Determination of Pickering Nanoemulsion by Eudragit Rl-100 Nanoparticle as Oral Drug Delivery for Poorly Soluble

Objective: The purpose of this research study was to develop Ketoprofen-loaded Pickering nanoemulsion with the help of polymeric nanoparticles [NPs]. The pickering nanoemulsion formulation Results: All the prepared formulations show particle size in between 100-500nm hence it indicats formation of nanoemulsion. The zeta potential is -46mv which indicates good stability of formulation. The In vitro drug release shows maximum drug release i.e. 96.93% in 10 hrs which shows that the release of drug is prolonged due to formation of Polymer NPs. Conclusion: Thus the drug release was significantly controlled and slowed down when nanoemulsion is formulated by using NPs in comparison with control. These results fulfilled the objective of the study. This study opens new prospects on the formulation of Pickering nanoemulsion.


INTRODUCTION
Pickering's solid particle-stabilized emulsions were discovered by Ramsden and Pickering within the 1900s and developed slowly until the1980s [1,2]. Compared to surfactant-stabilized emulsions, Pickering emulsions have shown the benefits of strong stabilization and really good coalescence resistance happens to the formation of a dense shell of solid particles irreversibly adsorbed around water droplet emulsions [3][4][5]. Pickering emulsions have generated considerable interest in research in several areas particularly within the cosmetic and pharmaceutical fields or food applications, where surfactants often have undesirable effects (such as irritation and hemolytic behavior) [6]. As a new drug delivery system, Pickering emulsions could facilitate dermal drug delivery increase the oral absorption of poorly water-soluble drugs and improve drug stability [7]. Recently, various Pickering nanoemulsions have been described. They have average droplet diameters within the range of 50-300 nm. The adsorption of solid particles at the water/oil interface is irreversible and strong. Classical emulsion using surfactants has shown some undesirable characteristics in pharmaceutical applications. In most applications, Pickering particles can be substituted for surfactants in the classical emulsion [8][9][10][11]. Pickering emulsion using solid particle as an emulsifier may provide a way to avoid some undesirable side effects that can come along with using surfactant [12,13].
Ketoprofen is a non-steroidal anti-inflammatory drug. It belongs to BCS class II drug, that is high permeability low solubility [14]. It is used for the relief of pain and inflammation in rheumatoid arthritis, osteoarthritis and ankylosing spondylitis. It is administered orally and externally applied as a gel. The oral bioavailability is up to 60% [15]. The pickering nanoemulsion formulation is developed using Eudragit RL 100, which has the greater ability to stabilize the formulation as well as which better controls the release of drug upon oral administration.

Materials
Ketoprofen was gifted from BEC Chemicals Pvt. Ltd, Roha, Raigad, Captex -300(MCT) was purchased from Abitech corp. Mumbai India, Tween 80 and Eudragit RL-100 polymer are purchased from Modern Science Nashik and all other chemical used were of analytical grade.

Preformulation
Studies of Ketoprofen and Polymer [16,17] Preformulation parameters are check for Ketoprofen and eudragit and it was characterized by organoleptic properties solubility melting point and compatibility studies by using FTIR Techniques.

FTIR Analysis
The dry sample of the drug was mixed with KBr within the ratio of 1:99.
The sample was triturated and eventually placed in a sample holder and compressed employing a motorized pellet press at 15 tones pressure. The pellets were then scanned using an FTIR spectrophotometer (Shimadzu; IR Affinity-1S) over frequency ranges from 4000 to 400 cm -1 , The spectra were analyzed by comparing the standard ranges of functional groups [18].

Drug-Excipient compatibility Studies
A compatibility study was carried out in order to establish, that there was no interaction between the drug and excipients used in the formulation. The drug and physical mixtures of the drug: polymer (1:1) were filled in the vial and sealed.
The sealed vials were kept at a specific temperature (in a desiccator) for 45 days. After 1 month mixture evaluated by FTIR. The preliminary compatibility was carried out by FTIR (Shimadzu: IR Affinity-1S). The FTIR spectrum was recorded from 4500cm -1 . Infrared spectra of pure drug and physical mixture of drug with polymers were obtained by using standard KBr sample [18].

Step -I
Preparation of nanoparticles by Nano precipitation method in which the organic phase and aqueous phase were mixed at different ratios. The organic phase is Eudragit RL100 and acetone and the aqueous phase is 1% of tween 80 in water. Then the Nano dispersion was prepared by adding of organic phase at a constant rate of 0.5 ml/min in an aqueous phase under gentle agitation. After that acetone was removed from it by a Rotary evaporator. Then Nano dispersion is concentrated by evaporating a significant amount of water. After that kept it for washing in spectra pore regenerated cellulose tubing with water by the gentle stirring for 48 hrs. Formula for preparation given in Table 1.

Step -II
Preparation of Pickering Nano emulsion by using this washed Nano dispersion with an oil phase containing Ketoprofen + Captex 300 (MCT) at different ratios. The premix of Drug + Captex is formulated using a mechanical mixer. Operating at 5000 rpm for 10 min, at 25 0 C. Then Nano emulsion is generating by using probe sonication. Formula for preparation given in Table 2.

Physical Appearance
The physical appearance of Pickering nanoemulsion was observed visually.

PH Determination
The pH of the Pickering NE formulation was determined by using a pH meter. For pH determination, 1% Pickering naoemulsion formulation in deionized water was prepared and pH was determined [19].

Viscosity Determination
The viscosity is measured to determine the rheological properties of formulation by Brookfield viscometer at different rpm. The torque is set for 95% for all formulations so that we get constant readings of all formulations [20].

% Drug Entrapment Efficiency
To determine the percent drug entrapment drug efficiency, take 2ml of Pickering nanoemulsion of Ketoprofen and centrifuged at 12,000 rpm for 30 min. after centrifuge supernatant is obtained and diluted it with water (1:1) and after that analyzed by the UV-Spectrophotometry 257-260 nm [21, 18,22].
The % entrapment efficiency is calculated by following equation. (1)

In -vitro Drug Release of Pickering Nanoemulsion
The dissolution study is carried out using USP dissolution apparatus type-II (Paddle apparatus), The drug release study of Premix, Nanoemulsion formulation and Marketed film coated tablet formulation is measured by the dialysis tubing method. The premix of drug and Captex -300 and prepared Pickering nanoemulsion 2ml (8mg/ml) is filled in two tubes of dialysis membrane -50 (at Av. flat width -24.26 mm and Av. Diameter 14.3 mm). In order to simulate pH changes along with GIT, the dissolution medium with 0.1 N HCl and pH 6.8 buffer were sequentially used. When performing the in-vitro release experiments, 0.1 N HCL medium was first used for 2hr which was then replaced by fresh 6.8 pH phosphate buffer for a further 10 hrs. Samples were withdrawn at regular time intervals. Samples were estimated using UV-Vis Spectrophotometer (Shimadzu 2450double beam UV-Vis Spectrophotometer) at respective wavelengths [23].

Particle Size Distribution and Zeta Potential Measurement
Particle size distribution, zeta potential, and Polydispersivity index of nanodispersion and Pickering nanoemulsion were determined by using a Malvern particle size analyzer. To check the stability of this nanoemulsion. The sample was diluted 3 times with distilled water before the experiment is performed at 25 0 C [19,24].

Transmission Electron Microscopy
Transmission electron microscopy was used to analyze the morphology of the nanoemulsion droplets.Samples were diluted 100 times with distilled water after dilution a drop of Samples were placed on the carbon-support than allowed to stand 5 minutes for drying and placed in the vacuum chamber for 30 minutes before analysis [23].

Stability Study
To evaluate the physical stability of the optimum formulated Pickering NE for ketoprofen, it was stored at 40 °C and 75% relative humidity (RH) in an oven for 90 days. The NE entrapment efficiency, Zeta Potential, Particle size were determined after one and three month's storage.

Preformulation Study
The Preformulation study has been performed to determine the physicochemical properties of a drug. The melting point of Ketoprofen was found to be 94-97 0 C. The solubility of the drug is checked in three different solvents the results are given in Table 3 The solubility of ketoprofen determine in three different solvents was found to be soluble in ethanol and phosphate buffer 6.8 and poorly soluble in water [16,17].

FTIR analysis
FTIR analysis was done to check the possible interaction between drug and polymer. The Pure Ketoprofen spectra show characteristic peak at 1650cm The comparative spectra given are in Fig. 1.

Physical appearance
The appearance of the formulated Pickering nanoemulsion is transparent white. The mage is shown in Fig. 2.

PH determination
PH of all formulation batches NE1-NE9 was determined by pH meter results are given in the Table 4.  Table 5.

4.2.4% Drug entrapment efficiency
The drug entrapment efficiency of all NE1-NE2 of the Pickering nanoemulsion formulation was determined for drug content in the formulation. The entrapment efficiency of all formulations is found to be in the range of 72.00 -92.20 %. From them, the Optimized formulation NE3 shows higher entrapment efficiency. Results are shown in Fig. 3.

In-vitro drug release study a) Drug release from Marketed tablet and Premix (Ketoprofen +Captex -300)
This study is performed to study the effect of nanoparticles containing nanodispersion on the formulation to prolong the drug release of the drug. This marketed film coated tablet and premix is considering as control. The release of drug in tablet gives 92.78% in 3 hrs. and premix gives 100% in 2.5 hrs. Results are shown in Table 6 Fig. 4.

b) In vitro Drug release of ketoprofen loaded Pickering nanoemulsion
In this study, the dissolution of optimized batch NE3 was performed for 10 hrs. The release of ketoprofen loaded Pickering nanoemulsion at pH 1.2 is slower and by increasing the pH 6.8 of the release medium drug release is increase. The release of drug from Pickering nanoemulsion was found to be only 15 % of drug release within 2 hrs. And after 5 hrs. 70 % of drug release and finally 96% of drug release from formulation at 9 hrs. By comparing the Pickering nanoemulsion with control Marketed Tablet formulation and premix of (Drug + Captex -300) it clearly found that the effect of Eudragit RL-100 NPs significantly better controls and prolongs the release of drug. This is in line with the objective of this study. The result was shown in figure no. 4 and Table 7.

Nanodispersion
The mean particle size was done with the help of a zetasizer. The average mean particle size was calculated. The average mean diameter of D1 to D4 was analyzed. The result of optimized batch D3 show in figure no 4. In which the particle size of polymer nanoparticles is 154 nm and the Zeta potential is -16mv which indicates moderate stability of the formulation. The result shown in figure no. 5 And 6 Polydispersivity index of NE3 Batch is 0.098 which indicates monodispersity.

Pickering nanoemulsion
The size of Pickering nanoparticles was determined of all batches of formulation The optimized batch NE3 of Pickering nanoemulsion gives the average particle size 220nm standard particle size varies from 100 -500 nm hence consider as nanoemulsion preparation Results of optimized formulation NE3 are shown in Figure no. 6.PDI is a very crucial parameter to check particle size distribution. The monodisperse formulations have less value for PDI the optimized formulation shows PDI between 0. 220 is showing monodisperse characteristics. Zeta potential is determine to check the stability of the formulation. Zeta potential is between ±40 to ±60mV having an ideal repulsive force to achieve good physical stability of the nano formulation. The zeta potential of optimized formulation NE3 was found to be -46mV indicate good stability of formulation. Results are shown in 3 Fig. 7 and 8.

Transmission electron microscopy
The morphological characterization of the nanoemulsion transmission electron microscopy study was performed. The TEM image shows that nanoemulsion droplets are well differentiated from each other. And conserving their shape even after vacuum establishment during TEM observation .I.e. not spread on carbon support with a sample of nanoemulsion [26][27][28]. This means that the interface is stabilized by aggregated NPs and this particle creates a rigid film that acts as a physical barrier against Flocculation and Coalescence. Fig.9 shows the TEM image of nanoemulsion.

Stability study
Stability study was done for optimized batch as per ICH guideline for novel drug delivery system The entrapment efficiency, Zeta Potential, Particle size, was calculated. The stability study showed no change in the Entrapment Efficiency, Zeta potential, Particle size after 1, 2 and 3 time periods. The results are shown in Table  8.

CONCLUSION
From all observations and results obtained it can be concluded that all the prepared formulations show satisfied organoleptic properties. The characterization of drug and excipient was done, all results are compared with the standards and from results, it was concluded that drug and excipient are pure and it of standard quality. The Particle size of the nanoparticles in nanodispersion is around 154 nm and nanoemulsion formulation is around 220 nm which showing high stability. Zeta potential of the formulation is around -46 indicating good stability of formulation. The in-vitro drug release assessment of marketed tablet, and premix compare with optimized formulations of nanoemulsion further confirmed the use of Eudragit nanoparticles in inhibiting the release of ketoprofen for prolong period of time as well as enhanced stability of formulation. Transmission electron microscopy shows the nanoemulsion droplets are well differentiating from each other [25,29]. Finally, we have found that release of model drug was significantly slowed down when nanoemulsion is formulated by using eudragit nanoparticle, in comparison with controls. The results of the stability study shows that formulation can be stabilized by the use of eudragit nanoparticles. This study opens new prospects on the formulation of Pickering nanoemulsion.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.