Nanostructured lipid carriers of olmesartan medoxomil with enhanced oral bioavailability

Accepted Manuscript Title: Nanostructured lipid carriers of olmesartan medoxomil with enhanced oral bioavailability Authors: Vikram Kaithwas, Chander Parkash Dora, Varun Kushwah, Sanyog Jain PII: DOI: Reference:

S0927-7765(17)30121-2 http://dx.doi.org/doi:10.1016/j.colsurfb.2017.03.006 COLSUB 8416

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

13-12-2016 1-3-2017 2-3-2017

Please cite this article as: Vikram Kaithwas, Chander Parkash Dora, Varun Kushwah, Sanyog Jain, Nanostructured lipid carriers of olmesartan medoxomil with enhanced oral bioavailability, Colloids and Surfaces B: Biointerfaces http://dx.doi.org/10.1016/j.colsurfb.2017.03.006 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.

Nanostructured lipid carriers of olmesartan medoxomil with enhanced oral bioavailability

Vikram Kaithwas1,2, Chander Parkash Dora1,2, Varun Kushwah1, Sanyog Jain1,*

1

Centre for Pharmaceutical Nanotechnology, Department of Pharmaceutics, National Institute of

Pharmaceutical Education and Research (NIPER), Sec-67, S.A.S. Nagar, Punjab-160062, India 2

Department

of

Pharmaceutical

Technology

(Formulations),

National

Institute

of

Pharmaceutical Education and Research (NIPER), Sec-67, S.A.S. Nagar, Punjab-160062, India

*Corresponding Address: Dr. Sanyog Jain, Associate Professor Centre for Pharmaceutical Nanotechnology, Department of Pharmaceutics National Institute of Pharmaceutical Education and Research (NIPER), S.A.S. Nagar, Punjab-160062, India Email: [email protected], [email protected] Telephone: +91 172 2292055

1

Graphical abstract

Highlights    

Nano lipid carrier of olmesartan medoxomil was prepared by systemic design approach. In vitro release showed the extended release of drug from nano lipid carriers. Enhanced in vitro cell uptake of nanocarriers was established. Nanoformulation showed improved oral bioavailability.

Abstract The current study explores the potential of nanostructured lipid carriers (NLCs) for oral bioavailability enhancement of olmesartan medoxomil (OLM) by systemic design approach. OLM-NLC was successfully prepared with optimized process parameters (i.e. amount of liquid 2

lipid, total amount of lipid, drug content and surfactant concentration) using the Box-Behnken design of experiments for different response parameters (i.e. particle size, Polydispersity index and entrapment efficiency). Further, optimized formulation was validated which depicted nano size, homogenous distribution with optimum entrapment efficiency. OLM-NLC was characterized by different techniques viz. differential scanning calorimetry (DSC), powder XRay diffraction (PXRD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM), which showed reduced crystallinity of the drug with smooth spherical appearance of nanoparticles. Formulation was found to be stable in simulated gastric fluids as no significant changes were found in size, PDI and entrapment efficiency. In vitro release showed extended release of OLM from OLM-NLC. In vitro cellular uptake study revealed 5.2 folds higher uptake of nanoparticles as compare to the free drug, when incubated with Caco-2 cells. In vivo performance showed that AUCtotal and Cmax of OLM-NLC was found significantly (P<0.01) higher as compare to the free drug. Overall, the present study successfully reports the improvement of oral bioavailability of olmesartan medoxomil.

Keywords: Olmesartan medoxomil; Nanostructured lipid carrier (NLC); Bioavailability; BoxBehnken Design; Caco-2 cells.

1. Introduction Among various antihypertensive agents, olmesartan medoxomil (OLM) is widely used to normalize blood pressure by owing antagonistic activity of AT1 subtype angiotensin II receptors [1]. However, it has unfavourable biopharmaceutical properties viz. poor aqueous solubility, high first pass metabolism and high P-glycoprotein (P-gp) efflux which leads to its poor bioavailability (~28%) [2, 3]. It has also some other disadvantages due to ominous breakage of OLM to active form, which causes sprue like enteropathy (severe diarrhoea) on long term conventional therapy [4]. It may also be due to direct physical contact of free olmesartan with intestinal villi that stimulate the cell mediated immune response [5]. Thus, there is an urge to develop strategies which could avoid these problems and thereby, maximize the therapeutic benefits. Though many delivery platforms such as, self emulsifying drug delivery systems (SEDDS) [6, 7], nanoemulsion [8], nanosuspension [9], gastroretentive systems [10], liquid solid 3

compact [11] PLGA nanoparticle [12] and solid dispersion [13] have been formulated to overcome the poor bioavailability problem of the drug, yet it remains a challenge for formulation scientist to overcome this issue. Previous attempts have concluded that lipid-based drug delivery systems are most promising platforms and have potential to alter the solubility of OLM like BCS class II drugs [14-18]. Among various lipid based delivery systems, solid lipid nanoparticles (SLN) and nanostructured lipid carrier (NLC) are two primarily lipidic systems which are mainly used nowadays as an alternative of conventional delivery systems [19]. SLN primarily comprises of pure solid lipid and aqueous surfactant phase, are widely used for increasing the bioavailability of poorly water soluble drugs, [20, 21]. However, some disadvantages like poor drug loading (drug expulsion phenomenon) and stability in gastrointestinal media are associated with SLN [22]. Further, the second generation lipid nanoparticle i.e. NLC has the potential to overcome the problem of poor drug loading efficiency of SLN [23]. It contains mixture of solid lipid and oils. Liquid lipids (oils) are the main contributing factor and are responsible for creating imperfections in solid lipid matrix that reduced the drug expulsion and increased the drug loading capacity [24]. Like others, designing NLC is a crucial step, as its physicochemical properties are affected by different formulations process and parameters. For optimizing any formulation, selection of one factor at a time (OFAT) screening approach is past now. Here, Response surface methodology (RSM) was selected as an optimization approach which briefly discussed the relationship between dependent and independent variables and their interactions with minimum experimentation [25, 26]. The objective of the present study is to design and assess the nanostructured lipid carriers (NLCs) as a potential oral formulation of OLM for enhancement of its oral bioavailability. In this study, OLM loaded NLCs (OLM-NLC) were prepared by hot high shear homogenization, optimized with Box-Behnken design and characterized by different techniques. Further, the formulation was evaluated for in vitro drug release and in vivo pharmacokinetic study. In addition, in vitro cell uptake of nanoformulation was discussed through Caco-2 cell lines studies.

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2. Materials and methods 2.1. Materials Olmesartan medoxomil was obtained as a gift sample from the Cadila Pharmaceuticals Ltd., (Ahmedabad, India). Precirol® ATO-5 (glyceryl distearate Type-1 EP) was obtained as a gratis sample from Gattefosse (Nanterre, France). Solutol® HS-15, Poloxamer-407 and Poloxamer-188 were kindly supplied by BASF (Ludwigshafen, Germany). PEG-40 stearate was purchased from Sigma-Aldrich Chemical Co. (Missouri, USA) and Capmul® MCM EP (Glyceryl Caprylate/Caprate) was obtained from Abitec Corporation (Ohio, USA). High-Performance Liquid Chromatography (HPLC) grade solvents such as acetonitrile and methanol were purchased from J.T. Baker (USA). Caco-2 (colorectal adenocarcinoma) cell lines were obtained from the cell repository facility of National Centre for Cell Sciences (NCCS), Pune, India. All other chemicals and reagents used were of analytical grade.

2.2. Methods 2.2.1. Screening of solid lipids Solubility of OLM was studied in different solid lipids with modification of earlier described method [6]. Briefly, 5 mg of OLM was taken in a flat bottom screw cap glass vial and lipid was added in the increment of 100 mg. Vials were heated on water bath above the melting point of solid lipid and vortexed. The solubility of OLM was estimated by the amount of solid lipid required to solubilize the drug in molten state. 2.2.2. Screening of liquid lipids (oils) The saturation solubility of OLM in different oils was determined by taking an excess amount of drug in oil (1 ml) while keeping at 120 rpm [27] and 40°C for 24 h in water shaker bath (EQUITRON®, Medica Instrument Mfg. Co., India). Further, it was centrifuged at 10,000 rpm for 10 min (SIGMA 3-30K, SIGMA Laborzentrifugen GmbH, Germany) and supernatant was separated, filtered (0.45 µm PVDF syringe filter, Millipore Millex-HV), diluted with methanol. Further, it was analyzed with reverse phase high pressure liquid chromatogarphy (RP-HPLC) consisting of Shimadzu high pressure chromatography with CBM-20A pump, SIL-20AC Auto sampler, RF-10AXL PDA detector (Shimadzu corp., Japan). A C18 LiChrospher® RP-18e (250 mm×4.6 mm, 5 µm) (Agilent Technologies, USA) analytical column was used for analysis. The optimized mobile phase was composed of acetonitrile and 10 mM Phosphate Buffer (pH 2.8)

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(45:55 %v/v), with 1.2 ml/min flow rate. Samples of 10 µl were analyzed for estimation of OLM at 250 nm (ʎmax) using PDA detector. 2.2.3. Screening of a binary mixture of solid and liquid lipid

The solid and liquid lipid with highest solubility of OLM were mixed in different ratio (i.e. 100:0, 95:5, 90:10, 85:15, 80:20, 70:30, 60:40) in order to establish the miscibility between them. Lipid mixtures were kept at 150 rpm and 85°C for 1 h using water shaker bath and then stored at room temperature for 24 h for further investigation [28]. Differential scanning calorimetry (DSC 821e, Mettler-Toldeo International Inc., Switzerland) was used to confirm the solid state of the cooled sample and the miscibility of the solid lipids with liquid lipids. DSC data was generated by heating samples (3-5 mg) in a crimped aluminum pan between 25° to 85°C with cooling rate of 10°C/min. An empty aluminum pan without a pinhole was used as the reference sample. The samples were continuously flushed with nitrogen at a flow rate of 50 ml/min. 2.2.4. Screening of surfactant For selection of appropriate surfactant, NLCs were prepared by high shear homogenizer (POLYTRON® System PT 4000, KINEMATICA AG, Switzerland) with previously selected lipids for screening of surfactants. The surfactant concentration (1% w/v) was kept constant and further, selected surfactant was optimized for suitable concentration. Particle size, polydispersity index (PDI), zeta potential and % encapsulation efficiency (EE) were the selection criterion to optimize the surfactant and its concentration. 2.2.5. Optimization of homogenization speed Optimization of homogenization speed was performed by preparing the NLCs at different homogenization speed (from 100000-15000 rpm). All the results were evaluated on the basis of particle size, PDI and % EE. 2.2.6. Preparation of OLM-loaded NLCs For the preparation of OLM-NLC, the lipid and aqueous phases were prepared separately. The lipid phase consisted of oils, solid lipid and drug, while the aqueous phase consisted of aqueous solution of selected surfactant. The two phases were heated separately to 75°C for 10 min. The aqueous phase was added slowly to the oil phase and mixed using a high-shear homogenizer for 10 min at optimized speed.

6

2.2.7. Experimental design Box-Behnken design (BBD) is a type of response surface design that does not contain an embedded factorial or fractional factorial design and it has treatment combinations that are at the midpoints of the edges of the experimental space and require at least three continuous factors [29]. These designs allow efficient estimation of the first- and second-order coefficients because BBD often have fewer design points [30]; they are less expensive than central composite designs (CCD) with the similar number of trials. The experimental design and statistical analysis were performed using the Design Expert® software 9.0.7.1 (State-Ease Inc., USA). In this study, BoxBehnken experimental design (four factors and three levels) was applied for optimization to assess the relationship between the independent variables like amount of liquid lipids (A), amount of total lipids (B), drug concentration (C), concentration of surfactant (D), and dependent (responses) variables, i.e., particle size (Y1), polydispersity index

(Y2), and entrapment

efficiency (Y3). To execute this design, total 30 formulations were prepared and evaluated (Table 1). 2.3. Lyophilization Developed formulations were freeze-dried using Vir Tis Lyophilizer (Wizard 2.0, USA) by previously developed stepwise freeze-drying cycle with slight modification [31, 32]. Freezedrying cycle comprised of freezing at -60°C for 8 h, primary drying at −60°C to 20°C for 42 h, and secondary drying at 25°C for 2 h. At each step, constant pressure of 200 mTorr was applied. Initial screening of different cryoprotectants i.e. dextrose, trehalose, mannitol, and inulin was performed at 5% w/v concentration. Further, the selected surfactant was optimized for varying concentration of 2.5−10% w/v. Freeze-dried formulation was examined for appearance of the cake, redispersibility index, and redispersibility time. 2.4. Characterization of OLM-NLCs 2.4.1. Particle size and polydispersity index (PDI) The particle size and PDI (polydispersity index) of the OLM-NLC were determined by dynamic light scattering using Zetasizer (Nano ZS, Malvern Instruments Ltd., UK) at a fixed angle of 90° and at room temperature. OLM-NLC samples were appropriately diluted with HPLC grade water to a suitable concentration before analysis. The particle size and PDI were measured by taking average of five measurements.

7

2.4.2. Scanning electron microscopy (SEM) The surface morphology of OLM-NLC was visualized by scanning electron microscopy (Hitachi S-3400N, Hitachi High-Tech Co., Japan) at an acceleration voltage of 10 kV. Before observation, the lyophilized nanoparticles were fixed on a bioadhesive carbon tape which had previously been secured on aluminum stubs and sputter coated (E-1010, Hitachi High Tech Co., Japan) with gold-palladium alloy to minimize the surface charging in an inert atmosphere. 2.4.3. Transmission electron microscopy (TEM) TEM analysis was also used to determine the nanoparticle morphology. Briefly, aqueous dispersion of nanoparticles was diluted (×100) with HPLC grade water, placed on formvarcoated copper grids, further negatively stained with 1% w/v Uranyl acetate for 10 min, and allowed to dry. Excess liquid was drained off, and the grid containing the nanoparticle sample as a dry film was observed with a transmission electron microscope (FEI Tecnai G2F20, Netherlands) at an accelerating voltage of 200 kV. 2.4.4. Differential scanning calorimetry (DSC) Thermal characteristics of pure OLM, Placebo-lipid matrix (Precirol ATO-5: Capmul MCM EP; 85:15), physical mixture, surfactant, cryoprotectant and OLM-NLCs were studied using DSC (DSC 821e, Mettler Toledo International Inc., Switzerland). The samples were accurately weighed (5-7 mg), hermetically sealed in aluminum pans and heated at a fixed rate of 10°C/min over a defined temperature range (25-200°C). Sealed empty aluminum pan was used as a reference. An inert atmosphere was maintained by purging with nitrogen gas at a flow rate of 50 ml/min [33]. Indium and zinc standards were used to calibrate the temperature and enthalpy scale, respectively. 2.4.5. Powder X-ray diffraction analysis (PXRD) The PXRD patterns of pure OLM, Placebo-lipid matrix (Precirol ATO-5: Capmul MCM EP; 85:15 ratio), physical mixture, surfactant, cryoprotectant and NLCs were recorded on X-ray diffractometer (D8 Advanced Diffractometer, Bruker AXS GmbH, Germany). The X-ray source was Cu Kα tube (wavelength 1.5406 Å) operated at 40 kV and 40 mA. The samples were scanned from 4° to 40° (2θ at a scan rate of 0.1° (2θ) /min). The obtained diffractograms were studied with DIFFRAC plus EVA (ver.9.0) diffraction software.

8

2.5. Stability in simulated gastro-intestinal fluids The NLC dispersion was incubated in simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 6.8). SGF was prepared by dissolving 100 mg of pepsin in 5 mL of water containing 0.35 ml concentrated HCl followed by addition of sodium chloride (100 mg) and volume adjustment (up to 50 ml) with water. Eventually, pH of the solution was modified to 1.2 by adding concentrated HCl. SIF was prepared by dissolving 340 mg of monobasic potassium phosphate in 10 ml of water followed by addition of 3.85 mL of 0.2M NaOH and 500 mg of pancreatin was added. Final volume was made up to 50 mL, and pH was adjusted to 6.8 by NaOH. To simulate the effect of bile salts, 3 mM sodium taurocholate was added additionally to the SIF. Each ml of formulation was added to 9 ml of simulated media followed by its incubation for 2 and 6 h in SGF and SIF, respectively. The overall effect was evaluated by measuring the effect on size, PDI, and entrapment efficiency. 2.6. In vitro drug release In vitro drug release of OLM from OLM-NLC was studied using the dialysis membrane method. Dialysis membranes (MWCO ~12 kD and 1kD, Sigma Aldrich Inc., USA) were initially activated as per the manufacturer protocol. Phosphate buffer saline (PBS) (pH 6.8) was taken as the release medium. Lyophilized powder of OLM-NLC equivalent to 2 mg of OLM was poured into dialysis bags. The dialysis bags were suspended in 15 ml of PBS at 37.0±0.5°C in a water shaker bath at 50 rpm. The samples (500 μl) were withdrawn at predetermined intervals up to 24 h and replaced with an equal quantity of fresh medium to maintain the sink conditions [34, 35]. Samples were analyzed using pre-developed RP-HPLC method. 2.6. Caco-2 Cell culture experiments Caco-2 cells were grown in tissue culture flasks (25 cm2) and maintained under 5% CO2 atmosphere at 37°C. The growth medium comprised of Dulbecco’s Modified Eagle’s culture medium (DMEM), 20% fetal bovine serum (FBS), 100U/ml penicillin and 100Ug/ml streptomycin (PAA, Austria). The growth medium was changed on every alternate day. The cultured cells were trypsinized with 0.25% trypsin-EDTA solution (Sigma, USA) once 90% confluency was attained. The cells were then seeded in appropriate cell culture plates for further studies at a density of 5×104 cells/well for analysis of both qualitative and quantitative cell uptake using confocal laser scanning microscopy (CLSM) (Olympus FV1000, Olympus Imaging America Inc., USA) and HPLC respectively. 9

2.6.1. Qualitative uptake Coumarin 6 (C-6) was used as a model dye to analyze the qualitative uptake of drug in Caco-2 cell lines. C6-NLCs were prepared in the similar manner as OLM-NLCs were formulated; the only difference was addition of C6 instead of OLM in the procedure discussed in the Section 2.2.6. Caco-2 cells were seeded at a density of 3×105 cells/well in 6 well plates (Costars, Corning Inc., NY, USA) and incubated overnight for cell attachment. The cells were then exposed to C6-NLC (equivalent to 1 μg/ml of C-6) for 2 h. Subsequently, the medium was removed and cells were washed 2-3 times with the PBS (pH 7.4). The cells were fixed with glutaraldehyde (2.5 % v/v) (Sigma, USA), washed and observed under the CLSM.

2.6.2. Quantitative uptake Caco-2 cells were seeded at a density of 1×105 cells/ well in 24 well cell culture plates (Costars, Corning Inc., NY, USA) and allowed to attach overnight. The cell culture medium was replaced with fresh medium containing varying concentration of free OLM, and OLM-NLC which were further incubated for 2h to evaluate the concentration dependent effect on cell uptake. After completion of incubation period, the medium was removed and cells were washed twice with PBS (pH 7.4). Similarly, time dependent cell uptake studies were also performed by incubating the Caco-2 cells with appropriate concentration of different formulations for varying time intervals (0.5, 1, 1.5, 2 and 4 h). Further cells were lysed with the 0.1% Triton X-100 followed by extraction with methanol to entirely solubilize the internalized OLM. The cell lysate was centrifuged at 20,000 rpm for 10 min and obtained supernatant was subjected to HPLC analysis for quantification of internalized drugs. 2.7. In vivo pharmacokinetics Male Sprague Dawley rats weighing 200−250 g were used for the study. All the animal study protocols were duly approved by the Institutional Animal Ethics Committee (IAEC), National Institute of Pharmaceutical Education and Research (NIPER), S.A.S. Nagar, Punjab, India. Throughout the experiments, the animals were housed in laminar flow at a temperature of 25±2°C and relative humidity of 50−60% under 12 h light/dark cycles Animals were randomly distributed into two groups each containing five animals. Free OLM and OLM-NLC were administered to the overnight fasted animals by oral gavage at an equivalent drug dose of 10 mg/kg body weight. The blood samples were collected from the tail vein under mild anesthesia 10

into heparinized microcentrifuge tubes. Plasma was separated by centrifuging the blood samples at 15,000 rcf for 10 min at 4°C. Methanol (250 μl) was added to plasma (125 μl) to precipitate the plasma proteins, followed by addition of candesartan (Internal standard; 50 μl of 10 μg/ml). The samples were further vortexed and centrifuged at 15000 rcf for 10 min. The supernatant was collected, filtered (0.45 µm PVDF syringe filter, Millipore Millex-HV), diluted with mobile phase and analyzed with RP-HPLC. A C18 LiChrospher® RP-18e (250 mm×4.6 mm, 5 µm) (Agilent Technologies, USA) analytical column was used for analysis. The mobile phase consisted acetonitrile and 10 mM Phosphate Buffer (pH 2.6) (90:10 %v/v), was run throughout column at 1.2 ml/min flow rate. Samples of 80 µl were analyzed for estimation of OLM at 254 nm (ʎmax) using PDA detector. The pharmacokinetic parameters of plasma concentration time data were analyzed by onecompartmental model using Kinetica software version 5.0 (Thermo Fisher Scientific Inc., USA). Pharmacokinetics parameters like total area under the curve (AUC0−∞), half life (t1/2) and peak plasma concentration (Cmax) were determined. The relative bioavailability of formulations after oral administration was calculated as follows (equation 1): Relative bioavailability =

AUC(NLC)

×100

AUC(drug solution)

(1)

2.8. Statistical analysis All the results are exhibited here, as mean ± standard deviation (SD). Data was analyzed using Student’s t-test or one-way ANOVA (GraphPad InStat Software Demo, USA); p-values <0.0001 was considered as statistically extremely significant. 3. Results 3.1. Preparation of OLM-NLC Since solubility of drug directly affects the entrapment efficiency, it always remains the leading factor in preparation of nanostructured lipid carriers. Screening of solid lipids was evaluated based on solubility of OLM in different solid lipids (Fig.S1 A). OLM exhibited highest solubility in Gelucire® 44/14 and Precirol® ATO 5 among all lipids. In addition to solid lipid, selection of suitable liquid lipid was equally important in NLC formulation. Therefore, solubility study of OLM in liquid lipid was studied to screen liquid lipids and it was found maximum in Capmul® MCM EP (Fig.S1 B). Different surfactants were screened at constant concentrations on the basis of particle size, PDI and %EE (Table S1). Amongst, all surfactant, PEG-40 showed highest particle size 11

(179.6±3.4 nm) and PI (0.826±2.7); indicating broad and heterogeneous particle size distribution. On the other hand, TPGS showed effective particle size reduction of nanoformulation with size of 151.2±5.10 nm and PDI of 0.419±1.1. Further, it was found that as the concentration of TPGS increases beyond 1%, size and %EE increases with heterogeneous particle size distribution (Table S2). On the basis of these results, the surfactant concentration was further optimized to get the optimum particle size, PDI and % EE. Pure Precirol ATO-5 showed endothermic peak at 66.16°C in DSC analysis (Fig.1). To study the thermal behavior of solid lipid after addition of liquid lipid, a placebo mixture of solid lipid and liquid lipid was prepared in the same ratio as it was used in the NLC formulation (Precirol ATO 5: Capmul MCM EP; 85:15 ratio) and subjected to DSC study. Fig.1. revealed approximately 12°C depressions in melting of Precirol ATO 5 after addition of Capmul MCM EP in 85:15 ratios as compared to pure Precirol ATO 5. Table S3 enlists the calculated DSC parameters. As we increased the liquid lipid content upto 15% (nearly showed linear trend) in the total lipid matrix, decreasing pattern of onset temperature and melting point was observed which indicated the good miscibility between two lipid phases. Loss of crystallinity was clearly visible from the decreased enthalpy (from 127.13 to 84.88 J/g), and increased width of the melting event (from 704.68°C to 224.07°C). The onset temperature and melting point was not further decreased when the liquid lipid was increased from 20% to 30% in the total lipid content, which might be due to saturation of oil in total lipid matrix and thus initiation of miscibility gap emerges between oil and solid lipid [37, 38]. Moreover, adding more than 30% of oil to solid lipid showed no significant effect on the melting point and width of the melting event. Altogether, a binary mixture consisting of 15% (w/w) Capmul MCM EP and 85% (w/w) Precirol ATO5 was selected as the most suitable combination of liquid and solid lipid for the formulation of OLM-NLC to enhance solubility of OLM by providing good miscibility of lipid mixture. Variation in homogenization speed significantly affected the particle size of nanoparticles. So, batches of nanoparticles were prepared at different homogenization speed for 10 min and result was analyzed based on particle size, PDI and % EE (Table S4).

3.2. Optimization of OLM-NLC The selected independent variables including the amount of liquid lipid, amount of total lipid, surfactant concentration, and drug content significantly affect the dependent variables including %EE, particle size, and polydispersity index (PDI) (Table 1). Polynomial equations 12

involving the main effect and interaction factors were determined based on estimation of statistical parameters such as multiple correlation coefficient, adjusted multiple correlation coefficient, and the predicted residual sum of squares. The statistical validation of the polynomial equations was established by ANOVA. Therefore, the optimum values of the variables were determined according to the obtained experimental data using the Design-Expert software, based on the constrained criterion of response variables (Table S5). Response surface analyses plotted in three-dimensional model graphs for depicting the effects of predetermined factors on the response variables (Fig.2 a-c), which is primarily based on the model polynomial functions. The response surface plots were also used to study the interactions of two independent variables on the responses or dependent variables, while keeping third factor at a constant level. When these plots were carefully observed, the qualitative effect of each variable on each response parameter could be visualized [39, 40]. The particle size varies from 323.1 nm (formulation 2) to 129.9 nm (formulation 26) for various factor level combinations (Table 1). The most significant factor contributing to the variation in particle size was drug content (C), as evident from the value of the coefficient. The factor (C) showed the positive effect on the particle size, which means an increase in the value of drug content results an increase in the value of particle size. In Fig.2 (a), the effect of varying amount of drug content (C) and surfactant concentration (D) on the particle size was studied when the liquid lipid amount and amount of total lipid were kept constant. The liquid lipid and amount of total lipid content have also a positive effect on particle size as it was statistically validated too (see supplementary data). PDI was varied from 0.25 (formulation 3) to 0.292 (formulation 24) for various factor levels combinations shown in Table 1. The independent factor affecting PDI was surfactant concentration (D). However, surfactant concentration and drug content were also affected the response in combination. In Fig.2 (b), the effect of varying the drug content and surfactant concentration on the PDI was studied when the amount of oil and total lipid were kept constant. Entrapment efficiency was changed from 15.13% (formulation 18) to 56.84% (formulation 6) for various factors level combinations, shown in Table 1. In Fig.2 (c), the effect of varying amount of drug content and emulsifier on the entrapment efficiency was studied when the total lipid and amount of liquid lipid were kept constant. The drug content had a significant and negative effect on %EE as it was shown by the negative value in the quadratic equation (see supplementary content). The result showed that the entrapment efficiency was decreased as the 13

amount of drug content was increased. Increasing the amount of drug content was found to decrease the %EE. The surfactant level (D) also has a significant and positive effect on the entrapment efficiency. In Fig.2 (c), the entrapment efficacy was significantly increased by increasing the amount of emulsifier and the amount of liquid lipid. The desirability function was probed using Design-Expert software to get the optimized formulation. The optimum formulation was builded on the defined criteria of maximum entrapment efficiency, particle size (120 nm-323 nm) and PDI (0.25-0.29). Therefore, three new batches of NLC were prepared (see Table S6 for details) from the design space as depicted in overlay plot (Fig. 2(d)) with the predicted levels to confirm the validity of the optimization procedure. It was observed that the experimental values were in the range of the predicted values within the 5% of prediction error. Therefore, the Box–Behnken design for optimization of OLMNLC was validated.

3.3. Characterization of nanoparticles Different formulations were freeze dried to provide them long-term stability (Table S7). On the basis of redispersibility index with different cryoprotectants, mannitol was selected as it was resulted in the formation of voluminous, easy to redisperse cake with redispersibility index close to one. However, other cryoprotectants resulted in either collapsed or hard to redisperse cake (Fig. S2 (II)) which upon reconstitution resulted in aggregation, as was evident by a high redispersibility index. Therefore, mannitol was selected, which was further optimized for suitable concentration (Table S8). Finally, 5% w/v of mannitol was selected as a cryoprotectant which showed redispersibility index close to 1. The SEM results indicated that the large and irregular shaped morphology of pure drug (Fig. 3(I) A) was found; whereas OLM-NLCs executed round and homogeneous with smooth surface (Fig. 3(I) B). After reconstitution, rounded shape morphology of NLCs (Fig. 3(I) C) was seen, which indicates the loss of crystallinity of OLM. Spherical morphologies were also shown in the TEM images of OLM-NLC (Fig.3 (II)). The DSC thermogram showed a melting peak of crystalline OLM at around 184°C (Fig. 4 (I)). For Placebo mixture and mannitol, the melting process took place with maximum peak at 57°C and 163°C, respectively. These individual melting peaks were present and had almost the same value in the thermogram of the physical mixture. However, the melting peak for the OLM was nearly absent in the lyophilized OLM-NLC. The absence of the crystalline peak of drug in 14

OLM–NLC suggested that OLM exists in amorphous or molecularly disperse form in lipid matrix. PXRD was used to study the diffraction pattern of OLM in free form as well as in NLCs. PXRD analysis made it possible to assess the length of the long and short spacing of the lipid lattice. Several characteristic diffraction peaks of pure OLM could easily be detected at 2θ scattered angle values of 7.3°, 9.2°, 14.6°, 19.7°, 20.6°, 21.9°, 23.4°, 25.3°, 27.6° (Fig. 4 II) and these all peaks were also appeared in the physical mixture but with lesser extent. However, characteristic peaks of OLM were disappeared in OLM-NLCs, which confirmed the absence of crystallinity of drug.

3.4. Stability in GI fluids The effect of simulated gastrointestinal fluids and intestinal fluid on the stability of OLMNLC formulations is depicted in Table S9. Results of NLC stability study revealed high stability of NLCs with insignificant (P > 0.05) change in size, PDI and %EE.

3.5. In vitro cell culture experiments In qualitative uptake study, CLSM images (Fig. 5 (I)) of Caco-2 cells which were incubated with C-6-NLCs and free C-6 (1µg/ml, 2h) show significantly higher fluorescence with C-6 loaded NLCs as compared to free C-6, which is suggestive of efficient internalization of NLCs by Caco-2 cells. Horizontal line analysis showed that green and white line overlapping in case of C-6-NLC while in case of free C-6, no overlapping was seen which indicated the co-localization of C-6-NLC within the cells. The uptake of OLM-NLC shows progressive increase with increase in the incubation time until 1 h (Fig 5 II), after which saturation was reached and process of efflux took over, resulting in the reduction of OLM concentration in the cells. An insignificant (P>0.05) change in the amount of OLM uptake was observed upon increasing the incubation time of OLM-NLC beyond 1 h. At highest tested concentration (20 μg/ml), the uptake of OLM-NLC was 5.2 fold higher as compared to free OLM (Fig 5 II).

3.6. In vitro release study Fig. 6 (A) shows the in vitro release profile of OLM formulations in simulated GI fluids. An insignificant difference (p>0.05) in the cumulative release of free OLM from both dialysis 15

membranes (MWCO ~12 kD and 1kD, Sigma Aldrich Inc., USA) was observed at all the tested time points, while the same showed a significant difference (p<0.001) in case of OLM-NLC; 48.64±7.29 % and 11.97±1.07 % of OLM was released from OLM-NLC after 8 h through membrane of 12kD and 1kD MWCO respectively.

3.7. In vivo pharmacokinetics The plasma concentration–time profiles after a single oral administration of OLM-NLC and free OLM suspension are shown in Fig. 6 (B). Table 2 presents the mean pharmacokinetic parameters calculated using analysis of experimental data with suitable database. It was shown that, OLM-NLC showed 4.84 fold higher AUCtotal as compare to free drug (80.61±24.58 vs. 16.64±6.70 µg/ml*h, p<0.01), while Cmax of OLM-NLC was 5.01 fold higher as compare to free drug (19.51±6.68 vs. 3.89±0.91 µg/ml, p<0.01). These findings suggest the augmentation of oral bioavailability of drug when it was incorporated in nanostructured lipid carriers. 4. Discussion Various solid lipids, oils and surfactants were screened to formulate the optimized OLMNLC. Preliminary solubility studies screened Gelucire 44/14 and Capmul MCM EP as solid and liquid lipid, as both exhibited highest solubility for OLM. However, Precirol® ATO 5 was selected as the final solid lipid, because the addition of Gelucire® 44/14 to liquid lipid lowers the melting point which was not suitable for oral delivery of NLCs [28]. It is well known fact that high solubility of drug in the lipid melt leads to higher drug loading. Generally, it was seen that as the lipid melt cools earlier, solubility of drug within lipid decreases. Therefore, chemical nature of lipid is more important in solubilization of drug. Lipids, which are mixture of mono-diand triglycerides and lipid containing fatty acid of different chain length provide more imperfection, offering space for higher drug loading [41]. Thus, higher solubility of OLM in Precirol ATO5 was observed which could be due to its chemical nature (mixture of mono-di- and triglycerides) and presence of higher relative amount of monoglycerides among all the solid lipids. From the prior art also, it was shown that more the monoglyceride content in the lipid, higher the polarity of lipid [42, 43]. In addition, monoglycerides possess surfactant properties [44] which may also contribute in dissolving the drug. Muller et al. have reported that the addition of oil or liquid lipid to solid lipid decreases crystallinity and thereby reduces leakage of entrapped drug [22]. Higher solubility of drug in

16

Capmul MCM EP could be attributed to its inherent self emulsifying property and its chemical nature. Further, it is a prerequisite for the development of NLCs that the solid and liquid lipid should be miscible at the specific concentrations used [22]. The use of DSC to assess the miscibility of Precirol ATO 5 and Capmul MCM EP was based on the fact that a depression in the melting point of Precirol ATO5 might be observed following the incorporation of Capmul MCM EP in the lamellar structure of the solid lipid [37, 38]. Simply, this depression in melting point and broadening of peak can be attributed to the dissolution of Capmul MCM EP in Precirol ATO5 with the formation of less distinct crystalline structure [36]. With DSC study, binary mixtures were screened on the basis of melting point (should above 40◦C). The depression in the onset, peak maximum, enthalpy, and the increase in the WME of Precirol ATO 5 was shown up to 15% (w/w) of Capmul MCM EP added to the solid lipid, which confirms the miscibility of two components in the selected concentration range of liquid lipid. Screening of a suitable surfactant for the formulation of NLC is critical for the development of a stable formulation, as it adheres on the oil–water interface, it lowers the interfacial energy and configures a mechanical barrier against the coalescence of oily droplets [45]. Since, there are always GI stability related issues of nanoparticles, we used surfactant that contains PEG group, as PEG layer have protein rejection property and thus improving the stability of nanoparticles in the GI tract by protecting them from enzyme attack [46]. OLM-NLCs were formulated by using high shear homogenization and optimized by applying Box-Behnken experimental design. Optimized formulation showed particle size < 158 nm, PDI <0.287 and entrapment efficiency 48.44%. Freeze drying also plays a crucial role in formulation of nanoparticles, as it improves the stability and thus shelf life of nanostructured lipid carriers [47]. Freeze dried product was appeared as fluffy cake with high redispersibility in water. The in vitro drug release profile of the developed OLM-NLC was evaluated in SGF (pH 1.2) for 2 h and in SIF (pH 6.8) for 8 h using dialysis membranes of different MWCO (1KD and 12 kD). During drug release from the NLCs, there was possibility of formation of various release components, i.e., molecular form of the free drug, drug-loaded mixed in the micelles, and other secondary structures of average size <100 nm . From the dialysis bag of MWCO 12 kD, drug loaded secondary components (size <100 nm) are easily passed. Under these unusual conditions, it was necessary to separate the molecular form of the drug from those secondary forms to assess the actual drug release pattern. A low molecular weight cutoff dialysis membrane (1 kD) was 17

used to assess the real release pattern, as it allows only molecular forms of drug not the secondary structures [48]. It also indicated the NLCs ability to maintain the solubilization of OLM in both physiological fluids (SGF and SIF), which could be due to PEG coating over the surface of nanoparticles [49, 50]. Further, Caco-2 cells were employed to study the uptake pattern of the developed OLMNLC formulations. Caco-2 cells are well established in vitro model that mimics intestinal absorptive epithelium and is always helpful for studying uptake and transport of drug or nanoformulations across the transepithelial barrier [51]. From CLSM studies, higher uptake was seen with OLM-NLC than free OLM, when incubated with Caco-2 cells for 1h (Fig. 5(I)). These findings were further supported by the quantitative uptake analysis (Fig. 5(II)), which revealed 5.2 folds appreciation in the uptake of OLM when incorporated in NLC compared to free drug. In vivo pharmacokinetic study showed the higher value of AUCtotal and Cmax of OLM-NLC as compared to free drug. These findings indicated the higher absorption of OLM when incorporated into NLCs. This could be due to more efficient uptake of nanoparticles through GI tract, increased GI permeability by TPGS [52] and higher dissolution [53]. Moreover, it might also be due to more adhesion to GI wall and avoidance of first pass metabolism [54]. Additionally, higher bioavailability of NLC might also be due to more lymphatic transport of hydrophobic triglycerides used in preparation of OLM-NLC [55].

5. Conclusion The present study illustrated the systemic QbD approach for development of optimized nanostructured lipid carrier formulation of Olmesartan medoxomil, for enhanced oral bioavailability and improved GI stability. OLM-NLC was developed utilizing excipients listed as generally regarded as safe in a quantity well below their Inactive Ingredients Guide limits. Successfully developed and optimized OLM-NLC exhibited nano size, homogenous size distribution, high entrapment, extended release with significant improvement in oral bioavailability. The cell line studies revealed higher uptake of OLM-NLC than free drug. In conclusion, the NLC formulation remarkably improved the oral bioavailability of OLM and demonstrated a promising perspective for oral delivery of poorly water-soluble drugs.

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Acknowledgments The authors are thankful to Cadila Pharmaceuticals Ltd. (Ahmedabad, Gujarat) for providing gift samples of Olmesartan medoxomil. Mr. Chander Parkash and Mr. Varun Kushwah deeply acknowledge University Grants Commission, New Delhi (UGC) and Council of Scientific & Industrial Research, New Delhi (CSIR), respectively for providing financial grants as Senior Research Fellows. Authors are thankful to Director, NIPER, S.A.S. Nagar for providing the necessary infrastructure facilities. The help and co-operation provided by Mr. Rahul Mahajan and Mr. Vinod for SEM and TEM analysis respectively, is duly acknowledged.

References [1] M. Destro, P. Preti, A. D'Ospina, N.N. Christian Achiri, A.R. Ricci, F. Cagnoni, Olmesartan medoxomil: recent clinical and experimental acquisitions, Expert Opin. Drug Metab. Toxicol., 5 (2009) 1149-1157. [2] H. Brunner, The new oral angiotensin II antagonist olmesartan medoxomil: a concise overview, J. Hum. Hypertens., 16 (2002) S13-6. [3] L.R. Schwocho, H.N. Masonson, Pharmacokinetics of CS‐866, a New Angiotensin II Receptor Blocker, in Healthy Subjects, J. Clin. Pharmacol., 41 (2001) 515-527. [4] H. Théophile, X.-R. David, G. Miremont-Salamé, F. Haramburu, Five cases of sprue-like enteropathy in patients treated by olmesartan, Dig. Liver Dis., 46 (2014) 465-469. [5] S.E. Dreifuss, Y. Tomizawa, N.J. Farber, J.M. Davison, A.E. Sohnen, Spruelike enteropathy associated with olmesartan: an unusual case of severe diarrhea, Case Rep. Gastrointest. Med., 2013 (2013) 618071. [6] S. Beg, G. Sharma, K. Thanki, S. Jain, O. Katare, B. Singh, Positively charged selfnanoemulsifying oily formulations of olmesartan medoxomil: systematic development, in vitro, ex vivo and in vivo evaluation, Int. J. Phar., 493 (2015) 466-482. [7] M.J. Kang, H.S. Kim, H.S. Jeon, J.H. Park, B.S. Lee, B.K. Ahn, K.Y. Moon, Y.W. Choi, In situ intestinal permeability and in vivo absorption characteristics of olmesartan medoxomil in self-microemulsifying drug delivery system, Drug Dev. Ind. Pharm., 38 (2012) 587-596. [8] B. Gorain, H. Choudhury, A. Kundu, L. Sarkar, S. Karmakar, P. Jaisankar, T.K. Pal, Nanoemulsion strategy for olmesartan medoxomil improves oral absorption and extended antihypertensive activity in hypertensive rats, Colloids Surf. B Biointerfaces, 115 (2014) 286294.

19

[9] H.P. Thakkar, B.V. Patel, S.P. Thakkar, Development and characterization of nanosuspensions of olmesartan medoxomil for bioavailability enhancement, J. Pharm. Bioallied Sci., 3 (2011) 426-434. [10] P. Sruthy, K. Anoop, Formulation and evaluation of olmesartan medoxomil floating tablets, Int. J. Pharm. Pharm. Sci., 5 (2013) 691-696. [11] S.T. Prajapati, H.H. Bulchandani, D.M. Patel, S.K. Dumaniya, C.N. Patel, Formulation and evaluation of liquisolid compacts for olmesartan medoxomil, J. Drug Deliv., 2013 (2013) 870579. [12] M. Anwer, S. Jamil, M. Ansari, M. Iqbal, F. Imam, F. Shakeel, Development and evaluation of olmesartan medoxomil loaded PLGA nanoparticles, Mater. Res. Innovat., 20 (2016) 193-197. [13] T. El-nawawy, A.M. Swailem, D. Ghorab, S. Nour, Solubility enhancement of olmesartan by utilization of solid dispersion and complexation techniques, Int. J. Nov. Drug Deliv., 2 (2012) 297-303. [14] V. Jannin, S. Chevrier, M. Michenaud, C. Dumont, S. Belotti, Y. Chavant, F. Demarne, Development of self emulsifying lipid formulations of BCS class II drugs with low to medium lipophilicity, Int. J. Pharm., 495 (2015) 385-392. [15] B. Krishnamoorthy, S.H. Rahman, M. Rajkumar, K. Vamshikrishna, M. Gregory, C. Vijayaraghavan, Design, formulation, in vitro, in vivo, and pharmacokinetic evaluation of nisoldipine-loaded self-nanoemulsifying drug delivery system, J. Nanopart. Res., 17 (2015) 1-11. [16] V. Makwana, R. Jain, K. Patel, M. Nivsarkar, A. Joshi, Solid lipid nanoparticles (SLN) of Efavirenz as lymph targeting drug delivery system: elucidation of mechanism of uptake using chylomicron flow blocking approach, Int. J. Phar., 495 (2015) 439-446. [17] P. Sassene, M. Michaelsen, M. Mosgaard, M. Jensen, E. Van Den Broek, K. Wasan, H. Mu, T. Rades, A. Müllertz, In Vivo Precipitation of Poorly Soluble Drugs from Lipid-Based Drug Delivery Systems, Mol. Pharm., 13 (2016) 3417-3426. [18] R. Sistla, N.R. Shastri, Modulating drug release profiles by lipid semi solid matrix formulations for BCS class II drug–an in vitro and an in vivo study, Drug Del., 22 (2015) 418426. [19] Ü. Gönüllü, M. Üner, G. Yener, E.F. Karaman, Z. Aydoğmuş, Formulation and characterization of solid lipid nanoparticles, nanostructured lipid carriers and nanoemulsion of lornoxicam for transdermal delivery, Acta Pharm., 65 (2015) 1-13. [20] H. Ji, J. Tang, M. Li, J. Ren, N. Zheng, L. Wu, Curcumin-loaded solid lipid nanoparticles with Brij78 and TPGS improved in vivo oral bioavailability and in situ intestinal absorption of curcumin, Drug Del., 23 (2016) 459-470. [21] H. Harde, M. Das, S. Jain, Solid lipid nanoparticles: an oral bioavailability enhancer vehicle, Expert Opin. Drug Del., 8 (2011) 1407-1424. 20

[22] R.H. Müller, M. Radtke, S. Wissing, Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) in cosmetic and dermatological preparations, Adv. Drug Del. Rev., 54 (2002) S131-S155. [23] R.H. Müller, U. Alexiev, P. Sinambela, C.M. Keck, Nanostructured Lipid Carriers (NLC): The Second Generation of Solid Lipid Nanoparticles, Percutaneous Penetration Enhancers Chemical Methods in Penetration Enhancement, Springer2016, pp. 161-185. [24] M. Muchow, P. Maincent, R.H. Müller, Lipid nanoparticles with a solid matrix (SLN®, NLC®, LDC®) for oral drug delivery, Drug Dev. Ind. Pharm., 34 (2008) 1394-1405. [25] D. Cun, D.K. Jensen, M.J. Maltesen, M. Bunker, P. Whiteside, D. Scurr, C. Foged, H.M. Nielsen, High loading efficiency and sustained release of siRNA encapsulated in PLGA nanoparticles: quality by design optimization and characterization, Eur. J. Pharm Biopharm., 77 (2011) 26-35. [26] M. Ferreira, L.L. Chaves, S.A.C. Lima, S. Reis, Optimization of nanostructured lipid carriers loaded with methotrexate: A tool for inflammatory and cancer therapy, Int. J. Pharm., 492 (2015) 65-72. [27] K. Liu, J. Sun, Y. Wang, Y. He, K. Gao, Z. He, Preparation and characterization of 10hydroxycamptothecin loaded nanostructured lipid carriers, Drug Dev. Ind. Pharm., 34 (2008) 465-471. [28] K.W. Kasongo, J. Pardeike, R.H. Müller, R.B. Walker, Selection and characterization of suitable lipid excipients for use in the manufacture of didanosine‐loaded solid lipid nanoparticles and nanostructured lipid carriers, J. Pharm. Sci., 100 (2011) 5185-5196. [29] B. Singh, R. Kumar, N. Ahuja, Optimizing drug delivery systems using systematic" design of experiments." Part I: fundamental aspects, Crit. Rev. Ther. Drug Carrier Syst., 22 (2005) 27105. [30] D. Maurya, Y. Sultana, M. Aqil, D. Kumar, K. Chuttani, A. Ali, A. Mishra, Formulation and optimization of alkaline extracted ispaghula husk microparticles of isoniazid-in vitro and in vivo assessment, J. Microencapsul., 28 (2011) 472-482. [31] A.K. Jain, N.K. Swarnakar, C. Godugu, R.P. Singh, S. Jain, The effect of the oral administration of polymeric nanoparticles on the efficacy and toxicity of tamoxifen, Biomaterials., 32 (2011) 503-515. [32] S. Jain, V.V. Rathi, A.K. Jain, M. Das, C. Godugu, Folate-decorated PLGA nanoparticles as a rationally designed vehicle for the oral delivery of insulin, Nanomedicine, 7 (2012) 1311-1337. [33] A. Patil-Gadhe, V. Pokharkar, Montelukast-loaded nanostructured lipid carriers: Part I Oral bioavailability improvement, Eur. J. Pharm. Biopharm., 88 (2014) 160-168.

21

[34] M. Vertzoni, N. Fotaki, E. Nicolaides, C. Reppas, E. Kostewicz, E. Stippler, C. Leuner, J. Dressman, Dissolution media simulating the intralumenal composition of the small intestine: physiological issues and practical aspects, J. Pharm. Pharmacol., 56 (2004) 453-462. [35] K.K. Upadhyay, A.N. Bhatt, A.K. Mishra, B.S. Dwarakanath, S. Jain, C. Schatz, J.-F. Le Meins, A. Farooque, G. Chandraiah, A.K. Jain, The intracellular drug delivery and anti tumor activity of doxorubicin loaded poly (γ-benzyl l-glutamate)-b-hyaluronan polymersomes, Biomaterials, 31 (2010) 2882-2892. [36] K. Jain, S. Sood, K. Gowthamarajan, Optimization of artemether-loaded NLC for intranasal delivery using central composite design, Drug Del., 22 (2015) 940-954. [37] A. Kovacevic, S. Savic, G. Vuleta, R. Müller, C.M. Keck, Polyhydroxy surfactants for the formulation of lipid nanoparticles (SLN and NLC): effects on size, physical stability and particle matrix structure, Int. J. Pharm., 406 (2011) 163-172. [38] C. Zhang, F. Peng, W. Liu, J. Wan, C. Wan, H. Xu, C.W. Lam, X. Yang, Nanostructured lipid carriers as a novel oral delivery system for triptolide: induced changes in pharmacokinetics profile associated with reduced toxicity in male rats, Int. J. Nanomedicine, 9 (2014) 1049-1063. [39] R.F. Gunst, Response surface methodology: process and product optimization using designed experiments, Technometrics, 38 (1996) 284-286. [40] C.B. Woitiski, F. Veiga, A. Ribeiro, R. Neufeld, Design for optimization of nanoparticles integrating biomaterials for orally dosed insulin, Eur. J. Pharm. Biopharm., 73 (2009) 25-33. [41] R.H. Muller, K. Mader, S. Gohla, Solid lipid nanoparticles (SLN) for controlled drug delivery-a review of the state of the art, Eur. J. Pharm. Biopharm., 50 (2000) 161-177. [42] N. Garti, K. Sato, Crystallization and polymorphism of fats and fatty acids, M. Dekker1988. [43] D. Small, The physical chemistry of lipids, from alkanes to phospholipids. Handbook of Lipid Res. 4, Plenum Press, New York, 1986. [44] L.B. Jensen, E. Magnussson, L. Gunnarsson, C. Vermehren, H.M. Nielsen, K. Petersson, Corticosteroid solubility and lipid polarity control release from solid lipid nanoparticles, Int. J. Pharm., 390 (2010) 53-60. [45] T. Kommuru, B. Gurley, M. Khan, I. Reddy, Self-emulsifying drug delivery systems (SEDDS) of coenzyme Q 10: formulation development and bioavailability assessment, Int. J. Pharm., 212 (2001) 233-246. [46] E. Roger, F. Lagarce, J.-P. Benoit, The gastrointestinal stability of lipid nanocapsules, Int. J. Pharm., 379 (2009) 260-265. [47] H. Shete, V. Patravale, Long chain lipid based tamoxifen NLC. Part I: Preformulation studies, formulation development and physicochemical characterization, Int. J. Pharm., 454 (2013) 573-583. 22

[48] S. Jain, A.K. Jain, M. Pohekar, K. Thanki, Novel self-emulsifying formulation of quercetin for improved in vivo antioxidant potential: Implications for drug-induced cardiotoxicity and nephrotoxicity, Free Radic. Biol. Med., 65 (2013) 117-130. [49] M. Garcıa-Fuentes, D. Torres, M. Alonso, Design of lipid nanoparticles for the oral delivery of hydrophilic macromolecules, Colloids Surf. B Biointerfaces, 27 (2003) 159-168. [50] T. Verrecchia, G. Spenlehauer, D. Bazile, A. Murry-Brelier, Y. Archimbaud, M. Veillard, Non-stealth (poly (lactic acid/albumin)) and stealth (poly (lactic acid-polyethylene glycol)) nanoparticles as injectable drug carriers, J. Control. Rel., 36 (1995) 49-61. [51] A.K. Jain, N.K. Swarnakar, M. Das, C. Godugu, R.P. Singh, P.R. Rao, S. Jain, Augmented anticancer efficacy of doxorubicin-loaded polymeric nanoparticles after oral administration in a breast cancer induced animal model, Mol. Pharm., 8 (2011) 1140-1151. [52] L. Zhou, Y. Chen, Z. Zhang, J. He, M. Du, Q. Wu, Preparation of tripterine nanostructured lipid carriers and their absorption in rat intestine, Pharmazie, 67 (2012) 304-310. [53] T.H. Tran, T. Ramasamy, D.H. Truong, H.-G. Choi, C.S. Yong, J.O. Kim, Preparation and characterization of fenofibrate-loaded nanostructured lipid carriers for oral bioavailability enhancement, AAPS PharmSciTech, 15 (2014) 1509-1515. [54] V.V. Kumar, D. Chandrasekar, S. Ramakrishna, V. Kishan, Y.M. Rao, P.V. Diwan, Development and evaluation of nitrendipine loaded solid lipid nanoparticles: influence of wax and glyceride lipids on plasma pharmacokinetics, Int. J. Pharm., 335 (2007) 167-175. [55] E. Ros, Intestinal absorption of triglyceride and cholesterol. Dietary and pharmacological inhibition to reduce cardiovascular risk, Atherosclerosis, 151 (2000) 357-379.

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Figure captions:

Fig. 1 DSC thermogram bulk solid lipid with increment amount of liquid lipid: 0, 10, 15, 20 and 30 in thermogram A, B, C, D, and E respectively Fig. 2 Response surface plot ((a) particle size, (b) PDI, and (c) %EE) showing the effect of Independent variables on dependent variables (d) Overlay plot Fig. 3 (I) SEM images of (A) Pure drug (B) OLM-lyophilized powder (C) OLM-lyophilized powder after reconstitution, (II) TEM images of OLM-NLC at different magnifications (A) × 29000 (B) × 100000 Fig. 4 (I) DSC thermograms of (A) OLM, (B) TPGS, (C) Mannitol, (D) Physical mixture, (E) lipid mixture, (F) OLM-NLC, (II) X-ray diffraction pattern of (A) Mannitol, (B) OLM, (C) TPGS, (D) Physical mixture and (E) OLM-NLC Fig. 5 (I) Qualitative cellular uptake of (A) Free Coumarin-6 and (B) Coumarin-6 loaded nanoparticles upon incubation at 1 μg/ml for 2h. In all the images, Figure (a) Images well produced under the green fluorescence channel; Figure (b) Corresponding differential interface contrast images of Caco-2 cells (c) Superimposition of figure (a) and figure (b). Figure (d) and (e) in all the images show horizontal line series analysis of fluorescence along the white line (II) Quantitative Caco-2 cell uptake study (A) Time and (B) Concentration dependent Fig. 6 (A) In vitro release profile of OLM-NLC and pure drug suspension, (B) In vivo pharmacokinetics profile after a single oral administration of OLM-NLC, free OLM suspension and marketed formulation

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Figure 1

25

Figure 2

26

Figure 3

27

Figure 4

28

Figure 5

29

Figure 6

30

List of tables Table 1 Box-Behnken experimental design Run

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Factor A Amount of liquid lipid (mg) 10 15 10 15 20 15 15 15 20 15 15 15 20 15 15 10 20 15 10 15 15 15 10 10 15 20 20 15 15 15

Factor B Total amount of lipid (mg) 125 100 75 100 75 100 100 100 125 100 100 75 100 125 125 100 100 100 100 75 125 125 100 100 100 100 100 75 75 100

Factor C Drug content (mg)

Factor D Surfactant concentration (%w/w)

Response(Y1) Particle Size (nm)

Response(Y2) PDI

Response(Y3) % EE

10 15 10 10 10 5 10 5 10 15 10 15 10 5 15 10 10 10 10 5 10 10 5 15 10 5 15 10 10 10

0.25 0.2 0.25 0.25 0.25 0.3 0.25 0.2 025 0.3 0.25 0.25 0.3 0.25 0.25 0.2 0.2 0.25 0.3 0.25 0.3 0.2 0.25 0.25 0.25 0.25 0.25 0.2 0.3 0.25

214 323.1 190.7 198 192.2 141.1 191.7 145.2 159.5 172.6 190.9 185.9 175.5 144.6 187 137.2 140.3 152 172.5 133.6 148.2 154.4 132.8 186.9 142.7 129.9 187.8 136.2 132.2 145.7

0.289 0.352 0.25 0.264 0.316 0.283 0.29 0.269 0.281 0.284 0.313 0.345 0.295 0.275 0.334 0.254 0.277 0.277 0.295 0.273 0.281 0.269 0.263 0.335 0.26 0.267 0.301 0.271 0.282 0.275

31.783 17.55 32.25 30.75 28.06 56.84 25.74 19.78 16.34 18.99 26.35 18.55 32.67 19.91 14.47 16.14 19.16 14.13 25.12 19.34 23.53 15.66 29.96 24.92 29.13 48.81 19.6 33.11 36.01 32.27

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Table 2 Pharmacokinetics parameters obtained after a single oral administration of different formulations Formulations OLM suspension OLM-NLC

Cmax (µg/ml) 3.89±0.91 19.51±6.68**

Tmax (h) 1.1±0.54 1.25±0.5

AUCtotal (µg/ml*h) 16.64±6.70 80.61±24.58**

Values are presented as mean ± SD (n = 5), ** p<0.001 as compare to the OLM suspension

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