**3. Results and discussion**

#### **3.1 Method development and release profile**

In order to obtain insight into the binding interaction of RxAcNPs with Lyz, the drug content, encapsulation efficiency (EE), and releasing percentage of RxAc were determined utilizing spectrophotometric techniques, with the mole ratio 5:1:1 of Cs, TPP, and Tween80, respectively. The maximum absorption wavelength was found to be 275 nm for RxAc. The drug content and encapsulation efficiency of RxAc in CsNPs based on the preparation of formulation are represented in **Table 2**. As listed in **Table 2**, the results displayed high encapsulation efficiency, which was 88.25 0.26%, and the total content of drug in the nanoform was 26.48 0.17 mg, which was nearly the total content of drug used in the preparation of the formulation matrix. These results revealed that the developed method is reliable and accurate to estimate the content of drug without interference of the formulation matrix or excipients. Additionally, it has the possibility to estimate the content of drug in the complex nanocarriers-based formulation.

The RxAc release profile from RxAc-loaded Tween80–Chitosan nanoparticles at different values of pH is illustrated in **Figure 2**. The drug released from the nanoparticles was little during the initial 2 hours (less than 25%). After 2 hours, the quantity of the released drug increased with time. The RxAc percentages released at the end of 24 hours. were 90.21 0.73, 85.83 0.54, 82.79 0.34, and 75.01 0.57% for pH 3.5, 6.6, 7.4, and 8.4, respectively (**Table 3** and **Figure 2**). Furthermore, as the pH decreased, the amount of released drug increased, showing

*Influence of Tween 80 Surfactant on the Binding of Roxatidine Acetate and Roxatidine… DOI: http://dx.doi.org/10.5772/intechopen.100734*


**Table 2.**

*Nanoparticle sizes and mass balance of the Roxatidine acetate used in nanoparticles formulation.*

**Figure 2.** *In vitro of RxAcNPs release profile at different pH values.*

that the drug release depends upon the pH of the media, as well as the nature of the polymer matrix [33, 34], which means that the developed method is suitable and effective for preparing the antiulcer drugs in nanoform.

The nanoparticles resulting from this developed method were used to investigate the applicability in simulation of studies of drug nanoparticles–protein interaction. The known concentrations (0–16 μM) of the RxAcNPs solution were added to the fixed concentration of Lyz (10 μM) to examine the binding interaction under physiological conditions.

#### **3.2 Characterization of Roxatidine acetate–loaded Tween80–chitosan nanoparticles**

Fourier transform infrared (FTIR) spectra of the CsNPs and RxAcNPs are shown in **Figure 3A**. Chitosan is known to possess amine groups on the glucosamine moiety whereas Roxatidine acetate is an amphoteric drug having hydrophobic and hydrophilic moieties (dOH, CO, and dNH groups). The characteristic absorption bands for Chitosan were observed at 1650, 1545 and 1420 cm<sup>1</sup> were corresponding to amide I, amide II and amide III, respectively. 1095 cm<sup>1</sup> was corresponding to CdN stretching, and 2936 cm<sup>1</sup> was corresponding to the asymmetric stretching vibration of methylene and 3350 cm<sup>1</sup> was due to the stretching vibration of NdH. The FTIR spectra of RxAcNPs were compared with the FTIR spectra of CsNPs. The spectra did not show any new band for characteristic peaks of RxAc in RxAcNPs spectra and the existing shift of bands indicating entrapment of RxAc within the chitosan matrix, suggesting no new chemical bond formation between RxAc and CsNPs. Consequently, this observation excluded the possibility of an interaction between the polymer and drug indicating


**Table 3.**

*Mass balance of Roxatidine acetate used in vitro release study at deferent pH.*

#### **Figure 3.**

*(a) FTIR spectra for Roxatidine acetate (RxAc), Chitosan nanoparticles (CsNPs), Roxatidine acetate loaded Chitosan nanoparticles (RxAcNPs), (b) PXRD spectra for Roxatidine acetate (RxAc), Chitosan nanoparticles (CsNPs), Roxatidine acetate loaded Chitosan nanoparticles (RxAcNPs).*

that RxAc was physically dispersed in the polymer [33–38]. As shown in **Figure 3B**, RxAc-loaded Tween80–Chitosan nanoparticles were examined using the PXRD technique. The peak at 11.72° represents the presence of Cs and at 17.65°, the presence of TPP is indicated. A synthesized nanoform was specified, illustrating the semicrystalline nature of RxAcNPs after the available analysis, which depends on the little sharp pattern of XRD; hence, the drug was just encapsulated in Tween80–Cs nanoparticles without any interaction. The peaks at 24.18° and 27.21° indicated the presence of RxAc [39–45]. As shown in **Figure 4A** and **Table 2**, the data of DLS showed that the particle size of RxAcNPs was 220 5 nm, which was almost in conformity with the data of SEM as shown in **Figure 4B**. The SEM micrograph of RxAcNPs clearly illustrates the presence of RxAc on the Chitosan surface, which clarifies the drug encapsulation in the nanoparticle surface. The data of SEM of Tw80–CsNPs before and after loading RxAc illustrated that the spherical shape of the nanoparticles of Tw80–CsNPs is slightly deformed because of the loading of RxAc, as shown in **Figure 4B**.

#### **3.3 Analysis of RxAc and RxAcNPs with Lyz**

#### *3.3.1 Fluorescence spectroscopy*

Fluorescence quenching of lysozyme is broadly used in measuring the binding affinity of protein and drug. Lyz has three main Trp residues located at its active

*Influence of Tween 80 Surfactant on the Binding of Roxatidine Acetate and Roxatidine… DOI: http://dx.doi.org/10.5772/intechopen.100734*

**Figure 4.** *(a) Particle size distribution of RxAcNPs, (b) SEM image of RxAc-loaded Chitosan nanoparticles (RxAcNPs).*

**Figure 5.**

*Fluorescence emission spectra of Lyz in the presence of (a) RxAc and (b) RxAcNPs at 298 K. CLyz : 10μM (a), CRxAc or RxAcNPs (b-i): 2, 4, 6, 8, 10, 12, 14 and 16 μM; native RxAc or RxAcNPs (j): 2 μM.*

binding site, i.e., Trp-62, Trp-63, and Trp-108. The intrinsic fluorescence of Lyz comes from tryptophan residues (Trp-62, Trp-63, and Trp-108) and to study the conformational changes of Lyz in the binding process of Lyz with drugs used to be a fluorescent probe [4, 46]. The effects of RxAc and RxAcNPs on Lyz fluorescence intensity are shown in **Figure 5A** and **B**, respectively. After being excited with a wavelength of 280 nm, Lyz has a fluorescence emission with a peak at 337 nm; the fluorescent intensity of Lyz decreased regularly with increasing concentrations of RxAc and RxAcNPs. Interestingly, a red shift of about 6 nm and 4 nm in the λmax were observed in Lyz-RxAc and Lyz-RxAcNPs systems, respectively. Moreover, 78% of the fluorescence emission was quenched by RxAc in case of the Lyz–RxAc system whereas 77% quenching of the emission was observed in case of the Lyz– RxAcNPs system, which sketches a picture as to how the quencher RxAc and RxAcNPs ingress the fluorophore and bring about the quenching. Further, it suggests a change in the surrounding environment of the fluorophores due to interaction with RxAc and RxAcNPs and that the binding regions of RxAc and RxAcNPs are in the vicinity of Trp residues. Considering the above observations, it could be

adjudged that RxAc and RxAcNPs bind to Lyz and quench its intrinsic fluorescence. The red shift in the λmax in Lyz–RxAc and Lyz–RxAcNPs systems indicated an increase in polarity and a decrease in hydrophobicity [47–49].

As we know well, the phenomena of fluorescence quenching are brought about by various intermolecular episodes, namely excited-state reactions, ground-state complex formation, energy-transfer molecular rearrangements, and collisional quenching [50–52]. There are two types of quenching that are Static quenching and dynamic quenching. In static quenching, a nonfluorescent fluorophore-quencher complex is formed, whereas in dynamic quenching, collision between the quencher and fluorophore during the lifetime of the excited state is established. The two types of quenching can be distinguished from each other by taking viscosity and temperature-dependent measurements [53]. In the present systems, the fluorescence-quenching mechanism has been studied using the well-known Stern–Volmer (S–V) Equation [48, 53, 54]:

$$\frac{F\_0}{F} = \mathbf{1} + K\_{sv}[\mathbf{Q}] = \mathbf{1} + K\_q \tau\_0[\mathbf{Q}] \tag{3}$$

where F0 and F are the protein fluorescence intensities in the absence and in the presence of the drug molecule (quencher), respectively, Ksv is the constant of Stern–Volmer quenching, [Q] is the concentration of the quencher, Kq is the quenching rate constant of the biomolecule, and τ<sup>0</sup> is the biomolecule average lifetime in absence of the quencher. A single type of quenching mechanism, either static or dynamic quenching mechanism, is included, when the plot of F0/F vs. [Q] is linear, whereas deviation from linearity suggests the presence of both quenching mechanisms. The value of Ksv is estimated from the plot of F0/F vs. [Q]. Considering the well-known connection between the quenching constant and the Kq quenching rate constant, and taking into account the fluorescence lifetime of the biopolymer as 10�<sup>8</sup> s, the Kq values can be calculated [9, 19, 55]:

$$K\_q = \frac{K\_{sv}}{\tau\_0} \tag{4}$$

**Figure 6A** and **B** show the plots of F0/F for Lyz versus [Q] of RxAc and RxAcNPs at 298, 304, and 310 K and pH 7.4, where [Q] ranges from 2 to 16 μM of RxAc and RxAcNPs. Plots in **Figure 6A** and **B** show that the results of Lyz–RxAc and Lyz–RxAcNPs systems agree very well with the Stern–Volmer equation, which indicates that a single type of quenching mechanism is involved, either static or dynamic [56–59]. The results listed in **Table 4** showed that KSV and Kq values of Lyz–RxAc and Lyz–RxAcNPs decreased upon increasing temperature and that the quenching of both systems follows the static quenching mechanism [53, 60]. The maximum scatter collision quenching constant (Kq) with the biopolymer is <sup>2</sup> � <sup>10</sup><sup>10</sup> L mol�<sup>1</sup> <sup>s</sup> �1 . The values of Kq of the protein quenching initiated by RxAc and RxAcNPs are greater than the constant of maximum scatter collision quenching, thus indicating that quenching is initiated from the formation of complex and not the dynamic collision [53].

#### *3.3.2 Binding interaction analysis*

The constant of binding (Ka) and the number of binding sites (n) of the interaction between RxAc/RxAcNPs and Lyz could be investigated from the logarithmic form of the Stern–Volmer equation: [48, 52, 53]

*Influence of Tween 80 Surfactant on the Binding of Roxatidine Acetate and Roxatidine… DOI: http://dx.doi.org/10.5772/intechopen.100734*

**Figure 6.** *Stern-Volmer plots for quenching of Lyz fluorescence by (A) RxAc (B) RxAcNPs at different temperatures.*


*R\*\* is the correlation coefficient of KSV*

#### **Table 4.**

*Quenching parameters of Lyz-RxAc and Lyz-RxAcNPs systems at different temperatures.*

$$\log \frac{F\_0 - F}{F} = \log K\_a + n \, \log \left[ Q \right] \tag{5}$$

From the plot of Log[(F0 – F)/F] vs. log [Q], the binding constant (Ka) and the number of binding sites (n) could be obtained, where the intercept yields the value of the binding constant (Ka) and the slope gives the number of binding sites (n) (listed in **Table 5**). The values of Ka were 10<sup>4</sup> L mol�<sup>1</sup> for Lyz–RxAc (**Figure 7A**) indicating a high affinity of the Lyz molecule for RxAc besides binding number up to 0.96; however, the binding affinity of Lyz for RxAcNPs (**Figure 7B**) was found lower, ranging up to the order of 10<sup>3</sup> L mol�<sup>1</sup> and binding number up to 0.91. All these results lead to the conclusion that binding is stronger between Lyz and RxAc than that between Lyz and RxAcNPs, which will definitely affect its free concentration and its bound concentration in the blood plasma [61, 62].

The drug bioavailabilities could be estimated from the binding affinity values. The nanoform of drug (RxAcNPs) has shown less binding affinity to Lyz, which


#### **Table 5.**

*Binding constant, number of binding sites and Thermodynamic parameters of Lyz-RxAc and Lyz-RxAcNPs systems at different temperatures.*

**Figure 7.** *Plots of log [(Fo-F)/F] versus log [Q] for (A) Lyz-RxAc and (B) Lyz RxAcNPs systems at different temperatures.*

indicates that the distribution and absorption of drug nanoparticles to various tissues will be higher, as the stability of the Lyz–RxAcNPs complex is lower compared to Lyz–RxAc complex [63, 64].

#### *3.3.3 The influence of Tween80 (Tw80) inclusion on the interaction of the Lyz*–*RxAc system*

The influence of Tween80 inclusion onto the interaction of Lyz–RxAc systems was studied by introducing Tween80 to the Lyz–RxAc system at room temperature (**Figure 8A** and **B**). The results of the Stern–Volmer constant (KSV) and binding constant (Ka) are shown in **Figures 9A,B** and **10A,B** and listed in **Tables 4** and **5**. We observed that the results of KSV and Ka in the presence of Tw80 were smaller than in its absence. These results indicated that the Tw80 helps to release RxAc from the Chitosan nanoparticles, due to a fraction of RxAc binding to it by weak

*Influence of Tween 80 Surfactant on the Binding of Roxatidine Acetate and Roxatidine… DOI: http://dx.doi.org/10.5772/intechopen.100734*

#### **Figure 8.**

*A,B: Fluorescence emission spectra of Lyz in the presence of RxAc and Tw80 at 298 K. CLyz: 10 μM (a), CTw80: 2 and 4 μM (b), CRxAc (c–k): 2, 4, 6, 8, 10, 12, 14 and 16 μM.*

**Figure 9.**

*A,B: Stern-Volmer plots for quenching of Lyz fluorescence by RxAc in the presence of Tw80 (2 and 4 μM) at 298 K.*

bonds; hence, Tw80 helps to release more drug to the tissues as compared to the drug released from plasma [65]. Furthermore, Tw80 encloses the RxAc molecule and obstructs it from colliding directly with the amino acid residues found in the binding sites of Lyz [66].

#### *3.3.4 The force acting between Lyz and RxAc/RxAcNPs*

The driving force of binding could be assessed from the thermodynamic law summarized by Ross and Subramanian. The stability of the protein–drug complex and the binding of drug onto protein are influenced by various types of noncovalent forces such as hydrophobic interactions, hydrogen binding, Van der Waals, and electrostatic forces. To get the thermodynamic parameters, the Van't Hoff equation has been used:

$$
\ln K\_a = \frac{-\Delta H^0}{RT} + \frac{\Delta S^0}{R} \tag{6}
$$

**Figure 10.** *A,B: Plots of log [(Fo-F)/F] versus log[Q] for Lyz-RxAc-Tw80 systems.*

$$
\Delta G^0 = -RT\ln K\_a = \Delta H^0 - T\Delta S^0 \tag{7}
$$

where Ka is the constant of binding at the corresponding temperature T, T is the absolute temperature, and R is the universal gas constant. The plot of lnKa versus 1/T allows the estimation of the enthalpy change (ΔH) and the entropy change (ΔS) [9, 67–69]. The enthalpy change (ΔH) and the entropy change (ΔS) can be obtained from the slope and the intercept of the Van't Hoff plots, respectively. From the thermodynamic viewpoint, Ross and Subramanian recommended that ΔH < 0 and ΔS < 0 suggest the van der Waals force and hydrogen bond formation, ΔH > 0 and ΔS > 0 show a hydrophobic interaction, and ΔH < 0 and ΔS > 0 propose electrostatic forces of interaction [53, 62–64].

As shown in **Figure 11A** and **B**, there is a good linear relationship between lnKa and 1/T, suggesting that ΔH is constant in the current temperature range. From **Table 5**, it could be seen that <sup>Δ</sup>H = � 71.80 kJ mol�<sup>1</sup> and <sup>Δ</sup>S = � 166.28 J mol�<sup>1</sup> <sup>K</sup>�<sup>1</sup> for the Lyz– RxAc system and <sup>Δ</sup>H = � 86.61 kJ mol�<sup>1</sup> <sup>K</sup>�<sup>1</sup> and <sup>Δ</sup>S = � 231.13 J mol�<sup>1</sup> <sup>K</sup>�<sup>1</sup> for the Lyz–RxAcNPs system. The negative values of ΔH and ΔS for interaction of Lyz with RxAc and Lyz with RxAcNPs indicate that hydrogen bonds and Van der Waals forces play a major role in the interaction of Lyz–RxAc and Lyz–RxAcNPs systems, and the binding reaction is exothermic and enthalpically driven. The negative values of ΔG for both systems at different temperatures (298, 304, and 310 K) mean that the binding processes are spontaneous in both systems.

#### *3.3.5 Fluorescence resonance energy transfer (FRET)*

Fluorescence resonance energy transfer is a nondestructive spectroscopic method and an investigatory tool that can monitor the proximity and relative angular orientation to study energy transfer from donor to acceptor. A transfer of energy could be carried out through direct electrodynamic interaction between the primarily excited molecule and its neighbors [70, 71]. The fluorophores of donor and acceptor can be entirely nonattached or attached to the same macromolecule [72]. In the present case, Lyz is the donor and RxAc and RxAcNPs are the acceptors. According to this theory, the efficiency (E) of energy transfer from Lyz to RxAc or RxAcNPs and the distance (r) of binding between Lyz and RxAc or RxAcNPs could be calculated by Eq. (8) [70, 73]:

*Influence of Tween 80 Surfactant on the Binding of Roxatidine Acetate and Roxatidine… DOI: http://dx.doi.org/10.5772/intechopen.100734*

**Figure 11.** *Vant-Huff Plot for (A) Lyz-RxAc and (B) Lyz-RxAcNPs systems at different temperatures.*

$$E = \frac{R\_0^6}{R\_0^6 + r^6} = \left| 1 - \frac{F}{F\_0} \right. \tag{8}$$

where E could be determined experimentally from the donor emission in the absence (F0) and presence of the acceptor (F), normalized to the same donor concentration, r is the actual distance between the donor (Lyz) and the acceptor (RxAc/RxAcNPs), R0 is the critical distance when the efficiency of transfer is 50%, which depends on the quantum yield of the donor, the extinction coefficient of the acceptor, the overlap of donor emission and acceptor absorption spectra, and the mutual orientation of the chromophores. R0 can be defined by Eq. (9) [70, 74]:

$$R\_0^6 = 8.8 \times 10^{-25} K^2 N^{-4} \Phi J \tag{9}$$

where K<sup>2</sup> is the spatial factor of orientation related to the geometry of the donor and acceptor of dipoles, N is the refractive index of the medium, Φ is the fluorescence quantum yield of the donor, and J is the effect of spectral overlap between the donor emission spectrum and the acceptor absorption spectrum, which could be calculated by Eq. (10)

$$J = \frac{\sum F(\lambda) \,\,\varepsilon(\lambda)\lambda^4 \Delta\lambda}{F(\lambda)\Delta\lambda} \tag{10}$$

where F(λ) is the donor fluorescence intensity at wavelength λ and ε(λ) is the molar absorption coefficient of the acceptor at wavelength λ. The efficiency of transfer (E) could be obtained using Eq.8, where F and F0 are the relative fluorescence intensities in the presence and absence of acceptor [56]. For Lyz, K<sup>2</sup> = 2/3, N = 1.36, and Φ = 0.15 [62, 75].

The overlap of the absorption spectrum of RxAc and RxAcNPs with the fluorescence emission spectrum of Lyz are shown in **Figure 12A** and **B**, in the wavelength range of 280–310 nm and 280–308 nm, respectively. The fluorescence emission from both systems at an excitation wavelength of 280 nm is mainly from the Lyz molecule as both RxAc and RxAcNPs are nonfluorescent at the excitation wavelength. However, at this excitation wavelength, RxAc and RxAcNPs do show weak

#### **Figure 12.**

*Spectral overlap between fluorescence emission spectrum of Lyz and absorption spectrum of (A) RxAc and (B) RxAcNPs when the molar ratio of Lyz and RxAc or RxAcNPs is 1:1. [Lyz]: 10 μM, [RxAc or RxAcNPs]: 2 μM at 298 K.*


#### **Table 6.**

*Energy transfer parameters for Lyz-RxAc and Lyz-RxAcNPs interactions at 298 K.*

absorption, which suggests the probability of energy transfer from Lyz to RxAc/ RxAcNPs. Using Eqs. (8)–(10), the parameters related to energy transfer from Lys to RxAc or RxAcNPs are calculated and are presented in **Table 6**. The values of R0, r, J, and E were found to be 3.40 nm and 4.66 nm, 6.67 <sup>10</sup><sup>14</sup> cm3 L mol<sup>1</sup> and 0.23 for Lyz–RxAc, whereas the corresponding values were 3.44 nm, 4.53 nm, 7.18 <sup>10</sup><sup>14</sup> cm3 L mol<sup>1</sup> and 0.24 for Lyz–RxAcNPs, respectively. The obtained result indicates that RxAc and RxAcNPs are strong quenchers and these may situate in the close proximity of Lyz. The values of binding distance (r) between the donor and acceptor for all the systems are in the range of 2–7 nm, denoting that the energy transfer is possible between Lys and RxAc or RxAcNPs. The values of R0 and r are also in the academic range, which proves that nonradiative energy transfer occurs between Lyz and RxAc/RxAcNPs. Furthermore, the results also suggest that static quenching is responsible for the quenching of fluorescence emission as the binding involved energy transfer from Lyz to RxAc/RxAcNPs [48, 59, 76, 77].

#### *3.3.6 Conformational changes of lysozyme*

Synchronous fluorescence spectroscopy is a kind of important method and a proficient technique, which is utilized to evaluate the conformational changes and provides the information regarding the molecular environment in the vicinage of the chromophore molecule [78, 79]. Because of its sensitivity, spectral simplification, spectral bandwidth reduction, and shunning of different perturbing effects, it can be used as an ideal and a very useful method to study the microenvironment of Trp residues by measuring the possible shift in wavelength emission maximum (λem) [48, 80]. The polarity changes around the chromophore molecule, i.e., the Lyz conformation, may be due to the shift in the position of emission maximum.

*Influence of Tween 80 Surfactant on the Binding of Roxatidine Acetate and Roxatidine… DOI: http://dx.doi.org/10.5772/intechopen.100734*

#### **Figure 13.**

*Synchronous fluorescence spectrum of (A) Lyz-RxAc and (B) Lyz-RxAcNPs systems at 298 K : (Δλ = 15 nm), C(Lyz) = 10 μM; C(RxAc or RxAcNPs) (b-j): 2, 4, 6, 8, 10, 12, 14 and 16 μM.*

As is well-known, the spectra of synchronous fluorescence show Trp residues of Lyz at the wavelength interval (Δλ) of 60 nm, while at the wavelength interval (Δλ) of 15 nm, the spectra of synchronous fluorescence show Tyr residues of Lyz [81].

At Δλ = 15 nm, in the Lyz–RxAc and Lyz–RxAcNPs systems in the investigated concentration range, the maximum emission wavelength keeps its position without any shift (**Figure 13A** and **B**), which indicates that there is no change in the microenvironment of the Tyrosine residues in both systems, whereas over the investigated concentration range at Δλ = 60 nm, it can be seen that the maximum emission wavelength moderately shifts from 279 to 274 nm in the Lyz–RxAc system and from 279 to 275 nm in the Lyz–RxAcNPs system toward blue wavelengths. On looking through the synchronous spectra for the Lyz–RxAc and Lyz–RxAcNPs systems (**Figure 14A** and **B**), the shift effect shows that the conformation of Lyz has changed. The blue-shift effect indicates that the microenvironment around the Tryptophan residues is disturbed and shows a decrease in the polarity and an increase in the hydrophobicity around Tryptophan residues.

#### *3.3.7 UV–vis absorbance spectroscopy*

UV–Vis spectroscopy is a simple technique and an effective method that can help to know the structural changes in the system and to explore the formation of complex and the change in hydrophobicity [82].

In the present study, we have observed the change in the UV absorption spectra of Lyz–RxAc and Lyz–RxAcNPs systems (**Figure 15A** and **B**), which indicated that the interaction between Lyz and RxAc/RxAcNP molecules may lead to change in the conformation of Lyz. It was evident that the UV absorption intensity of Lyz increased regularly with the variation of RxAc and RxAcNP concentrations. The maximum peak positions of Lyz–RxAc and Lyz–RxAcNPs were shifted slightly toward a longer wavelength region (279–284 nm and 279–283 nm, respectively). The change in λmax is observed possibly due to complex formation between Lyz and RxAc/RxAcNPs. The red shift in the absorbance spectra also indicated that the polarity of amino acid microenvironments increased with the addition of RxAc or RxAcNPs [83–86], which is in good agreement with the quenching and Synchronous fluorescence spectroscopy and thermodynamic analysis results.

#### **Figure 14.**

*Synchronous fluorescence spectrum of (A) Lyz-RxAc and (B) Lyz-RxAcNPs systems at 298 K : (Δλ = 60 nm), C(Lyz) = 10 μM; C(RxAc or RxAcNPs) (b-j): 2, 4, 6, 8, 10, 12, 14 and 16 μM.*

**Figure 15.**

*UV–Vis spectra of Lyz in the presence of (A) RxAc and (B) RxAcNPs at 298 K. CLyz: 10 μM (a), CRxAc or RxAcNPs (b-j): 2, 4, 6, 8, 10, 12, 14 and 16 μM.*

#### *3.3.8 Circular dichroism spectroscopy*

The technique of far-UV Circular dichroism spectroscopy (CD) is an important and powerful technology technique utilized to probe the secondary and tertiary structures of the protein/biopolymer [87–89]. The method is used to explore the biopolymer conformational changes upon binding of RxAc and RxAcNPs to Lyz, due to its simplicity and reliability. The CD spectra of Lyz with various concentrations of RxAc and RxAcNPs have been shown in **Figure 16A** and **B** at room temperature. The results of CD spectra of Lyz show two negative bands at 208 nm (π ! π\* transition) and 229 nm (n ! π\* transition), which are attributed to the α-helical structure of protein [67] whose magnitude reveals the amount of α-helicity in Lysozyme and they arise due to π–π\* and n–π\* transitions of the peptide bond of α-helix [84, 89–91]. The CD data have been observed in terms of mean residue ellipticity (MRE) in deg cm�<sup>2</sup> dmol�<sup>1</sup> according to the following Equation [92–97]:

*Influence of Tween 80 Surfactant on the Binding of Roxatidine Acetate and Roxatidine… DOI: http://dx.doi.org/10.5772/intechopen.100734*

**Figure 16.**

*The CD spectra of (A) Lyz-RxAc and (B) Lyz-RxAcNPs systems. Lyz concentration was kept fixed at 10 μM (a). In Lyz-RxAc and Lyz-RxAcNPs systems, RxAc or RxAcNPs concentration was fixed at 40 (b) and 80 μM (c).*

$$MRE = \frac{obsCD(m \text{ deg})}{Cp \times n \times l \times 10} \tag{11}$$

where Cp is the molar concentration of protein, n is the number of amino acid residues of the protein (129 for Lyz), and l is the path length in cm. The α-helical content of Lyz is calculated from the MRE value at 208 nm, using the following Equation [92–97]:

$$a-helix\left(\%\right) = \frac{-MRE\_{208} - 4000}{\\$3,000 - 4000} \times 100\tag{12}$$

where MRE208 is the observed mean residue ellipticity (MRE) value at 208 nm, 4000 is the MRE of the β-form and random coil conformation cross at 208 nm, and 33,000 is the MRE value of a pure α-helix at 208 nm.

In order to study the influence of RxAc and RxAcNPs on the secondary structure of the Lyz, the CD measurements of Lyz in the absence and presence of RxAc and RxAcNPs were performed. From **Figure 16A,B** and **Table 7**, the α-helicity for free Lyz was 43.34%, while the addition of RxAc and RxAcNPs to the Lyz solution caused an increase in the negative peak ellipticities, probably as a consequence of the formation of complex between Lyz and RxAc/RxAcNPs. The CD data in the


**Table 7.** *α-helicity (%) of Lyz at different concentrations of RxAc and RxAcNPs at 298 K.* wavelength range of 200–250 nm are used to evaluate the change of the secondary structure in Lyz. The results showed that interaction with RxAc and RxAcNPs caused only an increase in the band intensity of Lyz without any significant shift of the peaks and the helical content decreased to 38.73% and 31.23%, respectively. The decrease in the α-helical content of lysozyme represents the unfolding of protein due to interaction with RxAc or RxAcNPs. The unfolding of protein changes the absorbance value, which in turn alters the ellipticity value. The results showed that Lyz was induced to adopt a more loose conformation of the extended polypeptide. The conformational transition probably resulted in the exposure of the hydrophobic cavities to more hydrophilic environment, which is favorable for the interaction between Lyz and RxAc or RxAcNPs. The CD results also corroborate the conclusion of fluorescence and UV studies [77, 98].
