DOE , formulation , and optimization of Repaglinide nanostructured lipid carriers

Shady A. Swidan1*, Zeinab N. Mansour2, Zeinab A. Mourad2, Nahla A. Elhesaisy2, Nada A. Mohamed2, Mohamed S. Bekheet2, Mohamed A. Badawy2, Mai M. Elsemeiri2, Aya E. Elrefaey2, Amera M. Hassaneen2 1Department of Pharmaceutics, Faculty of Pharmacy, The British University in Egypt, El-Sherouk City, Egypt. 2Undergraduate student of the 5th year, Faculty of Pharmacy, The British University in Egypt, El-Sherouk City, Egypt.


INTRODUCTION
Diabetes mellitus (DM) is an important chronic disease, which may cause severe complications if it is not properly controlled (He et al., 2015).Those severe complications include cardiovascular problems, high blood pressure, impaired lipid profile, nephropathy, neuropathy, and retinopathy.This makes this disease the seventh worldwide leading cause of death (Kramer et al., 2013).DM has two types and type II DM is the most common type and accounts for more than 90% of the cases (Zhu et al., 2013).
Different classes of oral hypoglycemic agents are used for the treatment of type II DM.Repaglinide (RPG), (S) (+)-2 ethoxy-4- (2[[3-methyl-1[2-(1-piperidinyl)phenyl]-butyl]amino]-2 oxoethyl) benzoic acid, is a new potent blood glucose lowering agent (Figure 1) (Akhtar et al., 2013).It belongs to the class of carbamoyl-methyl benzoic acids.It acts by stimulation of the release of insulin from the pancreatic cells by blocking of ATPsensitive potassium channels in the plasma membrane (Hu et al., 2000).Although RPG has an excellent antidiabetic effect, it suffers from certain problems, which diminish its activity and require frequent administration.RPG has a short half-life (about 1 hour), it suffers from a significant first-pass effect and it has a low oral bioavailability of about 50% (Kassem et al., 2017).
In order to increase the patient compliance and enhance its oral bioavailability, different RPG nanoparticles were formulated such as nanoemulsions (Akhtar et al., 2016), self-nano emulsifying systems (Kamble et al., 2013), nanocrystals (Gadadare et al., 2015), and solid lipid nanoparticles (Ebrahimi et al., 2015).One of the most promising novel nano-based techniques is nanostructured lipid carriers (NLC).NLC is a new improved generation of solid lipid nanoparticles (SLN), in which a spatially incompatible liquid lipid is added to the solid lipid to form less ordered lipid nanoparticles (Swidan et al., 2016).It overcomes the disadvantages of SLN, including the low entrapment efficiency and drug loading, especially upon storage due to the drug expulsion from the highly ordered solid lipid crystals.The presence of the liquid lipid disrupts the symmetry of the lipid crystals and shows imperfection of the crystals, thus allowing more drug to be incorporated and lower amount to leave the crystals upon storage while maintaining the solid state at both room and body temperatures (Weber et al., 2014).
Several methods can be used for the preparation of NLC such as microemulsion, solvent diffusion, solvent injection, double emulsion, and emulsification ultrasonification (Das and Chaudhury, 2011).In this study, emulsification-ultrasonification method was used as it has a number of advantages; it avoids the use of organic solvents, which may cause toxicity, it is also simple and leads to the formation of small and uniform nanoparticles.
Different factors affect the shape, size, and the function of NLC such as types of solid lipids and liquid lipids, the ratio of both lipids to each other and to the drug, the type and concentration of surfactants, and other different factors related to the preparation technique (Tan et al., 2010;Yang et al., 2014).
In this study, the objective was to apply response surface methodology optimization model to optimize RPG loaded NLC.Two formulation factors were selected as independent variables.The prepared formulae were characterized by the determination of particle size (PS), polydispersibility index (PDI), and zeta potential (ZP).Entrapment efficiency (EE%), drug loading (DL%), and in vitro RPG release were evaluated and the optimized formula was evaluated with the same experiments and the crystallinity of the optimized form was studied using differential scanning calorimetry (DSC).The shape and morphology of the optimized formula were examined using a transmission electron microscope (TEM).

Materials
RPG and the solid lipid glyceryl monostearate (GMS) are kind gifts from EIPICO pharmaceutical company.Oleic acid (OA) was purchased from Chemajet (Egypt).Polyoxyethylenesorbitan monooleate (Tween 80)-the used surfactant-was purchased from Acros (Netherlands).Potassium dihydrogen phosphate and sodium hydrogen phosphate were purchased from Lobechem (India).All other chemicals and solvents are of pharmaceutical grade and were used with no further modifications.

Experimental design
Two independent variables were selected for the study, the ratio of the liquid lipid to the solid lipid and the concentration of surfactant.Each factor has three levels: high, medium, and low.The dependent variables or the responses to be determined are the mean PS, ZP and EE%.Values and constraints of both independent variables and responses are listed in Table 1.A 3 2 factorial design was done and the composition of the nine resulted formulae is listed in Table 2.The optimization was done by response surface methodology using Statgraphics ® centurion XVII software.

Preparation of RPG loaded (NLCs)
The RPG-NLCs formulae were prepared by emulsification and ultrasonification technique (Das et al., 2011) with few modifications.The organic phase-composed of the calculated weights of RPG, the liquid lipid OA, and the solid lipid GMS-was melted by heating up to 5°C above the melting point of GMS.The aqueous phase was prepared by dissolving tween 80 in water and heated to the same temperature as that of molten lipid phase.The preheated aqueous solution was added to the molten lipid phase and homogenized using WiseTis homogenizer HG-15D (Diahan sci, South Korea) for 5 minutes at 10,000 rpm.The coarse hot O/W emulsion obtained was sonicated using a probe sonicator for 1 minute with a pause for 30 seconds for two cycles at 50 W (Vibra cell, Sonics, USA).The obtained NLCs were cooled to room temperature and stored at 4°C.

Particle size analysis
The mean PS of the RPG-NLC and the particle size distribution expressed by the PDI of the prepared NLCs were determined using the dynamic light scattering (DLS) technique with Nanotrac particle size analyzer (Microtrac, USA).RPG-NLC samples were diluted by distilled water and the concentration of samples was adjusted for the optimal measurement condition.Three measurements were averaged for the mean PS and PDI and the standard deviation (SD) was calculated.

Zeta potential analysis
The measurement of the electric charge on the surface of the nanoparticles is a good indication of the physical stability of the most colloidal system (Dubey et al., 2012).The surface charge of the prepared RPG-NLC was measured by determining ZP on the nanoparticles surface.ZP was measured using Zetatrac W3547 (Microtrac, USA).Conductivity was in the range of 24-42 μs/cm and the applied field strength was 10 kV/m with zeta run time of 30 seconds.

Determination of entrapment efficiency and drug loading of RPG-loaded NLCs
Both EE% and DL% were calculated by measuring the amount of free non-encapsulated RPG in the prepared RPG-NLC.Samples were centrifuged using cooling centrifuge (Centurion Scientific Ltd., UK) as mentioned by Chalikwar et al. (2012) with small modifications.Briefly, 1.5 ml of the NLCs dispersion is centrifuged while the speed of centrifuge was kept at 15,000 rpm for 60 min at temperature of 4º C.After suitable dilution, the concentration of RPG in the supernatant was measured using spectrophotometer (Jenway 6305 spectrophotometer, China) at λ max 237 nm.EE% and DL% were calculated using the Equations 1 and 2. The experiment was done in triplicates and the average and SD were calculated.

In vitro release study
The in vitro RPG release from the RPG-NLC test was done using dialysis bag diffusion technique (Ebrahimi et al., 2015).Dialysis bags used was with a molecular weight cut off 12,000-14,000 were soaked in the release medium for 24 hours before use.Volume equivalent to 1 mg of RPG of RPG suspension and RPG-NLC was added into a thoroughly sealed by double-folding on both sides dialysis bag, which was immersed in 50 ml of simulated intestinal fluid (phosphate buffered saline, PBS, PH 6.8) at 37°C ± 0.5°C.Then, it was placed on shaking water bath at 50 rpm (Wise ® bathwater bath, Wised B, Germany) for 24 hours.At predefined time intervals, 1 ml sample was withdrawn from dissolution medium and the amount of RPG released was analyzed spectrophotometrically at λ max 237 nm.The volume of dissolution medium was kept constant by the addition of replacement volumes of PBS after each sampling.

Optimization and preparation of the optimized formula
The relationship between responses and formulation variables of all prepared RPG-NLC were treated using a three-level factorial design model, which is a response surface technique done by the use of Statgraphics ® Centurion software.These high, medium and low levels of variables X 1 and X 2 were selected from different preliminary experimentation.The optimization of variables was performed using the desirability function to obtain the levels of X 1 and X 2 , which minimize Y 1 while maximizing Y 2 and Y 3 .

Characterization of the optimized formula
The optimized prepared formula was characterized by determining PS, PDI, ZP and its morphology and size were studied using TEM technique.Then, the EE% and DL% were also calculated and the in vitro release behavior was studied and the best fitted kinetic model for the in vitro release was investigated.The crystallinity and polymorphism of the optimized formula were studied using the DSC technique.

TEM
The TEM measurement was done using the negative staining method as reported by Yu and Huang (2013), using electron microscope JTEM-1010, (JEOL ® , Tokyo, Japan).One drop of the dispersion of the RPG-NLC was put on a copper grid coating and the excess droplets were wiped using filter paper.After 5 minutes, one drop of uranyl acetate solution (2% w/v) was then dropped onto the grids.After the samples were negatively stained and air-dried at room temperature, they were ready for the TEM investigation done at 74 kV.

Kinetic study of the in vitro drug release of the optimized RPG-NLC formula
The in vitro release of the RPG from the optimized formula was evaluated in comparison with the pure RPG.The dissolution medium and the conditions of the experiment were not changed from the conditions mentioned previously for the in vitro release of the nine prepared formulae.
RPG release data of the optimized formula were fitted to several mathematical models to know the RPG release mechanism from RPG-NLC in the simulated intestinal fluid.The models studied are zero-order model (percentage cumulative drug released vs. time), first-order model (log percentage cumulative drug remained vs. time), Higuchi diffusion model (percentage cumulative drug released vs. square root of time), and Hixon-Crowell model (cube root of percentage cumulative drug remaining vs. time) and the best-fitted model is the model with the highest correlation coefficient (r).

Differential scanning calorimetry analysis
DSC analysis was done on the optimized formula, which was lyophilized to study the physical state and polymorphism of the RPG-NLC.The DSC measurements were performed using differential scanning calorimeter, Shimadzu DSC-50 (Japan).The four studied powders were the pure GMS, pure RPG, physical mixture in the ratio (1:1), and the lyophilized optimized RPG-NLC formulae.In brief, samples of weight 2-5 mg were heated, then scanned between 20°C and 250°C.The heating rate was of × 100 × 100 10°C minute −1 and the measurements were done under nitrogen gas flow (30 ml minute −1 ) (Mazumder et al., 2017).

Particle size analysis
Size of the nanoparticles plays a key role in the evaluation of the efficiency of nanoparticles for RPG delivery.The mean PS and PDI of RPG-NLC is listed in Table 3.The mean PS ranged from 182.7 ± 07.9 in formula F9 to 452.0 ± 66.1 in formula F2, while the narrowest PS distribution was in F7 with PDI of 0.09 ± 0.06, while the widest distribution with PDI of 0.54 ± 0.01 was in F5.The polynomial equation obtained by the linear regression of the data of particle size is shown in Equation 3Y Where Y 1 , X 1 , and X 2 are the particle size, liquid lipid concentration, and surfactant concentration, respectively.
As seen from the data illustrated in Figure 2A, the smallest PS obtained when the liquid lipid ratio was 45% with the higher concentration of tween 80.This was in agreement with what stated in Kumar et al. (2013), who found that the highest percentage of tween 80 surfactant gave the lowest PS.Concerning the percentage of liquid lipid, Teeranachaideekul et al. (2008) found that the increase in the amount of liquid lipid should result in an increase in the mean PS of NLCs.Different findings were mentioned by Abdelbary and Haider (2011) who stated that there is no significant effect of liquid lipid to solid lipid ratio on the size of NLC formulae.

Zeta potential
RPG-NLC with high ZP is likely of better stability upon storage than the formulae of ZP value close to 0 mV.It is known that ZP values of more than ±30 are considered a good indication for the stability of the nanoparticles (Honary and Zahir, 2013).As shown in Table 3, Formula F7 showed the highest negative ZP value of −44.4 ± 6.25, while F2 showed the smallest value of −07.9 ± 0.93.The linear regression of the zeta potential data of the prepared formulae resulted in Equation 4 Where Y 2 , X 1 , and X 2 are the zeta potential, liquid lipid concentration, and surfactant concentration, respectively.
As seen from Figure 2B, no significant effect of Tween 80 concentration on the ZP value was observed.This might be due to the nature of Tween 80 as a non-ionic surfactant.It was noticed that formulae with higher content of OA in the RPG-NLC resulted in an increase in the ZP values and vice versa.This was suggested to be due to the presence of the carboxylic (COO -) group in the OA.This was in complete agreement with the finding Teeranachaideekul et al. (2007) who stated that because of the negative charged medium chain triglycerides at their (COO -) group, increasing the % of medium chain triglycerides increases the ZP of NLC, and in the absence of the solid lipid, the nanoemulsion showed the highest ZP values.

Entrapment efficiency and drug loading of RPG-loaded NLCs
The percentages of RPG entrapped in all RPG-NLC are listed in Table 3. From table 3 and figure 2C, it is clear that high values of EE% achieved and most of the RPG was entrapped in the nanoparticles.The lowest EE value was 74.7% ± 0.82% in formula 3 with 30% OA and 2% Tween 80, while the highest EE% value was 85.5% ± 0.95% in F4 which contains the lowest concentration of both liquid lipid and surfactant.As there is no change in the total amount of the lipids or the RPG, the ranking of the DL% of the RPG-NLC formulae was the same as in EE%.F4 showed the highest DL% with 2.14% ± 0.13%, while F3 had DL% of 1.87% ± 0.06%.It was found that high EE% and DL% values were obtained with low surfactant concentration.The polynomial equation of the entrapment efficiency is shown in Equation 5Y 3 = 110.927− 0.850667 * X 1 − 14.82 * X 2 + 0.00557037 * X 1 2 + 0.326222 * X 1 * X 2 − 1.18667 * X 2 2 (5) Where Y 3 , X 1 , and X 2 are the entrapment efficiency, liquid lipid concentration, and surfactant concentration, respectively.
The highest three formulae in both EE% and DL% were with 1% Tween 80.This may be due to the enhanced solubilization caused by Tween 80 in the aqueous phase.Luo et al. (2006) found the same findings and stated that when the amounts of the emulsifiers increased, the EE% decreased due to the solubilization effect of the emulsifiers which enhance the solubility of the drug in the aqueous phase, not in the lipid nanoparticles.Same findings were reported by Venkateswarlu and Manjunath when they used poloxamer 188 as surfactant; as the percentage of poloxamer 188 increased, the EE% was decreased marginally.This thought to be due to increased solubility of the drug used in the aqueous phase as the percentage of poloxamer 188 increased (Venkateswarlu and Manjunath, 2004).

In vitro RPG release from RPG-NLC nanoparticles
The in vitro release of RPG from the NLC compared to pure RPG powder was evaluated for 24 hours in simulated intestinal fluid (pH 6.8).The concentrations of RPG were calculated using constructed calibration curve on RPG concentration range 5-50 µg/ml.The curve was linear and obeys the Beer's Lambert law over the whole concentration range, the equation describes the curve is (y = 0.02x − 0.0125) and the r = 0.9996.The cumulative percentage released of RPG was illustrated in Figure 3.As seen from the graph, the release of RPG from all RPG-NLC formulae   was biphasic with the initial rapid release and sustained release in the second phase.The faster initial release was derived from the diffusion of RPG located at the surface of the nanoparticle, while the slower later one from that incorporated in the NLC core and released in a prolonged pattern via the erosion or degradation of the matrix (Zeng et al., 2012).
It is also clear that all formulae showed the slower release of the drug compared to the pure RPG.After 4 hours, the pure RPG dispersion released about 100%, while all prepared formulae released much smaller amount of RPG within the same period.Formula F3 showed the lowest RPG release with only 18.125% ± 1.15% of RPG, while F2 released the highest percentage of RPG 53.875% ± 3.3%.After 24 hours, the release of RPG from F4 was only 55.90% ± 3.6%, while the fastest RPG release from RPG-NLC formulae was about 98.88% ± 4.7% from formula F7.It is clear that the fastest release obtained in the formula with higher liquid lipid content, while the slowest release was when the OA is only 15%.A possible explanation is that the presence of a high percentage of the lipid in the liquid state led to thermodynamic instability which enhances the release of the drug from the nanoparticles.

Optimization of the independent variables
The optimization of the concentration ratio of the liquid lipid to the solid lipid and the surfactant concentration was done using response surface methodology and through the determination of the desirability function.
This statistical function is considered one of the most important functions to optimize variables statistically.It is a function based on the concept that the quality of a formula that has many features is very unacceptable if one of them is outside of a desirable limit.It aims to adjust the formulation or process variable to values that ensure compliance with the criteria of all of the involved responses and so to provide the optimum value of compromise in the desirable joint response.This can be achieved by converting the multiple responses into a single response, then combining the individual responses into a composite function followed by its optimization (Vera Candioti et al., 2014).
All values of the optimized variables, predicted and observed responses are listed in Table 4.The optimum values of variables X 1 and X 2 were 45% and 1.476%, respectively.The predicted responses were 242.5 nm, −41.1 mV, and 81.1% for mean PS, ZP, and EE%, respectively.The measurements done after the formulation of the optimized formula-as observed responses-were 228 nm, −38.2 mV, and 80.2%, respectively.Both ZP and EE% showed a better observed response, while the predicted PS was smaller than that actually measured for the optimized formula, the mean PS and size distribution of the optimized formula is shown in Figure 4.All responses were close to the expected values and were within the statistically accepted deviation range.The desirability function for the optimization process equals 0.751189 (Figure 2D), this value is high enough to ensure acceptable values for the three studied responses.

TEM
The TEM photograph of the optimized RPG-NLC formula is shown in Figure 5 with magnification power 80,000 ×.From the photograph, it is clear that the NLC vesicles are separate entities.All particles appeared in the photograph were in the nanometer range.The TEM investigation also revealed that the nanoparticles of the RPG-NLC formula were homogeneous ellipsoidal or spherical in shape.This might indicate homogeneity and good uniformity of the release of the entrapped RPG.It was noticed that the vesicles look smaller in size when measured using TEM technique compared to that measured by DLS technique.As the preparation of the TEM sample involves drying, this leads to shrinkage of the nanoparticles so they appear smaller compared with the DLS measurement, in which the hydration of samples maintains the size of the nanoparticles (Sanad et al., 2010).

Kinetic study of the in vitro drug release of the optimized RPG-NLC formula
The in vitro release data of the optimized formula showed slower RPG release than all previously prepared NLC formulae.Optimized formula released only 17.13% ± 0.53% of RPG after 4 hours, while after 24 hours, 44.38% ± 2.1% of RPG released.This pattern of release proved that the optimized formulae achieved the controlled prolonged release pattern that allows sustained RPG release, hence better control of DM can be obtained.
Different kinetic models were applied to the RPG release data.RPG release from the optimized formula at pH 6.8 did not show proper fitting to zero order, first order, and Hixon Crowell models and the r values were 0.967, −0.979, and 0.976, respectively.The best-fitted model to describe the kinetics of the release and hence describe the release mechanism was Higuchi diffusion model with the highest r value equals 0.992.Graphical representation of the application of Higuchi diffusion model to the release data is shown in Figure 6.The fitting of this model suggests that the major mechanism of the RPG release was by diffusion as this model best applied to systems shows release by diffusion through the lipid matrix (Siepmann and Peppas, 2011).This was in complete agreement with the findings of Sangsen et al. (2014), which prepared curcumin loaded NLC.

Differential scanning calorimetry analysis
DSC is an accurate, rapid, and reliable method for the characterization of crystallinity of the drug.It also gives a strong indication on the possible interactions that might occur between the drug and all additives, which is reflected in the thermogram by the appearance of new peaks, displacement of the endothermic peaks, or change in their shape or enthalpy (Maiti et al., 2007).
The thermograms of pure RPG, pure GMS, physical mixture of both, and the RPG-NLC optimized formula are shown in Figure 7.As seen from the figure, RPG showed a sharp The GMS showed a sharp single endothermic peak at 69.1°C.The physical mixture of the drug and the lipid showed two endothermic peaks at approximately the same temperatures for the pure powders.A slight widening in the peak of the RPG was noticed which might be an indication of the presence of slight affinity between RPG and GMS, but both are still in the crystalline state and no interaction was noticed.Complete disappearance of the melting peak of the RPG in its melting temperature range was noticed, while a slightly displaced peak for GMS appeared.This finding suggests the conversion of the crystalline RPG to its amorphous state.This signifies the possibility of interaction between the drug and the lipid and indicates a better solubility of the RPG which affects its release from the nanoparticles (Kassem et al., 2017).

CONCLUSION
In the current study, the RPG-NLC formulae were successfully prepared using emulsification-ultrasonification method.High RPG entrapment was achieved in the prepared nanosized vesicles.The liquid lipid and surfactant concentrations are crucial for optimizing size, charge, drug loading, drug entrapment, and RPG release from RPG-NLC.RPG-NLC achieved sustained controlled biphasic release in vitro, while the release kinetics of the optimized formula obeyed Higuchi diffusion kinetics model.RPG was in an amorphous state in the NLC, which allows better solubility and    controlled release.Further studies on the in vivo pharmacodynamics, pharmacokinetic, and therapeutic effects must be done.Finally, NLC is a promising nanocarrier for the delivery of RPG orally.

Figure 3 .
Figure 3.The in vitro release of RPG from the prepared RPG-NLC formulae compared to the pure RPG powder in the simulated intestinal fluid dissolution medium.

Figure 4 .
Figure 4. Particle size distribution of the optimized RPG-NLC formula.

Figure 6 .
Figure 6.Higuchi diffusion model representation for the in vitro release of RPG from the optimized RPG-NLC formula.

Table 1 .
Values of independent and dependent variables.

Table 2 .
Composition of the prepared NLC formulae.

Table 4 .
The predicted and the observed response for the optimized RPG-NLC formula.
Purvis et al. (2007)131.3°C.This indicates that pure RPG is in the crystalline state and this value was in agreement withPurvis et al. (2007).