SCREENING STUDY FOR FORMULATION VARIABLES IN PREPARATION AND CHARACTERIZATION OF CANDESARTAN CILEXETIL LOADED NANOSTRUCTURED LIPID CARRIERS

Objective: The current study inspects the screening of the formulation components further, evaluates the physicochemical properties of the nanostructured lipid carriers (NLCs) for the antihypertensive drug as Candesartan Cilexetil (CC). The sequence screening of all excipients required for the preparation of NLCs should be performed. Methods: The prepared formulations were investigated for the different quality issues. The screening studies were performed to select the appropriate one of solid lipid, liquid lipid and surfactant. Also, investigation of physical compatibilities of solid lipid with liquid lipid and the ratios of them were evaluated. Furthermore, the physical characterization and quality issues of developed formulations were described and determined. Firstly, the solubility of CC in different solid and liquid lipids is the major parameter for the selection of the best one. Results: Precirol ATO 5, Compritol ® 888 ATO and Glyceryl Monostearate (GMS) were showed the maximum solubility of the CC (1000±4.12 mg, 1500±4.15 mg and 1750±3.16 mg), respectively. Hence, they were selected as the solid lipids for the development of NLCs. Liquid lipids Transcutol HP (30±2.21 mg/ml), Labrasol ALF (25±1.32 mg/ml) and Capryol 90 (18±1.34 mg/ml) were observed to have good affinity for the drug on systematic screening of different liquid lipids. All designed formulations observed in nanometer size of particles ranged from (408.9±11.5 to 114.6±8.3 nm) with high encapsulation efficiency around 99%.Also, the obtained results revealed that the ZP of the various formulations was consistently negative surface charge in between ((-13±2.3 to27.3±3.7 mV). Conclusion: Finally, formula number nine of CC (CC-NLC9) which composed of GMS (solid lipid), Capryol 90 (liquid lipid) and Lutrol® F127: Cremophore® RH (surfactants combination) was selected as the best formulation after the rank order for further investigations in the next work. The current work clarifies a sequence steps for selection of excipients for NLCs by employing simple experiments.


INTRODUCTION
Candesartan Cilexetil (CC) is prodrug of candesartan, angiotensin II type 1 (AT1) receptor antagonist, widely used in the management of hypertension and heart failure 1 . Candesartan Cilexetil is radially hydrolyzed to active form Candesartan during absorption from gastro intestinal tract 2 . Candesartan Cilexetil own great drawbacks which influence on its oral efficacy and therapeutic application such as very low aqueous solubility and first pass metabolism. Consequently, it has very low oral bioavailability not exceed 15% 3 . To repair previously mentioned drawbacks and to enhance oral bioavailability, lipid-based drug delivery systems like nanostructured lipid carrier (NLCs) second type of lipid nanoparticles system can be employed. Lipid nanoparticles systems (LNs) which have to generation's first, solid lipid nanoparticles (SLN) and second, nanostructured lipid carrier (NLCs) can improve the lymphatic transport of the lipophilic drugs as CC and hence, increase its oral bioavailability 4 . LNs systems were recorded as an advanced drug carrier system than polymeric nanoparticles 5, 6 . Advantages of nanostructured lipid carriers (NLCs) over the advantages of polymeric nanoparticles because of the lipid component matrix and its properties, which is physiologically tolerated. Resulted in avoidance of acute and chronic toxicity. In addition . Nanostructured lipid carriers (NLCs) composed of both solid and liquid lipids in certain proportion. Therefore, they offer various advantages over solid lipid nanoparticles (SLN) such as higher encapsulation efficiency, smaller size and low polymorphic changes 8 . Generally, nanostructure lipid carriers (NLCs) are nano-drug delivery carrier, which own the advantages of polymeric nanoparticles, emulsion, and liposomes. Furthermore, (NLCs) are essentially composed of a biocompatible lipid core with entrapped lipophilic drugs and surfactant at the outer shell. The major aim of this workwas to select a proper excipient for the development of NLCs using Candesartan Cilexetil (lipophilic anti-hypertensive agent) as a model drug. The screening studies were performed to select the appropriate one of solid lipid, liquid lipid and surfactant.
Also, investigation of physical compatibilities of solid lipid with liquid lipid and the ratios of them were evaluated. Furthermore, the physical characterization and quality issues of developed formulations were described and determined. Therefore, this study can offer the sequence steps for the development of NLCs and evaluation of their quality characteristics.
All the above materials were in analytical grade and were used without further purification.

Selection of solid lipid
The solid lipids screening was carried out by quantification of the saturation solubility of CC in different solid lipids which were determined by the test tube method. Precisely weighted amount of the CC (100 mg) putted in the test tube then the solid lipid was added in increments of (250 mg) to the test tube which could be heated to 4-5°C above the melting point of the solid lipid by saving in a controlled temperature water bath (Water path 4050, Romo, Cairo, Egypt). The quantity of solid lipids required to solubilize the drug in the molten state was recorded. The full dissolution state was completed by the formation of a clear, transparent solution 9 .

Selection of liquid lipid
Screening of liquid lipids were achieved by determination of saturation solubility of CC in various oils which was performed by adding an excess amount of drug in small glass vials contain fixed volume (5 ml) of different liquid lipids. The vials were strictly closed and incubated in adjusted mechanical shaker (Oscillating thermostatically controlled shaker, Gallent Kamp, England) for 72 h at 37 0 C with continuous agitation at 100 rpm 10 . Then the mixtures of liquid lipids and CC were centrifuged at high speed using (Biofuge Primo centrifuge maximum 17.000 rpm, England) centrifuge at 10,000 rpm for 15 min. The supernatant was separated and dissolved in an appropriate amount of methanol and the drug solubility was determined spectrophotometrically using UV-Vis spectrophotometer (Ultraviolet spectrophotometer, Shimadzu 1800, Japan) at λ max 254nm.

Physical compatibility of solid and liquid lipid
The miscibility of Selected Solid lipids and liquid lipids which possess the maximum affinity for the drug could be achieved. Constant ratio 1:1 of solid lipids and liquid lipids were mixed and melted in different glass tubes. The molten binary lipid mixture was permitted to solidify at room temperature. After that, the glass tubes were determined visually for the absence of divided layers in congealed lipid mass. Furthermore, the miscibility between solid lipid and liquid lipid was inspected by smearing a cooled sample of congealed lipid mixture onto a filter paper, followed by visual observation to clear the presence of any residue of oil on the filter paper. A binary mixture distinguished a melting point over 43 0 C which did not reveal any residue of oil droplets on the filter paper was selected for the development of CC-loaded NLCs 4 .

Selection of a binary lipid phase ratios
The ratio of selected solid lipids and liquid lipids was determined based on the melting point of the binary lipid mixture. Selected solid and liquid lipids were blended in the ratio varying from 90:10 to 10:90, then the binary Lipid mixtures were exhibited to be melted and stirred at 200 rpm for 1 h at 5°C above the melting point of solid lipid using hot plate magnetic stirrer (Magnetic stirrer, Wise-stir, Model MSH-20D, Hot plate stirrer, Korea) then kept aside to solidify at room temperature. The capillary method was used to ISSN: 2456- 8058 10 CODEN (USA): UJPRA3 determine the melting points of the congealed lipid mixtures 11 .

Selection of surfactant
The surfactant used for fabrication of NLCs should be screened selected depending on its ability to emulsify solid-liquid binary lipid mixture, binary lipid mixture (100 mg) was dissolved in 3 ml of methylene chloride and added to10mL of 5% surfactant solutions then stirred by applying magnetic stirrer. The organic layer was evaporated at 40 0 C and the remaining suspensions were diluted with 10-fold distilled water. The transmittance percent of the resultant samples was determined using UV-Vis spectrophotometer at 510nm 12 .

Fabrication of nanostructured lipid carriers (NLCs)
CC nanostructured lipid carriers (CC-NLC) were prepared by hot homogenization-ultrasonication technique but with some few modifications. Briefly, a weighted amount of selected solid-liquid binary lipids mixture (5% w/v) was melted at 5°C above the melting point of solid lipid. A known concentration of CC (5% w/v of lipids) was dissolved in the prepared oil phase (5% w/v mixture of solid and liquid lipid). The aqueous phase containing selected surfactant (2.5% w/v) was heated to the same temperature was added drop by drop to the lipid phase under magnetic stirring at 1500 rpm for 5 min. After that, homogenization of the resultant pre-emulsion was performed at high speed of mixing about 20,000 rpm using an Ultra-Turrax T25 homogenizer (WiseMix™ HG15A, Daihan Scientific, Seoul, Korea) for 10 min 11 . The resultant o/w nanoemulsions were subjected to probe sonication (ultrasonic processor, GE130, probe CV18, USA) at 60 % amplitude for 10 min. The obtained NLC dispersion was left beside to reach room temperature.

Physicochemical characterization of CC-NLCs Particle size and polydispersity index
The mean diameter and polydispersity index of particle of nanostructured lipid carriers loaded with CC was determined using a Zetasizer Nano-ZS (Malvern Instruments, Worceshtire (UK), equipped with a 10 mW He-Ne laser employing the wavelength of 633 nm and a back-scattering angle of 90° at 25°C. Before Photon correlation spectroscopic (PCS) analysis, CC-NLCs formulations should be diluted with a certain amount of double-distilled water (1:200) to get appropriates cattering intensity. The analysis, of Particle size was determined using Mie theory with the refractive index and absorbance of lecithin at 1.490 and 0.100, respectively 13 .

Zeta potential analysis
The zeta potential of NLC formulations was measured via electrophoretic mobility measurements using a Zetasizer Nano-ZS (Malvern Instruments, Worceshtire (UK). The zeta potential was calculated by applying the Helmholtz-Smoluchowski equation (n = 3) 14 .

Encapsulation efficiency (EE) and loading capacity (LC)
The encapsulation efficiency and loading capacity of CC into NLC formulations were measured by the indirect method by measuring the concentration of the free CC. Initially, 2 ml of NLCs formulations were centrifuged at 100,000 rpm for 1 h at 4 0 C to evaluate the un entrapped CC using cooling ultracentrifuge (Beckman Instruments TLX-120 Optima Ultracentrifuge). The aqueous layer was aspirated and filtered using Millipore® membrane (0.2μm) and diluted with an appropriate amount of methanol and measured by UV-Vis spectrophotometer (Shimadzu, the model UV-1800 PC, Kyoto, Japan) at 254 nm to measure the free amount of CC. Consequently, encapsulation efficiency and loading capacity of CC into NLCs were determined through the following equations 15, 19 .
Where, Wi= Weight of initial drug, Wf= Weight of free drug LC% = Wd Wn x100 Where, Wd= Wt. of drug in nanoparticles Wn=Wt. of nanoparticles In-vitro drug release study The in vitro release of CC from CC suspension and CC-NLCs was performed by a dialysis bag diffusion technique. The receptor compartments consist of the following release media: 500 ml Hydrochloric acid solution (0.1 N) of pH 1.2 and phosphate buffer solution (PBS) of pH 6.8 and again, in the same previous media but with addition Polysorbate 20 (0.35%-0.7%w/w) to confirm more achieve sink conditions of dissolution media 16 . The donor compartment is cellulose membrane dialysis bags (MWCO-12 000, Sigma, USA) were soaked in dissolution media overnight prior experiment. One milliliter of freshly prepared CC-NLC and CC suspension (equivalent to 2.5 mg of CC) were diluted with 5 ml of dissolution media and which tightly closed from two sides by a thermo-resistant thread. The bags were immersed in the Dissolution apparatus, (sixspindle dissolution tester, Pharmatest, type PTWII, Germany) automatically adjusted at 37±2 °C and 100 rpm. Two-milliliter sample was aspirated at a predetermined time interval (0.5, 1, 2, 4, 6, 8, 10, 12 and 24 h) and the same volume of media was added to maintain sink condition. The release of free CC from NLC was compared to that from suspension. The aspirated samples were measured using UV-Vis spectrophotometer at 254 nm.

RESULTS AND DISCUSSION Selection of Solid Lipid
The efficient solubility of the drug in the solid lipid reflects the capacity of NLC formulations to accommodate high amount of specific drug 17 . Initially, Candesartan Cilexetil (CC) solubility in various solid lipids should be performed to select the appropriate ones, which allowed accommodation of high amount of the drug leading to maximizing an essential qualification of a carrier system as the loading capacity and encapsulation efficiency of the prepared NLC formulations. Figure 1

Figure 1: Solubility study of CC in different solid lipids
These results related to the imperfect structure of matrix of Gelucire ® 44/14, Precirol ® ATO 5, Compritol ® 888 ATO and Glyceryl Monostearate (GMS) molecules, which are formed due to its chemical nature (mono-, di-, and triglyceride contents) and its composition that containing different length of chain of fatty acid that offer loosely porous structural features that make the drug easier to modify and more soluble 18

Figure 2: Solubility study of CC in different liquid lipids
The variety of Precirol ATO 5 ® fatty acid (C 16 and C18) content with subsequent loosely porous structure and its higher relative monoglycerides content in between different solid lipids used (more lipid monoglyceride, more lipid polarity) 21 . In addition, monoglycerides possess emulsification properties 22 which can also improve the drug solubility, such

Figure 3: Mean particles size and polydispersity index of CC-NLCs formulations
The solubilization ability of Transcutol ® HP (30±2.21 mg/ml), Labrasol ® ALF (25±1.32 mg/ml) and Capryol TM 90 (18±1.34 mg/ml) for CC was attributed to their intrinsic self-emulsifying property and their chemical structure (PEG-medium chain triglycerides) because of the affinity of a broad range of hydrophilic and lipophilic drug molecules to be encapsulated into lipid carriers, increased with PEG-glycerides than that glycerides free from PEG moieties such as (Labrafac TM PG, Labrafac TM LipophilandLabrafac TM CC) due to their known surfactant properties 29 .

Figure 4: Zeta potential of CC-NLCs formulations
The high solubilizing effect of Transcutol® HP for CC is consistent with Cirri et al., 12 . Furthermore, presence of Caprylic acid (C8) in oil composition had great impaction on drug solubility, where the oils of the more Caprylic acid content were found to be the higher solubilizing one for drug such as (Caprylic acid content in Labrasol ® ALF and Capryol 90) are 80 and 90%, respectively, 30 . This phenomenon may be attributed to the Caprylic acid polarity making it more efficient solubilizing one for the poorly water-soluble drug. Thus, Transcutol® HP, Labrasol® ALF and Capryol TM 90 were selected as a liquid lipid for further investigation because of the high solubilizing extent of CC after discarding of peppermint oil due to the low of its flashpoint (66.1°C) than the temperature that needed during the formulation preparation process.

Physical Compatibilities between Solid lipid and Liquid lipid
An essential for the improvement of a stable NLC development and permits taking into account that the fluid lipid is completely entrapped inside solid lipid matrix thus, physical compatibility between solid lipids and liquid lipids must be achieved.  On the other hand, no presence of more than one layer was observed in the solidified mass and no residue of liquid lipid droplets on the filter paper of (Precirol ® ATO 5-Labrasol® ALF) mixture indicate that formation of homogenous mixture. These results are consistent with S. Doktorovova et al., 31 38 . The combination of two or more surface-active agents exhibits to form blended surfactant films at the surface of the particle size. The formed blended surfactant films were produced in sufficient amount to cover the surface of particles successfully and produce nanoparticles with small size as well as keeping storage stability by production of requisite viscosity 34 . In present art, it was observed in  36,39 who discovered that an increased concentration of lipid leads to an enormous increase of particle size. As formulations are designed to be orally used, surfactants have been established at a pleasant 2.5% concentration (w/w) 42 . The composition of the formulation is given in the Table 3. Preparation of CC-NLCs were performed using homogenization followed by probe sonication technique. The influence of the lipids and surfactants variation on the particle size and the PDI was studied.
Also, other physical characterization should be achieved for every formulation to select the best one for further investigations.

Physicochemical characterization of CC-NLC formulations Particle size, polydispersity index (PDI)
Determination of physical properties as particle size and PDI are essential for predicting the stability of NLCs formulations. Particle sizing is a significant method for confirming nanosized particle manufacturing. Also, the smallest particle size, more absorbable and uptake through the gastrointestinal tract then, efficiently phagocytosed by the reticuloendothelial system. Therefore, the accuracy in particle size evaluation was necessary. Usually, the recommended particle size requisite for transportation through the intestine should not be more than 300 nm 43 . As represented in Table 4 and Figure 3 the observations revealed that all the designed formulations were showed in the nanometer range (<408 nm). It can be concluded that particle size of Precirol® ATO 5 nanoparticles (F1 to F3), Compritol® 888 ATO nanoparticles (F4 and F5) and GMS nanoparticles (F6 to F9) ranged from (280.6±11.8 to 118.6±8.1 nm), (283±9.9 and 196.5±10.2 nm) and (408.9±11.5 to 114.6±8.3 nm), respectively. The obtained results were clearly distinguished that formulations that contain more than one surfactant give the small particle size than that contain one surfactant as in F3 and F9 (118.6±8.1 and 114.6±8.3 nm) this behavior was attributed to the same reason discussed ISSN: 2456- 8058 15 CODEN (USA): UJPRA3 above under the screening study of surfactants combination. Also, these results were in accordance with the following reported studies 34 .
On the other hand, the largest particle size was exhibited in GMS formulations which contain surfactant Lutrol® F68 alone or in combination with other surfactants as in F6 and F8 (408.9±11.5 and 392.1±13.8 nm). This observation may be attributed to the tendency of GMS nanoparticles to form a gel after 24 h storage at room temperature due to polymorphic transitions in GMS after cooling at room temperature. Furthermore, the interaction between GMS and Lutrol® F68. The polymorphic transitions in the lipids after cooling to the room temperature and the interaction between surfactant and lipid are known to cause gel formation and subsequently influence the PS in NLC and SLN dispersions 44 . The polydispersity index as an indicator of the size distribution width of the particle. The PI value that reflects dispersion quality typically varies between 0 and 1. Most researchers recognize PI values ≤0.3 as optimum values; however, values ≤0.5 are also acceptable 45 . Table 4 and Figure 3 give an overview of the results of polydispersity index measurements. The prepared NLC dispersions had a PI value ≤0.35±0.01 due to the preparation method used indicating a homogenous and narrow size distribution of nanoparticles of NLCs.

Zeta potential measurement
The main parameter which influences the storage stability of colloidal nanocarrier is zeta potential, which measures the nanoparticle's surface charge and provides the repulsion degree between the nanoparticles preventing its agglomeration 46 . From the factors which mainly influence zeta potential of lipidbased nanoparticles structure of solid and liquid lipid and the medium composition 47 . Also, it depends on higher steric stabilization and lowers an electrostatic stabilization of nonionic surfactants which perfectly forming a coat around the particles of NLCs. Result in surface coverage of NLC decreases the electrophoretic mobility of nanoparticles and thus lower the zeta potential values 3, 15 . This phenomenon explains the higher stability of NLC formulations despite having a lower zeta potential value.
Zeta potential values ofall designed formulations are shown in Table 4 and represented in Figure 4.  48 and Compritol® 888 ATO composed of glyceryl tribehenate (28%-32%), glyceryl dibehenate (52-54%) and glyceryl monobehenate (12-18%) 49 , both of them being glycerol esters of long chain-length fatty acids (C18, C16) and (C22) respectively. So, that they provide neither charge nor polarity that participates to zeta potential. whereas, GMS composed of triacylglycerols (5-15%), diacylglycerols (30-45%) and monoacylglycerols (40-55%) 19 . In such a case due to the high content of partial emulsifying glycerides (mono and diglycerides) of GMS and the presence of non-esterified hydroxyl groups of glycerol, this molecule showed some of the polarity that participates to zeta potential. On the other hand, the liquid lipids were used in developed NLCs composed of diacylglycerol of medium-chain-length fatty acids. Liquid lipids provide the majority impaction and contribute to zeta potential due to its polarity which results from a free hydroxyl group of the glycerol that not subjected to the esterification process and the chain length of the fatty acids.

Entrapment efficiency, drug content and drug loading of CC-NLCs
The quantity of drug encapsulated in the nanoparticles and the drug content in the lipid matrix is a further significant consideration for the optimization of NLC. The quantity of drug encapsulated in the lipid matrix depends on many factors as: the type of lipids used, physicochemical properties of the drug, miscibility and solubility of drug in the molten lipid 52 , physical and chemical nature of the lipid matrix and crystalline state of lipid matrix and also surfactant was found to affect encapsulation efficiency 15 . Encapsulation efficiency and loading capacity of all NLC formulations are showed in the Table 5 and demonstrated in Figure 5.  (F4 and F5) showed higher entrapment efficiency around 99% than that of GMS nanoparticles (F6 to F9) around 95%. Such a fact was attributed to the chemical composition of each one. Where, the imperfect and less ordered matrix structure of Precirol® ATO 5 and Compritol® 888 ATO molecules, which are formed from a combination of mono-, di-and triglyceride that expected to exhibit lower crystallinity and more structure porosity which allows higher solubility and easier accommodation of more drug molecules 17, 26 . Also, Precirol® ATO 5 is a di-glyceride with two different chain length fatty acids palmitic and stearic acid (C16 and C18); therefore, it is expected to have less ordered lipid network compared to GMS, and thus lead to the more drug molecules could be entrapped 18, 26 . Further, subsequent to cooling, Precirol®ATO 5 and Compritol®888 ATO recrystallize in a progression of polymorphs. In like manner, with respect to the conditions utilized during the preparation, CC could be homogenously dispersed 15 . Regarding the type of surfactant, it was clearly observed that NLCs formulation prepared using Lutrol® F68 higher E.E. than that prepared using other surfactants. This behavior repeated with every nanoparticle prepared using Lutrol® F68 alone (F1, F4 and F6) or in combination with Cremophore® EL (F8). This fact might be attributed to the high value of the hydrophilic-lipophilic balance of Lutrol® F68 (HLB ~ 29) compared to other surfactants.

In-vitro release study
In-vitro release study was achieved for all formulations in addition to pure CC suspension. The release condition monitored in 0.1 M HCl (pH 1.2) and PBS (pH 6.8) and at the same conditions with adding tween 20 (0.35%-0.7%w/w) to achieve "sink" conditions during a dissolution test for all formulations 54, 55  found that all formulations exhibit a lack of drug release within 24 h. The in-vitro release of the best formula (CC-NLC9) was reached to less than 5% as showed in (figure 6) but, CC suspension showed almost complete drug release (100%) within 8 h. As CC had solubility equal to 11 μg/mL in 0.1 M HCl and 1 μg/mL in PBS (pH 6.8). The very difficult release of CC results from its poor aqueous solubility and high lipophilicity (log p~6.2). Thus, CC possesses a high affinity to the lipids consequently the drug becomes more entrapped and retained inside the core of the lipid matrix preventing it from the release. Furthermore, the high efficient solubility and compatibility of CC with the lipid components as previously discussed before under screening studies 56,57 . These observations are in line with the study reported by Zhang et al., 56 . Previous studies ascertained that NLCs must be absorbed into the blood or lymphatic system after duodenal administration to rates. Consequently, lack of in-vitro release of CC from NLCs suggesting that NLCs could be absorbed via the enterocytes after oral administration, the most sought after therapeutic effect which required confirmation through employing more investigations in this work. The rank order was performed for all prepared NLCs formulations (F1 to F9) in order to choose the best formula based on the previously measured characterization as Particle size, polydispersity index (PDI), Zeta potential, encapsulation efficiency and loading capacity of CC-NLCs wherein the formula F9 was chosen as the best formula for further investigations.

CONCLUSION
Nanostructured lipid carriers (NLCs) are adaptable nanoparticles with multipurpose applications. However, quality and successful incorporation of CC into NLC to develop more efficient formulation based on proper selection of the components and optimization. The current work clarifies a sequence steps for selection of excipients for NLCs by employing simple experiments. Screening studies were performed for whole excipients to select appropriate ones to prepare CC loaded NLCs. Furthermore, the developed formulations were subjected to physicochemical characterization. The resulted formulations appeared in nanoparticles size with high encapsulation efficiency.

AUTHOR'S CONTRIBUTION
The manuscript was carried out, written, and approved in collaboration with all authors.

CONFLICTS OF INTEREST
There are no any conflicts of interest.