**Abstract**

The drug binding to protein is an attractive research topic. In order to assess the release of RxAc-CsNPs and their binding with lysozyme under physiological conditions, nanocomposite materials based on chitosan (Cs) and Roxatidine acetate (RxAc) in the presence Tween 80 (Tw80) surfactant were developed. The addition of Tw80 to CsNPs increased RxAc release in vitro. In this work, Stern–Volmer plot and thermodynamic results indicated that the mechanism of Lyz with RxAc and Lyz with RxAc-CsNPs was static mechanism and the main forces in both systems were hydrogen bonding and Van der Waals forces, which indicated that the binding reaction in both systems is spontaneous, exothermic and enthalpically driven. Synchronous fluorescence and CD results indicated that the RxAc and RxAc-CsNPs cause change in the secondary construction of Lyz. It was also found that the addition of Tw80 affects the binding constant of drug with protein. Finally, the molecular docking results have also been in accordance with the results of other techniques. Hence, the developed RxAc loaded Chitosan nanoparticles could be used as an effective strategy for designing and application of the antiulcer drugs. Altogether, the present study can provide an important insight for the future designing of antiulcer drugs.

**Keywords:** Roxatidine acetate, lysozyme, tween 80, chitosan nanoparticles, spectroscopy, molecular docking

### **1. Introduction**

The interactions of proteins with chemicals have prompted increasing research interest in recent years. Proteins are remarkable biomolecules presenting different functions and roles. Some are specific to their biological actions whereas some are selective toward the binding site [1, 2]. Conformational changes of protein may influence its transportation, function, assembly, potential cytotoxicity, and tendency to aggregate [3, 4]. Furthermore, it has been indicated that the serum

albumin conformation will be changed upon binding with ligands or molecules, and the change shows its influence on the secondary and tertiary structures of albumins and their biological function as a carrier protein [5, 6]. Some diseases (such as Alzheimer's disease, Parkinson's disease, and amyloid disease) are related to protein misfolding [6]. The thermodynamic and kinetic study of proteins plays an important role in understanding biological functions ranging from genetic information to molecular diagnostics [7, 8]. The functions and structure of a protein are strongly related to each other and due to this, protein folding/unfolding has protruded as an important property in biochemistry and biophysics [9, 10]. Therefore, the studies of such chemicals and their bindings with proteins are of fundamental and imperative importance.

The binding of nanoscale materials with proteins has become the most common with the availability of organic polymers, inorganic nanoparticles, carbon nanotubes, etc.. Recently, the nanoparticle studies have opened new avenues to study biomolecular interactions with their applications as drug delivery, biocompatibility, diagnostics, and smart materials. The binding of nanocolloidal particles to proteins has also been formed, since the study of immunoprobes in the early 1970s [11]. Moreover, various studies of peptide or protein including lysozyme binding with nanoparticles of different sizes have been conducted. In the process, the proteins, generally, suffer a significant loss in enzyme activity and a partial loss of structure [12, 13]. Thus, it should be expected that the size of the particle plays a major role in changing protein structure and function [13]. However, no systematic study has been performed to date on the effect of Roxatidine acetate**–**Chitosan nanoparticles on the structure and function of Lysozyme. For this reason, we embarked on a study of protein binding with Roxatidine acetate**–**Chitosan nanoparticles.

Lysozyme (Lyz) (**Figure 1A**) is one of the important proteins that is found in the blood and has various functions but is similar in its tendency to bind ligands/drugs. Lyz is an antibacterial and antiviral protein found in various biological tissues and fluids, such as skin, liver, lymphatic tissues, tears, saliva, and blood of human and other animals [13, 14]. Lyz is unique in its ability to hydrolyze the β-1,4 glycoside bond between N-acetylglucosamine and N-acetylmumaric acid of the gram-positive bacteria, thus protecting the body against the bacterial invasion [15]. Some of its important biological roles also include antihistaminic, anti-inflammatory, and antineoplastic activity [15–18]. Lyz consists of 129 amino acid residues and contains six tryptophan (Trp) and three tyrosine (Tyr) residues [4, 15]. Three residues of Trp

**Figure 1.** *(A) Three dimensional structure of Lyz, (B) chemical structure of Roxatidine acetate.*

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

are placed at the binding sites, two sites in the hydrophobic cavity, while the last site is located independently from others [4, 15, 19–21], and the effectiveness of drugs depends on both pharmacokinetic and pharmacodynamic factors. Therefore, the studies on the interactions of drugs and Lyz are of importance in understanding the release disposition, transportation, and metabolism of drug as well as the efficacy process involving drug and Lyz. Lyz has been preferentially used as a model protein to study the protein folding/unfolding, dynamics, and ligand interaction due to its small size, abundance, high stability, and ability to bind and drug carrying capacity [22–24].

Roxatidine acetate (RxAc) (**Figure 1B**) is an antagonist of a histamine H:zreceptor, which rapidly turns into Roxatidine through esterases in the plasma, small intestine, and liver (its active metabolite) [25, 26]. Roxatidine is an active inhibitor of gastric acid secretion in humans and animals [3, 25] and does not overlap with other drugs in the hepatic metabolism and has no antiandrogenic influences as most other H:z-receptor antagonists [27]. Wide-scale experiments have illustrated that 150 mg of Roxatidine acetate per day is recommended as typical dosages of ranitidine and cimetidine in the patients for treatment of gastric ulcer or duodenal ulcer [25, 27] and that 75 mg of Roxatidine acetate as dosage in the evening is probably a standard amount for the prohibition of peptic ulcer recurrence [25, 28]. Primary studies also mention that Roxatidine acetate is perhaps useful in the treatment of stomach ulcer and reflux esophagitis and in the protection of pulmonary acid aspiration [25].

Spectroscopic techniques are mostly used to detect the accessibility of quenchers to fluorophore groups of albumin and help to understand the binding mechanism of albumin to small molecules and clarify the nature of the binding phenomenon [11, 29].

In the present study, the RxAc and RxAcNPs interactions with Lyz were methodically investigated and analyzed using diverse spectroscopic techniques to reveal the binding types and properties of RxAc and RxAcNPs with Lyz. The influences of RxAc and RxAcNPs on the conformation and microenvironment of Lyz were explored.

The aim of this study was the synthesis and characterization of Roxatidine acetate–loaded Chitosan in the presence of Tween80 (Tw80) surfactant (RxAcNPs) and to clarify the binding mechanism of RxAc and RxAcNPs with Lyz using multi-spectroscopic and molecular docking techniques and provide useful information for understanding the toxicological actions of RxAc and RxAcNPs at the molecular level.

#### **2. Experimental**

#### **2.1 Materials**

Lysozyme (from hen egg white) (Catalog number: L6876) was purchased from Sigma and was used as such. The Lysozyme solution was prepared in the 0.1 M phosphate buffer of pH = 7.40. The concentration of Lyz was determined using the extinction coefficient ϵ280 = 37,750 mol1 L cm1 [30]. Chitosan, Sodium tripolyphosphate (TPP), and Tween80 (Tw80) were also purchased from Sigma (India). NaCl (0.15 M) has been added to buffer solutions to control the ionic strength, as required. Roxatidine acetate HCl (RxAc) (≥ 98%) was purchased from Tokyo Chemical Industry Co., Ltd. (TCI), India. The stock solution of RxAc (0.3 mM) was prepared in ethanol and the final concentration of ethanol was below 2.3%, and the stock solution of RxAcNPs (0.3 mM) was also prepared in ethanol and

#### **Table 1.**

*Formula for preparation of Roxatidine acetate loaded Tween80-Chitosan nanoparticles.*

this concentration was used for all the spectroscopic measurements. RxAc and RxAcNPs were accurately weighed on Shimadzu AUY-220 microbalance of resolution 0.1 mg. All the reagents were of analytical grade. For all experiments, doubledistilled water was used.

#### **2.2 Synthesis of Roxatidine acetate nanoparticles**

Roxatidine acetate drug-loaded Tween80-Chitosan nanoparticles (RxAcNPs) were prepared through the ionotropic gelation technique. The principle of this method is interaction of the positive charge of Chitosan amino groups with the negative charge of TPP groups [31, 32]. As listed in **Table 1**, the solution of Chitosan was prepared by dissolving 0.5 g Chitosan (0.5%) in 100 ml of acetic acid (1% v/v). TPP solution (0.1%) was prepared through dissolving 100 mg of TPP in 100 ml of deionized water. 30 mg of Roxatidine acetate was added to the solution of TPP. The solution was stirred at 1500 rpm for 30 min using an ultrasonicator (vibronics), and the solution of TPP was added gradually with continuous stirring for 3 hours on a homogenizer. The mixture of Roxatidine acetate, 0.1% TPP, and 0.1% Tween80 was added gradually to Chitosan solution. Tween80 was added to make the prepared solution stabilized and to limit the nanoparticle growth and thus to obtain particles of reduced mean sizes [31–33]. The precipitate was stirred at 9000 rpm for 3 hours using an ultrasonicator (vibronics). After the addition of a drug–Tween80– TPP solution to the solution of Chitosan, the suspended solution of nanoparticles was centrifuged for 15 min at 10,000 rpm, and the Roxatidine acetate nanoparticles were obtained.

#### **2.3 Drug content and release profile of Roxatidine acetate nanoparticles**

To confirm the drug content, encapsulation efficiency, and release of RxAc, the conventional method and dialysis method were used for testing RxAc-loaded Tween80-CsNPs. The encapsulation efficiency and drug content were estimated according to the procedure reported by Cevher et al. [34]. After drug loading, the RxAcNPs were isolated from the suspension using centrifugation at 10000 rpm for 15 min. The quantity of free Roxatidine acetate in the supernatant was measured using the UV–Vis spectrophotometer (double beam Perkin Elmer λ-45) at 275 nm. RxAc release was investigated in vitro by dialysis using phosphate buffer (PBS) at different pH (3.5, 6.6, 7.4, and 8.4) and 298 K. 25 mg of Roxatidine acetate–loaded Tween80–Chitosan nanoparticles were added to 50 ml of each PBS buffer in different flasks and were shaken using a magnetic stirrer at 298 K. At various time intervals, 2 ml from the suspended solution of nanoparticles was taken and centrifuged at 10000 rpm for 15 min and the standard curve for RxAc was acquired by UV spectrophotometry. At 275 nm, the RxAcNPs entrapment efficient (EE),

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

drug content, and accumulated release percentage (%) at different pH were determined spectrophotometrically and were calculated using the following equations, as described previously [31–35]:

$$n\,\text{drug\ content}\,(\text{\%}) = \frac{weight\,\text{of\ drug\ in\ nonparticles}}{weight\,\text{of\ nonparticles}} \times 100\tag{1}$$

$$EE\left(\mathfrak{We}\right) = \frac{\left(Amount\text{ }dry\text{ }taken\text{ }for\text{ }formation\text{ }) - \left(amount\text{ }of\text{ }unentrapted\text{ }drug\text{ })\right)}{Total\text{ }amount\text{ }of\text{ }drug\text{ }in\text{ }formula\text{ }tion}$$

� 100

(2)

### **2.4 Characterization of Roxatidine acetate–loaded Tween80–Chitosan nanoparticles**

Characterization of RxAc-loaded Tween80–Chitosan nanoparticles comprised Fourier transform infrared spectroscopy using PerkinElmer Frontier equipment with a resolution of 4 cm�1 and a wavenumber range of 400–4000 cm�1 . The particle size was measured by Zetasizer Nano ZS (Malvern, UK), and scanning electron microscope (SEM) in a JEOL JSM-6510 with an accelerating voltage of 15 kV was used to visualize the shape of RxAcNPs. Powder XRD scanning (Lab-X, Shimadzu-XRD 6100 instrument, Japan) was performed to analyze the crystalline nature of RxAcNPs within the range of diffraction angle 2θ from 5° to 60.

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

#### *2.5.1 Fluorescence spectra study*

The spectra of fluorescence emission were collected on Hitachi F-2700 Spectrofluorimeter with a Xenon lamp, and the quartz cuvette of 1 cm path length was used. The slit widths of excitation and emission were set at 5 nm. The rate of scanning was set to 300 nm/min. The wavelength of excitation was set at 280 nm and emission wavelength at 290–500 nm. The synchronous fluorescence spectra were scanned from 260 to 330 nm (Δλ = 15 nm) and from 220 to 330 nm (Δλ = 60 nm). A buffer blank spectrum was subtracted from the measured spectra for fluorescence background correction. The concentration of Lyz was kept constant at 10 μM, while the concentrations of RxAc and RxAcNPs were varied. All the measurements were performed at pH 7.4.

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

Tween80 was utilized to improve the stability of the therapeutic molecule and its safety at its target site. The influence of Tw80 inclusion on the interaction of the Lyz– RxAc system was studied by keeping the concentration of Lyz at 10 μM and changing the concentration of RxAc (2–16 μM), while the concentrations of Tw80 were maintained at 2 and 4 μM.

#### *2.5.3 UV–Vis spectroscopic measurements*

The UV–Vis absorption spectra were recorded using a double-beam PerkinElmer λ-45 spectrophotometer. For the whole experiment, the quartz cuvette of 1 cm path

length was used. The concentration of Lyz was kept at 10 μM while the RxAc and RxAcNPs concentrations were varied. All the readings were recorded at room temperature.

### *2.5.4 Circular dichroism (CD) measurements*

Circular Dichroism (CD) spectra were carried out using a Jasco J-815 spectropolarimeter and using a quartz cell of 0.1 cm path length. Response time and data pitch were fixed at 1 s and 1 nm, respectively. CD spectra were measured in the far-UV region (200–250 nm) with a scan speed of 100 nm/min and two scans for each spectrum under constant nitrogen flow. For all the measured spectra, Phosphate buffer baseline subtraction (pH 7.4) was used. Concentration of Lyz for all runs was fixed at 10 μM, while the RxAc and RxAcNPs concentrations were 0, 40, and 80 μM. All the measurements were carried out at room temperature.

#### *2.5.5 Molecular docking of the Lyz*–*RxAc system*

Molecular docking study used software Autodock 4.2 and Autodock tools (ADT) using the Lamarckian genetic algorithm [29]. The crystal structure of Lyz (PDB ID: 2LYZ) was obtained from Brookhaven Protein Data Bank and three-dimensional structure of Roxatidine acetate (CID = 5105) was obtained from PubChem. All the ions and water molecules were removed, hydrogen atoms were added, and partial Kollman charges were assigned. The Autodock run was carried out through the following parameters: GA population size, 150; maximum number of energy evolutions, 2.5 106, and Grid box size 86 Å 80 Å 96 Å along x-, y-, and z axes covering the whole protein with a grid-point spacing of 0.375 Å. Discovery Studio 3.5 was utilized for identification and visualization of the residues involved.