**4. Results and discussion**

#### **4.1 Characterization of drug, excipients, and their interactions by FTIR**

FTIR spectroscopy was used to further characterize possible interactions between the drug and the excipients. The FTIR spectra of atenolol and sodium lauryl sulfate and also of the formulated nanocrystals and lactose were obtained at wavelength ranging from 4000 to 400 cm<sup>−</sup><sup>1</sup> . The spectra obtained from FTIR studies confirmed that there was no major shifting, as well as no loss of functional peaks between the spectra of pure atenolol and atenolol nanocrystals. Comparing the spectra of pure atenolol and atenolol nanocrystals, no difference was shown in the position and trend of the absorption bands. All the distinctive groups in the FTIR spectra of atenolol were found in all the spectra of atenolol nanocrystals, like the amide group (O〓C–NH2) extruding from the benzene ring. Apart from the amide functional group, the presence of the conjugating C〓C bond in the benzene ring, the methane (CH), methylene (CH2) methyl (CH3), and OH functional group were distinctly observed in the IR spectra (amide:1650 cm<sup>−</sup><sup>1</sup> ; CH:2880–2900 cm<sup>−</sup><sup>1</sup> ; CH2: 2916–2936 cm<sup>−</sup><sup>1</sup> ; CH3: 2850 cm<sup>−</sup><sup>1</sup> ; conjugating C〓C: 1640–1610 cm<sup>−</sup><sup>1</sup> ; OH:3200–3550 cm<sup>−</sup><sup>1</sup> ), thus providing evidence for the absence of any chemical incompatibility between SLS and atenolol.

#### **4.2 Particle size and zeta potential**

The particle size of the atenolol nanocrystal formulations shown in **Table 1** showed a narrow size distribution from 125 to 652 nm, where the intensity of 117.8 nm was 93% and that of 652.5 nm was only 7%. The effect of stirring speed had an enormous effect on particle size. Formulation prepared with 25,000 rpm had smaller size as compared to particle prepared with 15,000 rpm. Concentration of SLS also had an effect on the size distribution. Less concentration of SLS yielded smaller size particles. The formulated nanocrystals were positively charged (16–19 mV), which is desirable for good ocular interaction. Formulation F7 was not further considered due to its larger size (more than 650). This may be due to slow stirring speed.

#### **4.3 Production yield**

The date of percentage yield of the prepared nanocrystals (**Table 1**) showed that the atenolol-SLS nanocrystals (batch F5), prepared by drug:SLS ratio 4:1, had comparatively higher yield of production (90%). Stirring speed and concentration of SLS also had an effect on the production yield.

**81**

**Figure 1.**

*SEM images of atenolol nanocrystals.*

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

The powder X-ray diffractogram of pure atenolol powder from 5 to 50° 2θ showed numerous distinctive peaks at 2θ degree that indicated a high crystalline content. The samples were scanned for 2 θ values over a range from 5 to 50°C at a scan rate of 10°/min. The PXRD pattern of pure drug and atenolol nanocrystals were compared with regard to peak positions and relative intensities and presence and/or absence of peaks in certain regions. **Figure 2** represents the XRD photograph

The permeability study showed increased permeability, when the atenolol was converted into the nanocrystals. The diffusion study showed that the % permeability of nanocrystal formulations was much higher as compared to that of the pure drug. The formulation F5 showed the maximum % release of 90.88%, whereas the

The dissolution rate of pure atenolol was very poor and during a 120-min period, 51.64% of drug was released. The reason for the poor dissolution of pure drug could be poor wettability and poor solubility. In vitro release studies revealed that there was a marked increase in the dissolution rate of atenolol, in the range of 78.30–98.28%, from all nanocrystal formulation compared to pure atenolol. The results revealed that the nanocrystals with a ratio of drug to carrier, 4:1, were having a higher dissolution rate in comparison to all other ratios. This could be attributed to the hydrophilic character of the surfactant and to the amorphous state of the drug. Hence, the present study showed that nanocrystal formulation can

of different nanocrystal formulation and pure atenolol powder.

pure drug showed only 31.22% release **Figure 3**.

The SEM image, as shown in **Figure 1**, of the atenolol nanocrystals revealed that the particles were crystalline in shape. The average size of the atenolol nanocrystals was found to be less than 200 nm, which was further supported by the results of

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

**4.4 Scanning electron microscopy**

particle size analysis by Zetasizer.

**4.6 Permeability study**

**4.7 In vitro dissolution studies**

**4.5 X-ray diffraction (PXRD) studies**

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

#### **4.4 Scanning electron microscopy**

*Nanocrystalline Materials*

significance level.

CH2: 2916–2936 cm<sup>−</sup><sup>1</sup>

OH:3200–3550 cm<sup>−</sup><sup>1</sup>

stirring speed.

**4.3 Production yield**

**4. Results and discussion**

at wavelength ranging from 4000 to 400 cm<sup>−</sup><sup>1</sup>

incompatibility between SLS and atenolol.

of SLS also had an effect on the production yield.

**4.2 Particle size and zeta potential**

were distinctly observed in the IR spectra (amide:1650 cm<sup>−</sup><sup>1</sup>

; CH3: 2850 cm<sup>−</sup><sup>1</sup>

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

**4.1 Characterization of drug, excipients, and their interactions by FTIR**

FTIR spectroscopy was used to further characterize possible interactions between the drug and the excipients. The FTIR spectra of atenolol and sodium lauryl sulfate and also of the formulated nanocrystals and lactose were obtained

studies confirmed that there was no major shifting, as well as no loss of functional peaks between the spectra of pure atenolol and atenolol nanocrystals. Comparing the spectra of pure atenolol and atenolol nanocrystals, no difference was shown in the position and trend of the absorption bands. All the distinctive groups in the FTIR spectra of atenolol were found in all the spectra of atenolol nanocrystals, like the amide group (O〓C–NH2) extruding from the benzene ring. Apart from the amide functional group, the presence of the conjugating C〓C bond in the benzene ring, the methane (CH), methylene (CH2) methyl (CH3), and OH functional group

The particle size of the atenolol nanocrystal formulations shown in **Table 1** showed a narrow size distribution from 125 to 652 nm, where the intensity of 117.8 nm was 93% and that of 652.5 nm was only 7%. The effect of stirring speed had an enormous effect on particle size. Formulation prepared with 25,000 rpm had smaller size as compared to particle prepared with 15,000 rpm. Concentration of SLS also had an effect on the size distribution. Less concentration of SLS

yielded smaller size particles. The formulated nanocrystals were positively charged (16–19 mV), which is desirable for good ocular interaction. Formulation F7 was not further considered due to its larger size (more than 650). This may be due to slow

The date of percentage yield of the prepared nanocrystals (**Table 1**) showed that the atenolol-SLS nanocrystals (batch F5), prepared by drug:SLS ratio 4:1, had comparatively higher yield of production (90%). Stirring speed and concentration

. The spectra obtained from FTIR

; conjugating C〓C: 1640–1610 cm<sup>−</sup><sup>1</sup>

), thus providing evidence for the absence of any chemical

; CH:2880–2900 cm<sup>−</sup><sup>1</sup>

;

;

**80**

The SEM image, as shown in **Figure 1**, of the atenolol nanocrystals revealed that the particles were crystalline in shape. The average size of the atenolol nanocrystals was found to be less than 200 nm, which was further supported by the results of particle size analysis by Zetasizer.

### **4.5 X-ray diffraction (PXRD) studies**

The powder X-ray diffractogram of pure atenolol powder from 5 to 50° 2θ showed numerous distinctive peaks at 2θ degree that indicated a high crystalline content. The samples were scanned for 2 θ values over a range from 5 to 50°C at a scan rate of 10°/min. The PXRD pattern of pure drug and atenolol nanocrystals were compared with regard to peak positions and relative intensities and presence and/or absence of peaks in certain regions. **Figure 2** represents the XRD photograph of different nanocrystal formulation and pure atenolol powder.

### **4.6 Permeability study**

The permeability study showed increased permeability, when the atenolol was converted into the nanocrystals. The diffusion study showed that the % permeability of nanocrystal formulations was much higher as compared to that of the pure drug. The formulation F5 showed the maximum % release of 90.88%, whereas the pure drug showed only 31.22% release **Figure 3**.

### **4.7 In vitro dissolution studies**

The dissolution rate of pure atenolol was very poor and during a 120-min period, 51.64% of drug was released. The reason for the poor dissolution of pure drug could be poor wettability and poor solubility. In vitro release studies revealed that there was a marked increase in the dissolution rate of atenolol, in the range of 78.30–98.28%, from all nanocrystal formulation compared to pure atenolol. The results revealed that the nanocrystals with a ratio of drug to carrier, 4:1, were having a higher dissolution rate in comparison to all other ratios. This could be attributed to the hydrophilic character of the surfactant and to the amorphous state of the drug. Hence, the present study showed that nanocrystal formulation can

**Figure 1.** *SEM images of atenolol nanocrystals.*

**Figure 2.**

*XRD of atenolol nanocrystals F5 (A), atenolol pure drug (B), atenolol nanocrystals F4 (C), and nanocrystals F1 (D).*

be successfully used to enhance dissolution rate of poorly soluble drugs. **Figure 4** shows drug release profile of pure drug nanocrystal formulation [F5] and capsulated nanocrystals.

#### **4.8 Stability studies**

The physical appearance of the prepared nanocrystals after keeping them 1 month in stability chambers under various conditions was found to be white to off-white in color, odorless, and crystalline powder. FTIR spectroscopy was used to further characterize possible interactions between the drug and the excipients during the stability studies. There was no major change shown in the FTIR peaks. The prepared nanocrystal formulation was stable during the stability studies done for 1 month.

#### **4.9 In vitro study**

The results of in vivo study revealed an improvement in bioavailability of nanocrystal formulation; it was observed that after oral dosing of the drug and the equation 3, their

**83**

**Figure 3.**

**Figure 4.**

**4.10 Factorial analysis**

*of nanocrystals in phosphate buffer (pH 6.8).*

Applied 3<sup>2</sup>

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

individual kinetic curve exhibited double peaks. Thus, double peaks can be due to the existence of two absorption sites in the gut interrupted by a region of poor absorption [21]. A rapid attainment of peak plasma concentrations was observed that may be due to the burst release effect brought by the use of SLS for stabilization of nanocrystals. Same phenomenon was reported by Vergote et al. [22]. The AUC0–24 h, MRT, and Cpmax for test formulation were significantly higher at *p*¡0.05 compared to the drug (**Table 3**).

*In vivo dissolution cumulative of % release of atenolol pure drug, atenolol nanocrystals F5, and capsule dosage* 

*Permeability from Franz diffusion cell of atenolol pure drug and atenolol nanocrystal formulations.*

as well. Coefficient for more than one factor represents interaction of both factors.

factorial design yields coefficient for one factor and for two factors

*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*

**Figure 3.** *Permeability from Franz diffusion cell of atenolol pure drug and atenolol nanocrystal formulations.*

#### **Figure 4.**

*Nanocrystalline Materials*

**82**

lated nanocrystals.

**Figure 2.**

*F1 (D).*

**4.8 Stability studies**

**4.9 In vitro study**

be successfully used to enhance dissolution rate of poorly soluble drugs. **Figure 4** shows drug release profile of pure drug nanocrystal formulation [F5] and capsu-

*XRD of atenolol nanocrystals F5 (A), atenolol pure drug (B), atenolol nanocrystals F4 (C), and nanocrystals* 

in stability chambers under various conditions was found to be white to off-white in color, odorless, and crystalline powder. FTIR spectroscopy was used to further characterize possible interactions between the drug and the excipients during the stability studies. There was no major change shown in the FTIR peaks. The prepared nanocrystal formulation was stable during the stability studies done for 1 month.

The physical appearance of the prepared nanocrystals after keeping them 1 month

The results of in vivo study revealed an improvement in bioavailability of nanocrystal formulation; it was observed that after oral dosing of the drug and the equation 3, their

*In vivo dissolution cumulative of % release of atenolol pure drug, atenolol nanocrystals F5, and capsule dosage of nanocrystals in phosphate buffer (pH 6.8).*

individual kinetic curve exhibited double peaks. Thus, double peaks can be due to the existence of two absorption sites in the gut interrupted by a region of poor absorption [21]. A rapid attainment of peak plasma concentrations was observed that may be due to the burst release effect brought by the use of SLS for stabilization of nanocrystals. Same phenomenon was reported by Vergote et al. [22]. The AUC0–24 h, MRT, and Cpmax for test formulation were significantly higher at *p*¡0.05 compared to the drug (**Table 3**).

#### **4.10 Factorial analysis**

Applied 3<sup>2</sup> factorial design yields coefficient for one factor and for two factors as well. Coefficient for more than one factor represents interaction of both factors.


#### **Table 3.**

*Pharmacokinetic data of nanocrystal formulation and pure drug.*

Coefficient may be positive or negative for synergistic or antagonistic effect, respectively. The coefficients can be directly compared to assess the impact of factors on responses. Obtained polynomial Eqs. (3)–(5) for dependent variables are as follows:

$$\begin{array}{c} \text{Particles Size} = 4280.7153 - 6169.2601 \times \mathbf{x} - 0.2812 \times \mathbf{y} + 4894.7164\\ \text{ } \times \mathbf{x} \times \mathbf{x} + 0.1959 \times \mathbf{x} \times \mathbf{y} + 4.6667 E^{-8} \times \mathbf{y} \times \mathbf{y} \end{array} \tag{3}$$

$$\begin{array}{l}\text{Zeta potential} = \text{31.4304 - 43.3651 \times x - 0.0004 \times y + 67.3401} \\ \times \text{x} \times \text{x - 0.0004 \times x \times y + 1 \, E}^{-8} \times y \times y \end{array} \tag{4}$$

$$\begin{aligned} \text{Production Yield} &= -71.7445 - 370.4401 \times \mathbf{x} + 0.0192 \times \mathbf{y} + 606.0606 \\ &\times \mathbf{x} \times \mathbf{x} - 0.0009 \times \mathbf{x} \times \mathbf{y} - 4.2667 E^{-\top} \times \mathbf{y} \times \mathbf{y} \end{aligned} \tag{5}$$

Speed of homogenizer has a greater effect on the particle size (−0.2812), zeta potential (−0.0004), and production yield (0.0192), whereas amount of surfactant has a lesser effect on the production yield (−370.4401), zeta potential (−43.3651), and particle size (−669.2601).

It was clearly depicted from the magnitude of the coefficients that the amount of surfactant has a positive effect on all the three variables including particle size, zeta potential, and production yield; whereas, the magnitude of the coefficients for speed of homogenizer has an antagonistic effect on all the three variables.

Contour plots and surface plots as shown in **Figures 5** and **6** were plotted, which are very useful to study the interaction effects of the factors on the responses. The response surface depicts the effect of factor contributions at different levels on studied response. Three contour parameters were established for particle size. Drug entrapment and drug release percentage. The contour plots showed very clearly the relationship between the independent variables and the responses.

*P* value for the effect of drug surfactant ratio is statistically insignificant for particle size and production yield *p* has value greater that 0.05, but it is significant only for zeta potential (p = 0.009851) and particle size (p = 0.035269). Speed of homogenizer has an insignificant effect on zeta potential.

The goodness of fit of the R<sup>2</sup> model was checked by the determination coefficient (R<sup>2</sup> ). The values of the determination coefficients for particle size (R<sup>2</sup> = 0.78297), zeta potential (R2 = 0.80392), and production yield (R2 = 0.8988) indicated that over 95% of the total variations are explained by the model. The values of adjusted determination coefficients (adj R2 = 0.56594 for particle size, 0.60784 for zeta potential, and 0.79761 for production yield) are also very high (over 90% of the total variations), which indicates a high significance of the model.

A good way to check the model is to enter factor levels from the experimental design (observed response) and generate the predicted response. When we compare the predicted value with actual value, a discrepancy occurs which is called residual.

**85**

**Figure 7.**

**Figure 5.**

**Figure 6.**

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

*3D surface plots for the different variables (in order of particle size, production yield, and zeta potential).*

*3D contour plots for the different variables (in order of particle size, production yield, and zeta potential).*

*Plots of residuals for the three different variables (in order of particle size, production yield, and zeta potential).*

*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*

**Figure 5.** *3D surface plots for the different variables (in order of particle size, production yield, and zeta potential).*

#### **Figure 6.**

*Nanocrystalline Materials*

Nanocrystal formulation

as follows:

**Table 3.**

and particle size (−669.2601).

The goodness of fit of the R<sup>2</sup>

= 0.78297), zeta potential (R2

Coefficient may be positive or negative for synergistic or antagonistic effect, respectively. The coefficients can be directly compared to assess the impact of factors on responses. Obtained polynomial Eqs. (3)–(5) for dependent variables are

*Pharmacokinetic data of nanocrystal formulation and pure drug.*

**Formulation Cpmax (μg/mL) Tmax (hour) AUC0–24 (mAU) MRT (hour)** Pure drug 612.15 ± 10.6 5.2 ± 1.4 26927.8 ± 4.2 46.56 ± 3.1

*Particles Size* = 4280.7153 − 6169.2601 × x − 0.2812 × y + 4894.7164

*Zeta potential* = 31.4304 − 43.3651 × x − 0.0004 × y + 67.3401

*Production Yield* = −71.7445 − 370.4401 × x + 0.0192 × y + 606.0606

speed of homogenizer has an antagonistic effect on all the three variables. Contour plots and surface plots as shown in **Figures 5** and **6** were plotted, which are very useful to study the interaction effects of the factors on the responses. The response surface depicts the effect of factor contributions at different levels on studied response. Three contour parameters were established for particle size. Drug entrapment and drug release percentage. The contour plots showed very clearly the relationship between the independent variables and the

homogenizer has an insignificant effect on zeta potential.

values of adjusted determination coefficients (adj R2

Speed of homogenizer has a greater effect on the particle size (−0.2812), zeta potential (−0.0004), and production yield (0.0192), whereas amount of surfactant has a lesser effect on the production yield (−370.4401), zeta potential (−43.3651),

It was clearly depicted from the magnitude of the coefficients that the amount of surfactant has a positive effect on all the three variables including particle size, zeta potential, and production yield; whereas, the magnitude of the coefficients for

*P* value for the effect of drug surfactant ratio is statistically insignificant for particle size and production yield *p* has value greater that 0.05, but it is significant only for zeta potential (p = 0.009851) and particle size (p = 0.035269). Speed of

indicated that over 95% of the total variations are explained by the model. The

0.60784 for zeta potential, and 0.79761 for production yield) are also very high (over 90% of the total variations), which indicates a high significance of the model. A good way to check the model is to enter factor levels from the experimental design (observed response) and generate the predicted response. When we compare the predicted value with actual value, a discrepancy occurs which is called residual.

model was checked by the determination

= 0.80392), and production yield (R2

= 0.8988)

= 0.56594 for particle size,

). The values of the determination coefficients for particle size

× x × x + 0.1959 × x × y + 4.6667 *E*−8 × *y* × *y* (3)

957.51 ± 20.4 2.8 ± 0.5 75329.3 ± 6.3 84.64 ± 4.6

× x × x − 0.0004 × x × y + 1 *E*−8 × *y* × *y* (4)

× x × x − 0.0009 × x × y − 4.2667 *E*−7 × *y* × *y* (5)

**84**

(R<sup>2</sup>

responses.

coefficient (R<sup>2</sup>

*3D contour plots for the different variables (in order of particle size, production yield, and zeta potential).*

#### *Nanocrystalline Materials*

For statistical purposes, it is assumed that the residuals are normally distributed and independent with constant variance [21]. Residual versus predicted plots were constructed to check the statistical assumptions. In this experiment, there is no definite increase in residuals with predicted levels, which support the underlying statistical assumptions of constant variance. Moreover, the obtained plots of residuals that do not exhibit any systematic structure indicate that the model fits the data well (**Figure 7**). Plots of the residuals versus other predictor variables, or potential predictors that exhibit systematic structure indicate that the form of the function can be improved in some ways.
