**2. Materials and methods**

### **2.1 Materials**

Atenolol was supplied as a gift sample by Haustus Biotech Pvt. Ltd., Himachal Pradesh, India, and sodium lauryl sulfate, manufactured by Krishna Drug and chemical Pvt. Ltd., Gujrat, was supplied by Mahalakshmi Chemicals Ltd., Greater Noida, India. Triple distilled water was used throughout the experiments. All other chemicals were of reagent grade and used without further purification.

### **2.2 Preparation of atenolol nanocrystals**

Atenolol nanocrystals were prepared by high speed homogenization process using sodium lauryl sulfate as a surfactant [15–17]. The nanocrystals were prepared by adding the different compositions of the surfactant sodium lauryl sulfate and stabilizer (PVP K 30) as mentioned in **Table 1**. Atenolol (1000 mg) was dissolved in


**77**

axis [18].

*A Facile Method for Formulation of Atenolol Nanocrystal Drug with Enhanced Bioavailability*

solvent. Nanocrystals were collected and evaluated as required.

**2.3 Formulation of capsule dosage of atenolol nanocrystals**

80 ml of distilled water with sodium lauryl sulfate which resulted in a solution of 1 g concentration. The whole procedure was operated at 25 ± 2°C. The solution of drug and surfactant was placed under a high-speed homogenizer (T 25 digital ULTRA-TURRAX IKAR Werke Staufen/Germany) at different speeds (15,000–25,000 rpm) for 70 h. The resulting solution was placed in a tray dryer at 60°C to evaporate the

Atenolol nanocrystals were mixed with the same ratio of lactose powder, which shows no incompatibility with the drug. The mixture of 1:1 ratio of drug (atenolol nanocrystals) and lactose was prepared. Then, 100 mg of this mixture was filled

The nanocrystals of atenolol prepared by the abovementioned method was

Particle size of the prepared nanocrystals was determined using particle size analyzer (Malvern Instruments Ltd.). The prepared nanocrystals were dispersed in dimethyl sulfoxide and placed in cuvettes and the particle size in terms of average diameter (davg) was determined. Zeta potential was calculated by using Zetasizer

The morphology of the atenolol nanocrystals was examined by scanning electron microscopy (JSM 6390 India). The sample was mounted on to an aluminum stub and sputter coated for 120 s with platinum particles in an argon atmosphere. The coated samples were then scanned and images were analyzed at 500 or 1000

FTIR analysis of pure atenolol, mixture of atenolol and SLS and obtained

pellets using FTIR spectrophotometer (Perkin-Elmer BX II). The observed peaks

The crystallinities of atenolol and atenolol nanocrystals were evaluated by XRD measurement using an X-ray diffractometer (Bruker AXS, 08 Advance). All samples

For any formulation, it is always desirable to have a better production yield so that industrial production becomes feasible not only in terms of cost but also in

were measured in the 2θ angle range between 3 and 80° and 0.010 step sizes.

as thin KBr

nanocrystals and lactose was performed in the range of 4000–500 cm<sup>−</sup><sup>1</sup>

*DOI: http://dx.doi.org/10.5772/intechopen.88191*

**3. Characterization of nanocrystals**

characterized by the following techniques.

**3.1 Particle size and zeta potential analysis**

ZS 90 (Malvern Instrument Ltd. India) [18].

**3.3 Fourier-transform infrared (FTIR) analysis**

**3.2 Scanning electron microscopy**

were reported for functional groups.

**3.4 X-ray diffraction study (XRD)**

**3.5 Percentage yield of production**

into the capsules.

**Table 1.**

*Formulation composition of atenolol nanocrystal.*

*A Facile Method for Formulation of Atenolol Nanocrystal Drug with Enhanced Bioavailability DOI: http://dx.doi.org/10.5772/intechopen.88191*

80 ml of distilled water with sodium lauryl sulfate which resulted in a solution of 1 g concentration. The whole procedure was operated at 25 ± 2°C. The solution of drug and surfactant was placed under a high-speed homogenizer (T 25 digital ULTRA-TURRAX IKAR Werke Staufen/Germany) at different speeds (15,000–25,000 rpm) for 70 h. The resulting solution was placed in a tray dryer at 60°C to evaporate the solvent. Nanocrystals were collected and evaluated as required.

#### **2.3 Formulation of capsule dosage of atenolol nanocrystals**

Atenolol nanocrystals were mixed with the same ratio of lactose powder, which shows no incompatibility with the drug. The mixture of 1:1 ratio of drug (atenolol nanocrystals) and lactose was prepared. Then, 100 mg of this mixture was filled into the capsules.

## **3. Characterization of nanocrystals**

*Nanocrystalline Materials*

after storage [14].

**2.1 Materials**

**Formulation code**

**2. Materials and methods**

**2.2 Preparation of atenolol nanocrystals**

**Drug: surfactant**

*Formulation composition of atenolol nanocrystal.*

suffers from poor bioavailability after oral dosing due to stumpy permeability through GIT [6]. Approximately 50% of an oral dose is absorbed from the gastrointestinal tract, the remainder being excreted unchanged in the feces. Researchers have been endeavored to increase its permeability and bioavailability by different techniques including osmotic pump, cyclodextrin-based delivery systems, hydro-

In the present study, we had prepared nanocrystals of atenolol to improve its permeability and modify its solubility, because this method is less time-consuming, required no organic solvents or harsh chemicals like other nanodelivery systems, has a high product yield, has good product stability, and is cheap. High pressure homogenization method was employed to prepare nanocrystals [13]. In this method, high pressure was applied on liquid suspension to force it through a gap or narrow channel inside a pipe. Here, the medium was aqueous containing a hydrophilic surfactant SLS to prevent agglomeration of suspended particles and thus it helped in stabilization. The surfactant used in the study also prevented crystal growth (Ostwald ripening) that could change the dissolution and bioavailability of the drug

Atenolol was supplied as a gift sample by Haustus Biotech Pvt. Ltd., Himachal Pradesh, India, and sodium lauryl sulfate, manufactured by Krishna Drug and chemical Pvt. Ltd., Gujrat, was supplied by Mahalakshmi Chemicals Ltd., Greater Noida, India. Triple distilled water was used throughout the experiments. All other

Atenolol nanocrystals were prepared by high speed homogenization process using sodium lauryl sulfate as a surfactant [15–17]. The nanocrystals were prepared by adding the different compositions of the surfactant sodium lauryl sulfate and stabilizer (PVP K 30) as mentioned in **Table 1**. Atenolol (1000 mg) was dissolved in

F1 2:1 20,000 312.7 ± 2.0 18 ± 0.2 82 ± 1.0 F2 4:1 20,000 296.7 ± 0.6 20 ± 0.4 88 ± 2.0 F3 4:3 20,000 416.2 ± 0.5 16 ± 0.1 84 ± 1.0 F4 2:1 25,000 210.4 ± 1.0 16.5 ± 0.3 72 ± 0.5 F5 4:1 25,000 125.6 ± 0.5 19 ± 0.2 90 ± 0.8 F6 4:3 25,000 552.6 ± 0.7 17 ± 0.3 88 ± 1.0 F7 2:1 15,000 652.6 ± 2.0 18 ± 0.7 64 ± 1.0 F8 4:1 15,000 620.0 ± 2.5 20 ± 0.4 66 ± 2.0 F9 4:3 15,000 590.0 ± 1.8 19 ± 0.5 64 ± 1.0

**Particle size (nm)**

**Zeta potential (mV)**

**Production yield**

chemicals were of reagent grade and used without further purification.

**Speed in rpm**

philic matrices, transdermal delivery systems, and so on [7–12].

**76**

**Table 1.**

The nanocrystals of atenolol prepared by the abovementioned method was characterized by the following techniques.

#### **3.1 Particle size and zeta potential analysis**

Particle size of the prepared nanocrystals was determined using particle size analyzer (Malvern Instruments Ltd.). The prepared nanocrystals were dispersed in dimethyl sulfoxide and placed in cuvettes and the particle size in terms of average diameter (davg) was determined. Zeta potential was calculated by using Zetasizer ZS 90 (Malvern Instrument Ltd. India) [18].

#### **3.2 Scanning electron microscopy**

The morphology of the atenolol nanocrystals was examined by scanning electron microscopy (JSM 6390 India). The sample was mounted on to an aluminum stub and sputter coated for 120 s with platinum particles in an argon atmosphere. The coated samples were then scanned and images were analyzed at 500 or 1000 axis [18].

#### **3.3 Fourier-transform infrared (FTIR) analysis**

FTIR analysis of pure atenolol, mixture of atenolol and SLS and obtained nanocrystals and lactose was performed in the range of 4000–500 cm<sup>−</sup><sup>1</sup> as thin KBr pellets using FTIR spectrophotometer (Perkin-Elmer BX II). The observed peaks were reported for functional groups.

#### **3.4 X-ray diffraction study (XRD)**

The crystallinities of atenolol and atenolol nanocrystals were evaluated by XRD measurement using an X-ray diffractometer (Bruker AXS, 08 Advance). All samples were measured in the 2θ angle range between 3 and 80° and 0.010 step sizes.

#### **3.5 Percentage yield of production**

For any formulation, it is always desirable to have a better production yield so that industrial production becomes feasible not only in terms of cost but also in

terms of environmental protection. The production yield of prepared nanocrystals was calculated by the following Eq. (1):

$$Percentage\ yield = (B/A) \times 100\tag{1}$$

where B is the weight percentage of the final product obtained after drying, and A is the initial total amount of atenolol and sodium lauryl sulfate used for the preparation.

#### **3.6 In vitro release studies of atenolol nanocrystals**

The dissolution test was performed in the USP type II apparatus. Nanocrystals (100 mg) were accurately weighed and put into the pretreated dialysis membrane and sealed with clips. The release medium was phosphate buffer (pH 6.8) maintained at 37°C with agitation rate set at 50 rpm. The amount of drug was determined spectrophotometrically at λmax = 275 nm against suitable blank using a preconstructed calibration curve [19].

#### **3.7 In vitro release studies of capsule dosage form of atenolol nanocrystals**

The dissolution test was performed on the USP type I apparatus. Capsules containing nanocrystals (100 mg) were accurately placed into the basket of dissolution test apparatus. The release medium was phosphate buffer (pH 6.8) maintained at 37°C with agitation rate set at 50 rpm. The amount of drug released was determined spectrophotometrically at λmax = 275 nm.

#### **3.8 In vitro intestinal permeability studies of pure atenolol and atenolol nanocrystals**

The permeability studies of pure atenolol and atenolol nanocrystals were carried out using Franz diffusion cell. To check the intra duodenal permeability, the duodenal part of the small intestine was isolated from sacrificed goat and taken for the in vitro diffusion study. Then this tissue was thoroughly washed with cold Ringer's solution to remove the mucous and lumen contents. The sample solutions were injected into the lumen of the duodenum using a syringe, and the two sides of the intestine were tightly closed. Then the tissue was placed in a chamber of organ bath with continuous aeration and at a constant temperature of 37°C. The receiver compartment was filled with 30 mL of phosphate-buffered saline (pH 5.5). The permeability was tested for 60 minutes. The absorbance was measured using a UV-Vis spectrophotometer at a wavelength of 275 nm, keeping the respective blank. The percent diffusion of the drug was calculated against time and plotted on a graph.

#### **3.9 Stability studies of prepared nanocrystals**

The prepared nanocrystals were subjected to stability studies. The nanocrystals were placed in stability chambers for a month at different temperatures, like 4, 25, 37, and 60°C. After 1 month, the tested nanocrystals were subjected to FTIR to find the spectra and compare with the standard spectra of nanocrystals.

#### **3.10 In vivo studies**

To determine the in vivo pharmacokinetic parameters for optimized nanocrystal formulation, experimental rats were used. This investigation adhered to the Principles of Laboratory Animal Care. Female albino rats (0.20–0.25 Kg) were

**79**

**3.11 Statistical analysis**

**Table 2.**

*Design of experiment for 3<sup>2</sup>*

was considered significant at p ≤ 0.05.

were prepared with these variables.

**3.12 Experimental design and statistical analysis**

*A Facile Method for Formulation of Atenolol Nanocrystal Drug with Enhanced Bioavailability*

**Drug: surfactant (X1) Speed in rpm (X2)**

−1 1 0 1 1 1 −1 0 0 0 1 0 −1 −1 0 −1 1 −1 −1 = 2:1 −1 = 15,000 0 = 4:1 0 = 20,000 +1 = 4:3 +1 = 25,000

 *factorial analysis.*

divided in two groups, each containing six. They were fasted overnight and allowed to administer 0.5 mL aqueous dispersion of pure drug and the most successful formulation of nanocrystal (equivalent to 10 mg/mL atenolol) using oral feeding tube. Blood samples of 0.2 mL were withdrawn through the tail vein of rats after 0.5, 1, 1.5, 2, 2.5, 4, 6, and 24 h of sample administration. The withdrawn samples were centrifuged at 5000 rpm for 20 min. The plasma was separated and stored at −20°C until drug analysis was carried out using HPLC analytical method of analysis. The whole process was carried out according to the reported method by Anwar et al. [20].

Independent T-test was used to analyze data of two batches obtained in various experiments at the 0.05 level of significance by Origin 6.0 software. The difference

In this study, a 32 full factorial experimental design was introduced to optimize the formulation of nanoparticles. Initial studies were undertaken to decide on the factors and their levels in the experimental design. Based on the results obtained in preliminary experiments, surfactant amount and speed of homogenizer were found to be the major variables in determining the particle size and production yield. So, in this design, two factors, namely surfactant amount (X1) and speed of homogenizer (X2), were evaluated each at three levels and suitably coded (**Table 2**). The effect of these factors were evaluated on three dependent variables, namely particle size (Y1), zeta potential (Y2), and production yield (Y3). A total of 9 formulations

For the studied design, the multiple linear regression analysis (MLRA) method was applied using Statistica 10 (StatSoft Inc., USA) software to fit the full secondorder polynomial equation with added interaction terms. Polynomial regression

*Y* = *b*1 + *b*2*X*1 + *b*3*X*2 + *b*4*X*1*X*2 + *b*5*X*12 + *b*6*X*22 (2)

results were demonstrated for the studied responses using Eq. (2):

*DOI: http://dx.doi.org/10.5772/intechopen.88191*

*A Facile Method for Formulation of Atenolol Nanocrystal Drug with Enhanced Bioavailability DOI: http://dx.doi.org/10.5772/intechopen.88191*


#### **Table 2.**

*Nanocrystalline Materials*

was calculated by the following Eq. (1):

preconstructed calibration curve [19].

spectrophotometrically at λmax = 275 nm.

**3.9 Stability studies of prepared nanocrystals**

**nanocrystals**

**3.6 In vitro release studies of atenolol nanocrystals**

terms of environmental protection. The production yield of prepared nanocrystals

*Percentage yield* = (*B*/*A*) × 100 (1)

where B is the weight percentage of the final product obtained after drying, and A is the initial total amount of atenolol and sodium lauryl sulfate used for the preparation.

The dissolution test was performed in the USP type II apparatus. Nanocrystals

(100 mg) were accurately weighed and put into the pretreated dialysis membrane and sealed with clips. The release medium was phosphate buffer (pH 6.8) maintained at 37°C with agitation rate set at 50 rpm. The amount of drug was determined spectrophotometrically at λmax = 275 nm against suitable blank using a

**3.7 In vitro release studies of capsule dosage form of atenolol nanocrystals**

**3.8 In vitro intestinal permeability studies of pure atenolol and atenolol** 

The permeability studies of pure atenolol and atenolol nanocrystals were carried out using Franz diffusion cell. To check the intra duodenal permeability, the duodenal part of the small intestine was isolated from sacrificed goat and taken for the in vitro diffusion study. Then this tissue was thoroughly washed with cold Ringer's solution to remove the mucous and lumen contents. The sample solutions were injected into the lumen of the duodenum using a syringe, and the two sides of the intestine were tightly closed. Then the tissue was placed in a chamber of organ bath with continuous aeration and at a constant temperature of 37°C. The receiver compartment was filled with 30 mL of phosphate-buffered saline (pH 5.5). The permeability was tested for 60 minutes. The absorbance was measured using a UV-Vis spectrophotometer at a wavelength of 275 nm, keeping the respective blank. The percent diffusion of the drug was calculated against time and plotted on a graph.

The prepared nanocrystals were subjected to stability studies. The nanocrystals were placed in stability chambers for a month at different temperatures, like 4, 25, 37, and 60°C. After 1 month, the tested nanocrystals were subjected to FTIR to find

To determine the in vivo pharmacokinetic parameters for optimized nanocrystal formulation, experimental rats were used. This investigation adhered to the Principles of Laboratory Animal Care. Female albino rats (0.20–0.25 Kg) were

the spectra and compare with the standard spectra of nanocrystals.

The dissolution test was performed on the USP type I apparatus. Capsules containing nanocrystals (100 mg) were accurately placed into the basket of dissolution test apparatus. The release medium was phosphate buffer (pH 6.8) maintained at 37°C with agitation rate set at 50 rpm. The amount of drug released was determined

**78**

**3.10 In vivo studies**

*Design of experiment for 3<sup>2</sup> factorial analysis.*

divided in two groups, each containing six. They were fasted overnight and allowed to administer 0.5 mL aqueous dispersion of pure drug and the most successful formulation of nanocrystal (equivalent to 10 mg/mL atenolol) using oral feeding tube. Blood samples of 0.2 mL were withdrawn through the tail vein of rats after 0.5, 1, 1.5, 2, 2.5, 4, 6, and 24 h of sample administration. The withdrawn samples were centrifuged at 5000 rpm for 20 min. The plasma was separated and stored at −20°C until drug analysis was carried out using HPLC analytical method of analysis. The whole process was carried out according to the reported method by Anwar et al. [20].

#### **3.11 Statistical analysis**

Independent T-test was used to analyze data of two batches obtained in various experiments at the 0.05 level of significance by Origin 6.0 software. The difference was considered significant at p ≤ 0.05.

#### **3.12 Experimental design and statistical analysis**

In this study, a 32 full factorial experimental design was introduced to optimize the formulation of nanoparticles. Initial studies were undertaken to decide on the factors and their levels in the experimental design. Based on the results obtained in preliminary experiments, surfactant amount and speed of homogenizer were found to be the major variables in determining the particle size and production yield. So, in this design, two factors, namely surfactant amount (X1) and speed of homogenizer (X2), were evaluated each at three levels and suitably coded (**Table 2**). The effect of these factors were evaluated on three dependent variables, namely particle size (Y1), zeta potential (Y2), and production yield (Y3). A total of 9 formulations were prepared with these variables.

For the studied design, the multiple linear regression analysis (MLRA) method was applied using Statistica 10 (StatSoft Inc., USA) software to fit the full secondorder polynomial equation with added interaction terms. Polynomial regression results were demonstrated for the studied responses using Eq. (2):

$$Y = b\mathbf{1} + b\mathbf{2}X\mathbf{1} + b\mathbf{3}X\mathbf{2} + b\mathbf{4}X\mathbf{1}X\mathbf{2} + b\mathbf{5}X\mathbf{12} + b\mathbf{6}X\mathbf{2}\mathbf{2}\tag{2}$$

where Y is the dependent variable and b1 is the arithmetic mean response of the 9 trials. Coefficient b2 is the estimated coefficient for the factor X1, and coefficient b3 is the estimated coefficient for the factor X2. The main effects (X1 and X2) represent the average result of changing one factor at a time from its low to high value. The interaction terms (X1X2) show how the response changes when two factors interact. The polynomial terms (X12 and X22) are included to investigate nonlinearity. The values of correlation coefficients were set to be statistically significant at 95% confidential interval [21]. To analyze the significance level of all these data, ANOVA was used at 95% confidence interval at 0.05 significance level.
