar, V., 2011. Optimization and formulation design of gels of diclofenac and curcumin for transdermal drug delivery by Box–Behnken statistical design. J. Pharm. Sci. 100, 580–592. Das, S., Ng, W.K., Kanaujia, P., Kim, S., Tana, R.B.H., 2011. Formulation design, preparation and physicochemical characterizations of solid lipid nanoparticles containing a hydrophobic drug: effects of process variables. Colloids Surf. B Biointerfaces 88, 483–489. Dash, S., Murthy, P.N., Nath, L., Chowdhury, P., 2010. Kinetic modeling on drug release from controlled drug delivery systems. Acta Pol. Pharm. 67, 217–223. Garcia, J.S., Jain, N., Godley, A.L., 2010. An update on the safety and efficacy of DCB in the treatment of myelodysplastic syndromes. OncoTargets Ther. 3, 1–13. Gore, S.D., Jones, C., Kirkpatrick, P., 2006. Decitabine. Nat. Rev. Drug Discov. 5, 891– 892. Gualbert, J., Shahgaldian, P., Coleman, A.W., 2003. Interactions of amphiphilic calixarene-based, arene-based solid lipid nanoparticles with bovine serum albumin. Int. J. Pharm. 257, 69. Hatziantoniou, S., Deli, G., Nikas, Y., Demetzos, C., Papaioannou, G.T., 2007. Scanning electron microscopy study on nanoemulsions and solid lipid nanoparticles containing high amounts of ceramides. Micron 38, 819–823. Heaney, M.L., Golde, D.W., 1999. Myelodysplasia. N. Engl. J. Med. 340, 1649–1660. Issa, J.P.J., Kantarjian, H.M., Kirkpatrick, P., 2005. Azacitidine. Nat. Rev. Drug Discov. 4, 275–276. Jenning, V., Thunemann, A.F., Gohla, S.H., 2000. Characterization of a novel solid lipid nanoparticle carrier system based on binary mixtures of liquid and solid lipids. Int. J. Pharm. 199, 167–177. Jones, P.A., Taylor, S.M., 1980. Cellular differentiation: cytidine analogues and DNA methylation. Cell 20, 85–93. Kasongo, K.W., Muller, R.H., Walker, R.B., 2012. The use of hot and cold high pressure homogenization to enhance the loading capacity and encapsulation efficiency of nanostructured lipid carriers for the hydrophilic antiretroviral drug,

612

Y.R. Neupane et al. / International Journal of Pharmaceutics 477 (2014) 601–612

didanosine for potential administration to paediatric patients. Pharm. Dev. Technol. 17, 353–362. Liu, D., 2012. Recent advances in myelodysplasia: update from 2011 ASH annual meeting. J. Hematol. Oncol. 5, A4. Liversidge, G.G., Cundy, K.C., 1995. Particle size reduction for the improvement of oral bioavalability of hydrophobic drugs I. Absolute oral bioavailability of microcrystalline danazol in beagle dogs. Int. J. Pharm. 125, 91. Mehnert, W., Mader, K., 2001. Solid lipid nanoparticles production characterization, and applications. Adv. Drug Deliv. Rev. 47, 165–196. Mujtaba, A., Ali, M., Kohli, K., 2014. Formulation of extended release cefpodoxime proxetil chitosan-alginate beads using quality by design approach. Int. J. Biol. Macromol. 69, 420–429. Muller, R.H., Maassen, S., Weyhers, H., Mehnert, W., 1996. Phagocytic uptake and cytotoxicity of solid lipid nanoparticles (SLN) sterically stabilized with poloxamine 908 and poloxamer 407. J. Drug Targeting 4, 161. Muller, R.H., Mader, K., Gohla, S., 2000. Solid lipid nanoparticles (SLN) for controlled drug delivery—a review of the state of the art. Eur. J. Pharm. Biopharm. 50, 161–177. Muller, R.H., Runge, S., Ravelli, V., Mehnert, W., Thunemann, A.F., Souto, E.B., 2006. Oral bioavailability of cyclosporine: solid lipid nanoparticles (SLNR) versus drug nanocrystals. Int. J. Pharm. 317, 82–89. Narvekar, M., Xue, H.Y., Wong, H.L., 2012. A novel hybrid delivery system: polymeroil nanostructured carrier for controlled delivery of highly lipophilic drug alltrans-retinoic acid (ATRA). Int. J. Pharm. 436, 721–731. Neupane, Y.R., Sabir, M.D., Ahmad, N., Ali, M., Kohli, K., 2013. Lipid drug conjugate nanoparticle as a novel lipid nanocarrier for the oral delivery of decitabine: ex vivo gut permeation studies. Nanotech 24, 415102. Neupane, Y.R., Srivastava, M., Ahmad, N., Soni, K., Kohli, K., 2014. Stability indicating RP-HPLC method for the estimation of decitabine in bulk drug and lipid based nanoparticles. Int. J. Pharm. Sci. Res. 07, 294. Pardeikea, J., Webera, S., Haberb, T., Wagnerb, J., Zarflc, H.P., Plankb, H., Zimmera, A., 2011. Development of an itraconazole-loaded nanostructured lipid carrier (NLC) formulation for pulmonary application. Int. J. Pharm. 419, 329–338. Porter, C.J.H., Trevaskis, N.L., Charman, W.N., 2007. Lipids and lipid-based formulations: optimizing the oral delivery of lipophilic drugs. Nat. Rev. Drug Discov. 6, 231–248.

Pouton, C.W., 2006. Formulation of poorly water-soluble drugs for oral administration: physicochemical and physiological issues and the lipid formulation classification system. Eur. J. Pharm. Sci. 29, 278. Priyadarshani, A., Chuttani, K., Mittal, G., Bhatnagar, A., 2010. Radiolabeling, biodistribution and gamma scintigraphy of noscapine hydrochloride in normal and polycystic ovary induced rats. J. Ovar. Res. 3, 10. Radtke, M., Souto, E.B., Muller, R.H., 2005. Nanostructured lipid carriers: a novel generation of solid lipid drug carriers. Pharm. Technol. Eur. 17, 45. Rahman, Z., Zidan, A.S., Khan, M.A., 2010. Non-destructive methods of characterization of risperidone solid lipid nanoparticles. Eur. J. Pharm. Biopharm. 76, 127–137. Sabyasachi, M., Ranjit, M., Biswanath, S., 2014. Nanoreticulations of etherified locust bean polysaccharide for controlled oral delivery of lamivudine. Int. J. Biol. Macromol. 65, 193–199. Schubert, M.A., Muller-Goymann, C.C., 2003. Solvent injection as a new approach for manufacturing lipid nanoparticles—evaluation of the method and process parameters. Eur. J. Pharm. Biopharm. 55, 125–131. Shahgaldian, P., Silva, E.D., Coleman, A.W., Rather, B., Zaworotko, M.J., 2003. Paraacyl-calix-arene based solid lipid nanoparticles (SLNs): a detailed study of preparation and stability parameters. Int. J. Pharm. 253, 23. Silverman, L.R., Mufti, G.J., 2005. Methylation inhibitor therapy in the treatment of myelodysplastic syndrome. Nat. Clin. Pract. Oncol. 2, S12–S23. Snehalatha, M., Venugopal, K., Saha, R.N., 2008. Etoposide loaded PLGA and PCL nanoparticles II: biodistribution and pharmacokinetics after radiolabeling with Tc-99m. Drug Deliv. 15, 277–287. Srivastava, M., Kohli, K., Ali, M., 2014. Formulation development of novel in situ nanoemulgel (NEG) of ketoprofen for the treatment of periodontitis. Drug Deliv [Epub ahead of print]. Stresemann, C., Lyko, F., 2008. Modes of action of the DNA methyltransferase inhibitors azacytidine and DCB. Int. J. Cancer 123, 8–13. Uner, M., Yener, G., 2007. Importance of solid lipid nanoparticles (SLN) in various administration routes and future perspectives. Int. J. Nanomed. 2, 289–300. Xie, S., Zhu, L., Dong, Z., Wang, X., Wang, Y., Li, X., Zhou, W., 2011. Preparation, characterization, and pharmacokinetics of enrofloxacin-loaded solid lipid nanoparticles: influences of fatty acids. Colloids Surf. B Biointerfaces 83, 382.