*2.1.2 Characterization of CDs and CD/Ch composite films*

The size of the carbon dots was analyzed by Transmission Electron Microscopy (TEM).The TEM samples were made by placing a drop of nanoparticle ethanol suspension on acarbon-coated copper grid. The X-ray photoelectron spectra (XPS) were performed on aVGESCALAB 220-IXL spectrometer using an Al Kα X-ray source (1486.6 eV). The crystallinenature of the plain and CD loaded chitosan film was investigated by X-ray diffraction analysisusing a Rikagu Diffractometer (Cu radiation = 0.1546 nm) operating at 40 kV and 40 mA. The X-ray Photon Spectroscopy (XPS) was also carried out.

### *2.1.3 Protein adsorption study*

In order to study the adsorption of therapeutic protein Bovine serum albumin(BSA) on the filmsurface, the test films, namely Ch/CD(0) and Ch/CD(4), were cut into 1 x 1 cm<sup>2</sup> pieces andimmersed in BSA solution, prepared in phosphate buffer saline(PBS) at a concentration of 5 mg/ml, for a period of 24 h at 37 °C.

### *2.1.4 Antioxidant properties of hydrogel wound dressings*

In order to evaluate the anti-oxidant property of the carbon dots loaded wound dressing film, sample Ch/CD(2) was taken as a representative. We followed two methods to evaluate antioxidant property of the CDs-loaded chitosan film, namely, 2,2-diphenyl-1- picrylhydrazyl(DPPH) radical scavenging assay, and superoxide radical (O2. •−) scavenging activity assay. Inthe first method, definite quantity of powdered sample Ch/CD(2) was added in to methanolicsolution of DPPH radical (100 μM) and allowed to be kept in dark for a period of 18 h [15]. The DDPH

**99**

*Wound Dressing Application of Ch/CD Nanocomposite Film*

560 nm. The film free solution was taken as control.

solution, without containing film sample, was taken as control to calculate the

DPPH radical scavenging activity % 100

Where, A0 is the absorbance of the control and A1 is the absorbance of test

The Fenton reagent was employed to test the hydroxyl radicals scavenging capacity of plain chitosan film Ch/CD(0) and CDs-loaded sample Ch/CD(2), as described elsewhere [17]. In a typical experiment, pre-weighed quantity of grinded film was added in to a solution which contained 50 mL of 1.0 mM FeCl2, 100 ml of 1 mM 1,10-phenanthroline, 3.6 ml of 0.2 M phosphate buffer (pH 7.8), 175 mL of 0.18 M H2O2. The reaction was initiated by addition of pre-calculated quantity of hydrogen peroxide. The reaction mixture was incubated at room temperature for a period of 15 min under mild stirring so as to keep the grinded film powder constantly exposed to free radicals generated. Finally, the absorbance was recorded at

The activity of plain Ch/CD(0) and CDs-loaded sample Ch/CD(2) to scavenge superoxide free radicals was investigated in riboflavin/methionine-light system [18]. Results were expressed as percent inhibition of superoxide radicals. All the

The microwave assisted synthesis of carbon dots is an effective method to prepare CDs. When a solution of BTCA in aqueous medium, is allowed to get exposure of microwaves for a definite time, there occurs uniform heating and finally the volume of the solution is almost reduced to one tenth of the original volume. Now, the residue is taken out and is diluted by the addition of distilled water. The CDs solution, so obtained is centrifuged under high resolution speed of 10000 rpm and the supernatant is collected. The passage of laser beam through the solution is preliminary indication of the formation of carbon dots. The overall synthetic

As per conventional definition, carbon nanoparticles with a diameter of less than 10 nm are called carbon dots [19, 20]. Such small carbon dots are required for for cell imaging and other related biomedical applications [21]. However, in the present work, CDs with relatively larger size were required so that they could be used as efficient crosslinker to control the permeation properties of the chitosan

It can be seen that the particles are almost spherical and bear size in the range of 40 to 60 nm. The relatively bigger size range could be attributable to the fact that we did not use any stabilizer in the preparation of CDs and hence chances of formation of bigger carbon nanoparticles could not be ruled out. There are

experiments were carried out in triplicate and average data were given.

procedure and passage of laser beam through the solution are shown in

( ) *A A A* <sup>−</sup> = × 0 1 0

*DOI: http://dx.doi.org/10.5772/intechopen.95107*

percentage scavenging activity [16].

solution respectively.

**3. Results and discussion**

**Figure 1(a)** and **(b)** respectively.

**3.2 Size determination of carbon dots**

film. The results of TEM analysis are shown in **Figure 2**.

**3.1 Preparation of CDs**

solution, without containing film sample, was taken as control to calculate the percentage scavenging activity [16].

$$\text{DPPH radical scanning activity} \left(\% \right) = \frac{A\_0 - A\_1}{A\_0} \times 1000$$

Where, A0 is the absorbance of the control and A1 is the absorbance of test solution respectively.

The Fenton reagent was employed to test the hydroxyl radicals scavenging capacity of plain chitosan film Ch/CD(0) and CDs-loaded sample Ch/CD(2), as described elsewhere [17]. In a typical experiment, pre-weighed quantity of grinded film was added in to a solution which contained 50 mL of 1.0 mM FeCl2, 100 ml of 1 mM 1,10-phenanthroline, 3.6 ml of 0.2 M phosphate buffer (pH 7.8), 175 mL of 0.18 M H2O2. The reaction was initiated by addition of pre-calculated quantity of hydrogen peroxide. The reaction mixture was incubated at room temperature for a period of 15 min under mild stirring so as to keep the grinded film powder constantly exposed to free radicals generated. Finally, the absorbance was recorded at 560 nm. The film free solution was taken as control.

The activity of plain Ch/CD(0) and CDs-loaded sample Ch/CD(2) to scavenge superoxide free radicals was investigated in riboflavin/methionine-light system [18]. Results were expressed as percent inhibition of superoxide radicals. All the experiments were carried out in triplicate and average data were given.
