Formulation and Evaluation of Nanoparticulate Drug Delivery System of Benazepril HCl

The hypertension is a common cardiovascular disease affecting the worldwide population. Hypertension is also called high blood pressure a condition in which the force of the blood against the artery walls is too high (Tripathi, 2019). Usually, hypertension is defined as blood pressure above 140/90 mm of Hg, which is considered severe if the pressure exceeds 180/120 mm of Hg. High blood pressure frequently goes unreported. If left untreated, it can eventually lead to illnesses like heart disease and stroke (Michel and Hoffman., 2011). Hypertension could be primary or essential hypertension and secondary hypertension. Primary hypertension is the most common type and there are no specific underlying causes while secondary hypertension can be due to renal, vascular, endocrine disorders, etc. (Shanbhang and Shenoy, 2015). The Benazepril hydrochloride, an angiotensin-converting enzyme (ACE) inhibitor, is used in the treatment of hypertension and heart failure. The family of ACE inhibitors inhibits the angiotensin-converting enzyme, which is involved in the conversion of angiotensin I to angiotensin II. Angiotensin II has a strong direct vasoconstrictor impact that increases blood pressure while stimulating aldosterone production and secretion (Ameerah., 2018). Since ACE is comparable to bradykininase or kininase II, ACE inhibitors may reduce the degradation of bradykinin. Being a prodrug of the diacid benazeprilat, Benazepril hydrochloride is quickly absorbed and transformed into the active ACE inhibitor, Benazeprilat. The drug has a half-life of 10–11 hrs. It is approved for treating essential hypertension and may be effective for treating congestive heart failure (Revathi, 2012).

A nanoparticulate drug delivery system is used to solubilize and protect the drug and to enhance the absorption which ultimately enhances the bioavailability of the drug. A nanoparticulate drug delivery system also produces sustained or controlled release action for an extended period of time. (Poole and Owens, 2003) Benazepril HCl is an antihypertensive drug and it falls under BCS class I having high solubility and high permeability. In order to increase the bioavailability of Benazepril HCl, a Nanoparticulate drug delivery system was selected.

Hence this research topic was undertaken to formulate nanoparticles of Benazepril HCl by using ethyl cellulose as a polymer. The organic phase of Benazepril HCl drug and ethyl cellulose polymer was prepared in ethanol and the aqueous phase was prepared using polyvinyl alcohol as a stabilizer in distilled water, hence the emulsion was formed by adding an organic phase in the aqueous phase. Benazepril HCl nanoparticles were formulated by using an emulsion-solvent evaporation technique. Four formulations were formulated by altering different concentrations of polymer and stabilizer and evaluations were done by considering various evaluation parameters like its yield, particle size, entrapment efficiency, its release.

By using a nanoparticulate drug delivery system, the limitations of Benazepril HCl in terms of bioavailability were overcome. This was well studied by drug release study using dissolution test apparatus.

MATERIALS AND METHODS:

Materials

Benazepril Hydrochloride pure drug was received as a gift sample from Aurobindo Pharma Ltd., Hyderabad. Ethylcellulose was purchased from Yarrow chem products, Mumbai. Polyvinyl alcohol and Ethanol were taken from Laboratory Reagent, Loba Chemie Pvt. Ltd., Mumbai.

Preformulation study of drug

One of the crucial steps in the creation of any drug delivery system is the preformulation study of drug. It provides the details required to specify whether the medication release is diffusion or dissolution. Therefore, preformulation investigations of the drug Benazepril HCl for identification have been carried out in this research work, including melting point determination, FTIR study of the drug, DSC study of the drug, determination of λ max, construction of calibration curve, etc.

Determination of melting point

The melting point of Benazepril HCl was determined by a capillary method using Thiel’s tube. Precautions were taken to maintain the uniform heating to Thiel’s tube, in which the capillary containing the drug was placed. The melting point is the first indication of the purity of the sample (Abdine and Belal, 2004).

FTIR spectroscopy

Fourier transform infrared (FT-IR) study has been performed to identify the Benazepril HCl drug, excipients and their physiochemical compatibilities with the help of Shimadzu FT-IR spectrophotometer.

Differential Scanning Colorimetry

The purity and identification of Benazepril HCl were done by carrying out the thermal analysis of pure drug Benazepril HCl using the Setaram DSC instrument.

Determination of wavelength

Benazepril HCl has been quantitatively analyzed by various techniques. 10 mg of Benazepril HCl was accurately weighed and added to a 100 ml volumetric flask. Dissolved drug in a sufficient quantity of Phosphate buffer pH 7.4 and then final volume was made up. The prepared solution was scanned in a UV spectrophotometer in range of 200- 400 nm using Phosphate buffer pH 7.4 as blank and the λ _max was determined. The ultraviolet spectrum of Benazepril HCl in Phosphate buffer pH 7.4 solution was measured between 200-400 nm (Parmar and Chhalotiya, 2013).

Calibration Curve of Benazepril HCl

Calibration curve of Benazepril HCl in Ethanol

Benazepril HCl 10 mg was carefully weighed 100 ml in a volumetric flask before being diluted with ethanol to the proper concentration in order to create the standard stock solution (100 µg/ml). working standard solutions of suitable concentrations of the drug were prepared by diluting 1 to 10 ml stock solution up to 10 ml with ethanol to get a working standard solution of 10-100 µg/ ml(Nimaje et al., 2012).

Calibration curve of Benazepril HCl in Phosphate Buffer pH 7.4

Benazepril HCl 10 mg was carefully weighed 100 ml in a volumetric flask before being diluted with Phosphate Buffer pH 7.4 to the proper concentration in order to create the standard stock solution (100 µg/ml). working standard solutions of suitable concentrations of the drug were prepared by diluting 0.1 to 1 ml stock solution up to 10 ml with Phosphate Buffer pH 7.4 to get a working standard solution of 1-10 µg/ ml (Stanisz and Paszun, 2009).

Preparation of Nanoparticles

Benazepril HCl loaded polymeric nanoparticles were formulated by solvent evaporation technique. Four formulations were prepared. The organic phase was prepared by dissolving the drug and polymer in an organic solvent like ethanol. Then aqueous phase was prepared by dissolving the stabilizer in distilled water. The organic phase was added dropwise to the aqueous phase under a high share force of homogenizer to prepare an o/w emulsion. The rotary evaporator was used for the evaporation of the solvent. Then nanoparticles were collected by ultracentrifugation and washed with distilled water to remove stabilizer residue or any free drug. After this water was evaporated by lyophilization and dried nanoparticles were collected in the form of powder (Serrlola and Sailaja, 2014).

The comparative study was done for four formulations in order to know the best formulation for the preparation of Benazepril HCl nanoparticles. The prepared nanoparticles were evaluated for percentage yield, drug content, encapsulation efficiency, loading capacity and drug release and characterized for particle size and surface charge (Sharma et al., 2014).

Table No. 1: Polymers and stabilizers used for the formulations

Percentage yield

 It was calculated using the formula (Tabassum and Krishnasailaja, 2018)

Particle size determination:

The particle size of the nanoparticles of Benazepril HCl of each formulation was determined with the help of Horiba SZ-100. Approximately 1gm of the sample was selected and evaluated for its size in nanometers ( Reddy and Sailaja,2014)

Determination of Zeta Potential.

Zeta potential is highly useful for the assessment of the physical stability of colloidal dispersions. Zeta potential can be measured by the determination of the movement velocity of the particles in an electric field (electrophoresis measurements). Zeta limits ranged from – 200 mV to + 200 mV. Zeta potential was also measured with the Horiba SZ-100 instrument ( Parmar et al., 2011).

Determination of Drug Entrapment Efficiency :

The drug entrapment efficiency of nanoparticles of Benazepril HCl was determined by using Centrifuge. 100mg of nanoparticles were suspended in 10 ml of Phosphate buffer pH 7.4 and centrifuged for 2 hrs at the speed of 2000 rpm and filtered using Whatmann’s filter paper. 1ml sample was withdrawn from the filtrate and dilutions were made and the drug content was analyzed by using a UV spectrophotometer at 241 nm. Percentage drug content and drug entrapment efficiency were calculated by using the following formula: ( Mainardes and Khalil, 2014)

Free Drug content = Absorbance / Slope × Dilution factor /100

Scanning Electron Microscopy:

The shape and surface morphology of the optimized formulation of nanoparticles of Benazepril HCl were investigated and photographed with the help of Scanning Electron Microscopy.

In vitro Drug Release study:

The in vitro release test was carried out using USP type –I (rotating basket) dissolution apparatus (Tab machine dissolution system). A quantity of nanoparticles equivalent to a 20 mg dose of Benazepril HCl was filled into a capsule having size 3 and used for the dissolution test. The dissolution was performed under the following conditions, (Emami et al., 2014)

Apparatus                                            : USP type -I

Speed of basket                                            : 75 rpm

Volume of dissolution medium                     : 900ml

Dissolution medium use   : Phosphate buffer pH 7.4

Temperature of dissolution medium      : 37 ± 0.5⁰C

1 ml of an aliquot of the sample was withdrawn at a predetermined time interval and the dissolution medium was replaced immediately by an equivalent amount of fresh medium kept at the same condition, sample solution was filtered through filter paper having a pore size of 0.25µ. The filtrate was analyzed for absorbance by UV- visible spectrophotometer at 241 nm after diluting it from 1 to 10ml by using the same medium. Drug concentration in the sample was determined from the standard calibration curve. The dissolution test was carried out for 10 hrs and % cumulative drug release was calculated (Chourasiya et al., 2021).

Kinetics Studies

To study the release kinetics, data obtained from in-vitro release were plotted in various kinetic models (Supriya et al., 2018).

1. Zero Order Release Rate Kinetics:

To study the zero-order release kinetics the release rate data was fitted to the following equation,

C_t = C_o-K₀t

Where, Ct = Concentration of drug release

C0 = Initial amount of drug in the solution

K0 = Zero order rate constant (concentration/time)

t = Time (hrs.)

2. First Order Release Rate Kinetics:

To study the First order release kinetics the release rate data was fitted to the following equation,

Log Q_t = Log Q₀ – K₁t/2.303

 Where Q_t = Amount of drug dissolved in time t.

Q₀ = Initial amount of the drug in the solution

K₁ = First order release constant

3. Second Order Release Rate Kinetics:

To study the First order release kinetics the release rate data was fitted to the following equation,

1/Q_t = Kt + 1/Q₀

Where, Q_t = Amount of drug dissolved in time t.

Q₀ = Initial amount of the drug in the solution

K = second order release constant

4. Hixson-Crowell Cube Root Law Model:

To evaluate the drug release with changes in the surface area and the diameter of the particles/tablets, the data were also plotted in the Hixson-Crowell Cube Root Law,

W_o1/3 – W_t1/3 = K_hC t

Where, W₀ = Initial amount of the drug in the tablet.

W_t = Amount of drug remains in the dosage form

K_HC = Dissolution rate constant.

5. Korsmeyer and Peppas release model:

To study this model the release rate data are fitted to the following equation,

M_t / M_∞ = K.tⁿ

Where, M_t / M_∞ = the fraction of drug release

K = Release constant

t = Release time

 n = Diffusion coefficient for the drug release that is dependent on the shape of the matrix dosage form.

To characterize the release mechanism, the dissolution data (Mt /M∞<0.6) are evaluated. A plot of log (Mt / M∞ <0.6) versus log t will be linear with a slope of n and the intercept gives the values of log k. The antilog of log k gives the value of k. Log Cumulative percent drug release versus log time. Peppas used the n value in order to characterize different release mechanisms.

6. Higuchi Model:

Higuchi developed several theoretical models to study the release of water-soluble and low-soluble drugs incorporated in semisolids and/or solid matrices. Mathematical expressions were obtained for drug particles dispersed in a uniform matrix behaving as the diffusion media, and the equation is,

Q_t = K_H. t^1/2

Where, Q_t = Amount of drug released in time t

K_H = Higuchi Dissolution constant.

RESULTS

Characterization of Benazepril HCl

Physicochemical characterization and melting point determination

Benazepril HCl physicochemical properties have been studied. The substance was discovered to be white in color and smell-free. The typical drug’s melting point was discovered to be between 180-190⁰C . The manufactured drug formulation was in accordance with the USP specification as shown by the organoleptic properties and melting point being determined to be standard for drugs.

IR analysis of Benazepril HCl

FTIR of Benazepril HCl exhibited characteristic peaks of C=C stretching at wave number (cm−1 )1670.35, Primary or secondary OH in-plane bending peak at 1261.45, Aromatic C-H in-plane bending at 1246.02, Secondary amine CN stretching at 1184.29  (Figure 1).

Fig.1: FT-IR of Benazepril HCl

The purity and identification of Benazepril HCl were done by carrying out the thermal analysis of pure drug Benazepril HCl. The differential scanning Colorimetry for the pure drug Benazepril HCl was shown in the following Figure 2.

Fig 2 : DSC of Benazepril HCl

The drug Benazepril HCl used in the formulation has been scanned through the UV spectrum in Phosphate buffer pH 7.4  at 241 nm which showed maximum absorption at this wavelength (figure 3).

Fig 3: Spectrum of Benazepril HCl

Standard calibration curve of Benazepril HCl in ethanol and phosphate buffer pH 7.4. Standard calibration curve of Benazepril HCl in ethanol and phosphate buffer pH 7.4 have been developed at different concentrations of 10-100 µg/ml and 1-10 µg/ml  (Figures 4 and 5). A straight line has been drawn after taking the absorbances of different concentrations at 241 nm for the type of samples. The straight-line formulas for ethanol and phosphate buffer pH 7.4 were y=0.0189x+0.0119 (R2=0.999) and y=0.0337x+0.0074(R2=0.9982), respectively

Fig 4: Calibration curve of Benazepril HCl in Ethanol

Fig 5: Calibration curve of Benazepril HCl in Phosphate Buffer pH 7.4

The percentage yield of F1, F2, F3 and F4 were found to be 60.45%, 59.70%, 31.43%, and 66.44% respectively and illustrated below in Fig.6.

Fig 6: Graphical representation of percentage yield of formulated nanoparticles of Benazepril HCl

Fig 7: Graphical representation of particle size of formulated nanoparticles of Benazepril HCl

Fig 8: Practical size of optimized batch

Fig 9: Zeta potential of optimized batch

Fig 10: Graphical representation of % entrapment efficiency of formulated nanoparticles of Benazepril HCl

Fig 11: SEM images of formulation of Benazepril HCl nanoparticles

Fig 12: In vitro drug release study

Table no. 2: Results of drug release kinetic study