Design, Development and Optimisation of Carvedilol Microemulsion by Pseudoternary Phase diagram and Central Composite design

Aim of this study is to design and optimise carvedilol loaded microemulsion. Carvedilol microemulsion is prepared and optimised by Phase titration technique. The selections of material attributes for the formulation of microemulsion are chosen by building a pseudo ternary phase diagramwith various concentration oils and surfactant. Optimisation of Carvedilol microemulsion formulation technique was carried out by Central composite Design with two centre points by using selected critical material attributes like the different concentration of oils, surfactants and evaluated for its effect on Critical quality attributes like PS nm, ZP mV, EE %, PI, RI,% Drug Content and Self emulsi ication time (sec). From this optimisation study data, selected Carvedilol Microemulsion is subjected to measure for its in vitro drug release studies. CM9 formulation showed promising result in particle size, poly dispersibility index, entrapment ef iciency and drug release studies. All the above data, it has been accomplished that CM9 was selected as an optimised Carvedilol microemulsion formulation.


INTRODUCTION
Microemulsions [ME] are mixtures of hydrophobic, hydrophilic and amphiphilic sections that are macroscopically and optically isotropic.ME enhance the bioavailability of the drug. ME are homogenous, clear, thermodynamically constant dispersions of water (Danielsson and Lindman, 1981) and oil which is stabilised by a surfactant in combination to surfactant and their average droplet diameter lies in the range of within 10-140 micrometres (Aulton and Taylor, 2002;Lawrence and Rees, 2000;Shinoda and Lindman, 1987).
Direct (oil dispersed in water, o / w) and reversed (water dispersed in oil, w/o) are the two main types of microemulsions. ME is one of the potential and emerging drug carrier systems that help improve the release of drugs and improve the bioavailability of drugs that are poorly aqueous and soluble. The microemulsions demonstrate very rapid penetration of the active molecules, which is primarily due to the wide surface area of the internal phase, also decreasing the barrier property of their contents. The small droplets often have greater adherence in a regulated manner to membranes and transport drug molecules. MEs have been proposed to have a bene icial effect on drug absorption in a variety of ways, such as shielding the drugs from oxidation and enzymatic degradation and improving the membrane permeability and lymphatic transport. Also of importance is the composition of the interfacial layer of o / w microemulsions, since it can in luence the degree of solubilisation and the stabilisation of the structure. ow a day microemulsion is an emerging trade and having worldwide importance in a variety of technological applications (Sinko, 2012;Swarbrick, 2007).
Carvedilol is a non-selective antagonist of betaadrenergic receptors (β1, β2) and a regulator of alpha-adrenergic receptors (α1). Carvedilol binds to beta-adrenergic receptors on cardiac myocytes reversibly. Inhibition of these receptors restricts the sympathetic nervous system from responding, resulting in reduced heart rate and contractility. In patients with heart disease, where the sympathetic nervous system is activated as a compensatory mechanism, this action is advantageous. Carvedilol alpha1 receptor blockade induces blood vessel vasodilatation. This inhibition corresponds to a decrease in peripheral vascular resistance and induces an antihypertensive reaction. Due to carvedilol's blockade of β1 receptors in the heart, there is no re lex tachycardia reaction. Carvedilol is graded as a BCS Class-II compound of low aqueous solubility and reasonable biomembrane permeability (Laurence et al., 2008;Reiter, 2004). The central hypothesis of this research is to enhance the aqueous solubility the carvedilol formulating into microemulsion.

MATERIALS AND METHODS
Carvedilol obtained as a contribution sample from Aurobindo Pvt. Ltd, Hyderabad. Arachis Oil, Castor Oil, Palm Oil, Sun lower Oil, Olive Oil, Corn oil, Tween 20 and 80, Span 20 and 80, Sodium Lauryl Sulphate, Poloxamer 188 and Polysorbates are obtained from Abitec corporation and Loba Chemie Pvt Ltd, Mumbai, Maharashtra, India.

Solubility studies
By increasing the drug concentration, carvedilol's solubility has been evaluated in various oils such as Arachis Oil, Castor Oil, Palm Oil, Sun lower Oil, Olive Oil, and Corn Oil. Then it is allowed to dissolve until it is saturated to the equilibrium state in 10 ml of liquid. The solubility of the oil drug was estimated in mg/ml (Jada et al., 1990).

FTIR analysis
The chemical reaction between carvedilol and other ingredients such as oils, the surfactants used in the formulation, was calculated using FTIR tests. Studies have been carried out using the Potassium Bromide (KBr) pelletisation process for the carvedilol and microemulsion dispersion. Along with the KBr, narcotics (0.2 per cent) is grounded, and the mixture is then pressed with the aid of a mini KBr pellet press with a pressure of around 7 tons by shifting the press handle several times. The dispersion of the microemulsion was taken in a centrifuge tube and held for 40 minutes in a centrifuge at 15000 RPM., to eliminate the excess volume of oil, the supernatant solvent was siphoned from the centrifugal tube and traces were gathered and dried at 50 o C. Dried By ixing a resolution at 4cm −1 in the FTIR instrument (Bruker, Germany) itted with the OPUS Spectrum programme, the dried microemulsion dispersion was stacked like a sandwich between a plane KBr pellet and scanned over a wavenumber scale of 4000 to 500 cm −1 . Samples were placed on the sample stage with a force gauge of 100 N and the reproducible contact between the sample and crystal holder for scanning is ensured (Chaurasia, 2016).

Identi ication of Drug by IR-spectrophotometric method
Each chemical or drug's infrared spectrum contains details about the groups found in that unique chemical. A spectrophotometer for spectrum recording in the infra-red ield consists of an optical device capable of producing 400-4000cm −1 monochromatic light and a resolution of 2cm −1 in the scanning range. To analyse liquid and semi-solid samples for IR analysis, attenuated complete re lection (ATR) is used. A microemulsion drop is placed directly on the (ATR) stage and scanned from 4000cm −1 to 400cm −1 . Bruker FTIR and ATR spectrophotometer (Germany) used the opus programme to collect the infrared spectrum of the drug sample (Chaurasia, 2016).

Formulation of microemulsion
To formulate the successful formulation of the microemulsion is mainly depends on the selection of oil excipients. There are many oil-based excipients presently available commercially. The selected excipients must possess properties required for the microemulsion system. There must be compatibility between drug and oil excipients used in microemulsion formulation. The surfactant and co-surfactant must have high HLB value and must be transparent to form a microemulsion.

Phase titration method
The Spontaneous Emulsi ication Method (Phase Titration method) formulates microemulsions. It can be seen with the aid of phase diagrams, to prepare a microemulsion, a combination of fatty acids and oil is added a caustic solution. And a cosurfactant, and alcohol, is titrated with it until the system     (Muzaffar et al., 2013;Dhanapal, 2012;Naimish et al., 2000).
For a thorough analysis of the phase diagrams for the creation of microemulsions, these Smix ratios were chosen for increasing co-surfactant concentration concerning surfactant and increasing surfactant concentration concerning co-surfactant (Aboofazeli et al., 1994;Nguele et al., 2016).

Preparation of Microemulsion systems
As seen in Table 2, a series of microemulsions were prepared using Olive oil as the oil, Span 80 as the surfactant, and Poloxamer as the co-surfactant. The quantity of prescription was kept stable in all formulations. Drugs were put in a beaker, correctly measured, and oil, surfactant, and co-surfactant were applied. The components were combined with a magnetic stirrer by gentle stirring, and the resulting mixture was positioned for size reduction in ultra-sonication for around 10-15 minutes. Then, once the substance had fully dissolved, the mixture was heated at 40 0 C. Up for further application, the homogenous mixture was kept at room temperature (Wei et al., 2012;Sushma et al., 2013). Thermodynamic stability tests, such as heating cooling stage, freeze-thaw stage and centrifugation, have been carried out following completion of all formulations (Sushma et al., 2013;Cheong and Tan, 2010).

Phase separation study
Accurately about 1 ml of drug-loaded microemulsion was added to100 ml of distilled water in a beaker at 37 0 C and vortexed for 2 min. the mixture was stored at 37 0 C (Room Temperature) a period of 2 hrs and observed visually for any phase separation (Cheong and Tan, 2010).

Visual assessment
The microemulsion was diluted with distilled water (100 ml) and softly blended with a magnetic stirrer. Roughly 100 µl of medication was illed. It is necessary to sustain the temperature at 37 0 C. Slight white milk such as emulsion was the formulation CM1 was less transparent emulsion, which has a bluishwhite colour. The CM2-CM9 formulations have a smooth, slightly bluish appearance with strong stability (Cheong and Tan, 2010;Naz et al., 2020;Edris and Malone, 2012).

Transmission test
Light transmission from selected formulations of microemulsion as well as 50 times, 100 times and 200 times water dilution was tested by UV-Spectrophotometer at respective nm using water as a blank (Cheong and Tan, 2010;Naz et al., 2020;Edris and Malone, 2012).

Scanning electron microscopy
Scanning electron microscopy (SEM) is used to determine the Identi ied Optimised Microemulsions surface morphology, Using double adhesive tape, the samples were placed on alumina stubs and covered with gold in a HUS-5 GB vacuum evaporator. The sample was then observed in the Hitachi S-3000N SEM at 10KV acceleration voltage and 5000X magni ication (Aboofazeli et al., 1994;Nguele et al., 2016).

Particle size determination
The average particle size of the microemulsion was calculated by dynamic light dispersion (DLS) at a dispersion angle of 173 o , and the temperature of the sample holder (Nanopartica SZ-100 HORIBA Scienti ic, Japan) was approximately 25 o C.,o ensure that the light scattering rate remained inside the instrument limit, the dispersion assay remained diluted to 1:2500 v / v with double distilled water (Aboofazeli et al., 1994;Nguele et al., 2016).

Zeta potential
Zeta potential is the potential difference in the stationary phase of the luid bound to the distributed particles in the dispersion medium. Zeta potential is primarily based on the stability of colloidal dispersion; zeta potential was calculated using a Zetasizer (Nanopartica SZ-100 HORIBA Science, Japan). The Zeta potential was then directly calculated by the Smolochowski equation using the equation as follows (Wei et al., 2012;Sushma et al., 2013).

Determination of % drug content
The oil based dispersed system of the drug was analysed spectrophotometrically for the drug content at wavelength 241nm with a proper dilution of formulations taking dichloromethane as a blank (Cheong and Tan, 2010;Naz et al., 2020;Edris and Malone, 2012).

Lyophilization of Microemulsion
The procedure of lyophilisation is otherwise referred to as freeze-drying, involving freezing and then drying the microemulsion under high vacuum by applying ultra-low freezing temperatures in the range from -50 • C to -80 • C. The optimised microemulsion was added with 5% of Mannitol, which functions as cryoprotectants. This dispersion was frozen at approximately -80 • C and dried with Lyophilizer (Freeze Zone 2.5, Labconco, Labconco Company Czech Republic) at a chamber pressure of approximately 0.120 mBar for 48 hours. The dried microemulsion powder from the sample holders was extracted and processed for further use in desiccators (Bolzinger et al., 2008).

In-vitro drug release study
In-vitro drug release from ME was used to measure the consistency of emulsifying property. The usp06 station, dissolution apparatus used to study the drug release from the oil in an aqueous medium, hard gelatin capsule containing lyophilised microemulsion and marketed Carvil tablet (mfg.: Zydus Cadila Healthcare, batch no.: 2053297) was performed in basket type dissolution apparatus. 900 ml of phosphate buffer pH (7.4) was used as dissolution media. The bath temperature as well as bowl temperature, was maintained about 37±0.5 0 C and paddle allowed rotating 50 rpm. 5ml of the sample was withdrawn at time intervals 5, 10, 15, 20, 25, 30 min and dilution were made to 10ml. 5ml of fresh medium was replaced to dissolution jar. The diluted samples are analysed spectrophotometrically at 241nm for carvedilol, and their % drug release was calculated (Lipinski et al., 2012;Padula et al., 2018;Yang et al., 2017).

Solubility Studies
To prepare microemulsion, the assortment of excipients is signi icant criteria should pharmaceutically suitable, ideal and compatible with the formulation. In contrast to other oils, the solubility of carvedilol was found to be greater in olive oil. For the production of the ideal formulation, olive oil was chosen as the oil process. As the surfactant, Tween 80 was chosen, and Poloxamer 188 was picked as the aqueous process as a co-surfactant and double-distilled water. The results are shown in Figure 1 and Table 3.

Calibration curve of Carvedilol
The drug was observed for solubility, and the drug was identi ied with the help of UV and FTTR. the carvedilol exhibited absorption maxima at 241nm when 7.4 pH Phosphate buffer was used as a solvent which was same as mentioned in literature. The calibration curve was prepared diverse concentration of carvedilol like 0,5,10,15,20,25 µg/ml in 7.4pH phosphate buffer, and their absorbance was observed at 241 nm in UV spectrophotometer by keeping 7.4pH phosphate buffer as blank. XY linear plot was drawn by keeping concentration in µg/m on x-axis and absorbance on the y-axis, which shows a good linearity r 2 value as 0.998. The calibration data infer the good purity if carvedilol and suitable for formulation. The calibration curve was prepared diverse concentration of carvedilol like 0,2,4,6,8,10 µg/ml in 1.2pH 0.1N hydrochloric acid, and their absorbance was observed at 255 nm in UV spectrophotometer by keeping 1.2 pH 0.1Nhydrochloric acid as blank. XY linear plot was drawn by keeping concentration in µg/m on x-axis and absorbance on the y-axis, which shows a good linearity r 2 value as 0.997. The calibration data infer the good solubility and purity of carvedilol and suitable for formulation. The results are shown in Figure 2.

Compatibility studies by FTIR analysis -Carvedilol and its Microemulsion formulation
The  Figure 3 and Table 4.
The best Carvedilol microemulsion formulations have shown same spectra as an isolated drug which demonstrates that the chemical structure of the drug doesn't change after converting to microemulsion and shows there is no interaction between the drug and excipients. From the data obtained from FTIR spectra, it was concluded that when compared to the carvedilol, the preferred functional group frequencies in the pure drug were reproducible in carvedilol microemulsion. It was veri ied that the drug and excipients used were found to be compatible with each other in the formulations.

Construction of Pseudo-ternary phase diagram
Oil and precise Smix ratios as were extensively com-bined in various volume ratios from 1:9 to 9:1 in separate small glass test tubes for each phase diagram. Eleven different variations of oil and each Smix; 0:10, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, 10:0 were created to cover the available ratios for the sample to delineate the phase boundaries speci ically formed in the phase diagrams. Pseudo ternary step diagrams were developed using the water titration method to assess the presence of the microemulsion region. The olive oil was combined with various amounts of surfactant and cosurfactant (Smix) to create pseudo ternary phase diagrams, and the mixture was titrated with puri ied water until it turned turbid. Using data obtained from the aqueous titration process, pseudo ternary step diagrams were drawn. In the range of 5-95 per cent of the total volume at 5 per cent intervals, the quantity of water applied to offer water concentration. Visual evaluation was performed for every 5 per cent addition of water to the oil and Smix mixture, as seen in Table 5. The surfactant and cosurfactant ratios (Span 80 and Poloxamer 188) used for titration were 1:1,1:2,1:3, 1:4, 2:1,3:1 and 4:1, respectively.By using the phase diagram, the composition of the microemulsion, as well as the phase behaviour of mixture, can be determined. For each Smix ratio the pseudo ternary phase diagram constructed separately, O/W microemulsion regions could be identi ied, and microemulsion could be optimised. Pseudo ternary phase diagram construction was constructed by using a surfactant, cosurfactant was incorporated in the ratio 1:1, and the pseudo ternary diagram was constructed. It was found that the region of microemulsion existence was very less. The increase in surfactant 2:1 concentration resulted in the development of a greater microemulsion region. Further concentration changes from 2:1 to 3:1 and 3:1 to 4:1 culminated in a much larger rise in the area of the microemulsion. Further increase in the concentration of surfactant and co-surfactant ratio from 4:1 to 5:1, resulting in the formation of gel-like microemulsion with less area of formulation of the microemulsion. On the other hand the increase in the cosurfactant and surfactant ratio from 1:1 to 1:2, 1:2 to 1:3 and 1:3 to 1:4, resulting in less microemulsion when compared to surfactant: co-surfactant ratio (4:1). These pseudo ternary phase diagrams were constructed with a combination of oil and Smix ratio from 1:9 to 9:1 like 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1. Each ratio of Smix and oil ratio represents a separate phase diagram individually. On which ratio of oil and Smix gives the highest region of microemulsion was identi ied and select that ratio for main formulation optimisation of microemulsion preparation. In this study, we found that 4:1 (Surfactant: co-surfactant) ratio gives a high region area for microemulsion preparation, so 4:1 Smix took as the optimised ratio for further microemulsion formulation. The results are shown in Figure 4.

Thermodynamic stability studies
Thermodynamic stability tests, tests such as heating-cooling cycle, freeze-thaw cycle and centrifugation, were conducted after completion of all formulations. Of the nine formulations, six formulations were chosen based on heating, cooling and centrifugation. Based on thermodynamic stability tests, six formulations were found to have been introduced and chosen for further characterisation. The Carvedilol Microemulsion formulations were named as CM1-CM9, out of these formulations CM2 to CM7 shows good stability in Heating cooling cycles; CM1, CM8, CM9 shows phase separation in heating and cooling cycles; CM3 -CM8 shows no phase separation in Freeze-Thaw cycle; CM1, CM2, CM9 shows phase separation in Freeze-Thaw cycle; CM1-CM3 and CM7-CM9 show phase separation in Centrifugation; CM4-CM6 shows no phase separation in centrifugation. From the data shown in Table 6, it shows that CM4-CM6 shows good Stability studies in all type of Stability studies. The results show the Carvedilol microemulsion (CM2-CM9) was less clear and transparent.

Optimisation of Phase titration technique for the preparation of Carvedilol Microemulsion (Central composite design)
The central composite optimisation of carvedilol microemulsion and its result are shown in Tables 7  and 8; and Figures 5 and 6 revealed the effect of CMA on CQA during the preparation of Carvedilol microemulsion. It was inferred from the data that a clear association occurred between particle size and Smix. From the data, it was veri ied that there was a decrease in microemulsion particle size by growing the concentration of Smix. The 'P' value was found to be < 0.05 for ANOVA when the Smix vs PS was executed in nm, which suggests a massive change in PS when increasing the Smix concentration. Of all formulations (CM1-CM10), CM9 displayed a target particle size of approximately 1017.5±5.4 nm at a high +1 Smix range (i.e. 4:1 portion of span 80 ratio: Poloxamer 188). The increase in the concentration of Smix in microemulsion preparation revealed a parallel rise in the strength of the zeta with a decrease in the particle size of the microemulsion, which veri ied the strong stability of the non-sedimentary microemulsion. The 'p' value was found to be < 0.05 by de ining it in ANOVA; this indicated that there was a substantial improvement in ZP by increasing the concentration of Smix and oil. The CM9 formulation revealed that the zeta potential needed was approximate -64.4±1.4 mV at a high +1 Smix concentration level. A similar rise in the EE percentage indicated an increase in Smix concentration.CM9 formulation showed higher entrapment ef iciency of about 94.80± 2.0%. The other variables, like Polydispersity index (PI), Refractive index (RI), % Drug Content and Self emulsi ication time (sec), was determined for all the formulation. Among all the formulation, microemulsion CM9 shown least PI of 0.591 (acceptance criteria are <7 for monodisperse condition)which shows good dispersibility of microparticles in phase and so that sedimentation rate if less for CM9 microemulsion; Cm 9 shows least Refractive index of about 1.432±0.02 which con irms the good transparency of microemulsion; Cm9 shows maximum carvedilol content of about 96.80± 4.2%, it may result due to reduction in particle size which may enhance the encapsulation of drug in microparticles. The Self emulsi ication time for CM9 microemulsion was found very fast, i.e., 19 ± 0.26 sec, it may be due to least particle size and concentration on Smix on the interface of oil and water. So that the water consumption for the formation of microemulsion was less and the endpoint for microemulsion was found to be short. From the optimisation results, it was concluded that the optimal formulation was found to be CM9. The polynomial equations were derived from central composite design coef icient values, provided by the CMA-based adjustments in the CQA, as follows: From the optimisation, it was con irmed that all the practical values are correlated strongly with the predicted values, and the regression coef icient r2 value was found to be 0.995 when comparing the predicted vs observed values of PS, ZP and EE of carvedilol microemulsion CM9.

Scanning Electron Microscopy (SEM) Evaluation
Surface morphology and shape of microemulsion formulated with optimised parameters was observed for SEM studies.
The study revealed that the microemulsion was mostly spherical, the particle surface had a distinctive smoothness, and the particle size, as shown by SEM, was in the micrometric range. The results of Carvedilol microemulsion shown in Figure 7.

In vitro drug release study
The drug release pro ile for Lyophilized Carvedilol microemulsion particles was investigated in phosphate buffer (pH7.4) by packing in a gelatin capsule. It shows that droplet size decreased, the surface area increased, allowing more dissolution of the drug from microemulsion. The release of drug from formulation mainly depends on Smix: Oil ratio. When the concentration Smix increased in formulation result in smaller microdroplet was formed this result in enhancement of the dissolution pro ile of carvedilol. Thus, the carvedilol release from CM9microemulsion was found to be highest (100.74±2.80%) at30 min than other formulation and marketed Carvil tablet, as shown in Figure 8.
With an increase in the Smix ratio in CM9, the release of the drug increases, and the successful distribution of the drug is partly due to the size of the microparticles and the polarity of the resulting oil droplets, which makes for a higher rate of drug release into the aqueous process. The solubilised drug, which is independent of the lipid digestion process, does not precipitate in the lumen and undergo rapid absorption.

CONCLUSION
From the discusses optimisation study data, selected Carvedilol Microemulsion are subjected to measure for its in vitro drug release studies and CM9 formulation showed promising result in particle size, poly dispersibility index, entrapment ef iciency and in-vitro drug release studies. From all the above data, it has been accomplished that CM9 was selected as an optimised Carvedilol microemulsion formulation.