Evaluation of Antibacterial and In vivo Wound healing activity of Carbon Dot Nanoparticles

a Department of Biotechnology, M.S. Ramaiah Institute of Technology (Affiliated to Visvesvaraya Technological University, Belgaum) Bangalore-560054, India. b Department of chemistry, M.S. Ramaiah Institute of Technology (Affiliated to Visvesvaraya Technological University, Belgaum) Bangalore-560054, India. c Sree Siddaganga College of Pharmacy, Tumkur – 572102, India. d TheraIndx Life sciences Pvt Ltd, Bangalore -562123, India

Nanoparticles (NPs) are defined as particles having one or more dimensions in the order of 100 nm or less. Many researchers found application that Silver nano particles are toxic to bacteria and are currently used in everything from medical devices to sport socks and washing machines to detect microbial growth. Even though Ag NPscontaining dressings are declared to be safe for patients and non-cytotoxic [5], recent studies have shown possible toxic effects on human fibroblasts and keratinocytes [6]. Most of the antibiotic resistance mechanisms are irrelevant for nanoparticles (NPs) because the mode of action of NPs is direct contact with the bacterial cell wall, without the need to penetrate the cell; this raises the hope that NPs would be less prone to promoting resistance in bacteria than antibiotics. [7] Therefore, attention has been focused on new and exciting NP-based materials for various therapeutic purposes and different pharmacological evaluations [2]. C-dots are relatively nontoxic and easy to synthesize compared to conventional quantum dots which are based on inorganic semiconductors that contain heavy metals, such as Au, Ag, Cd, In, Sn etc. It is well-known that the heavy metals are toxic hence their application is limited. Management of under healing of wounds is a complicated and expensive program and research on drugs that increase wound healing is a developing area in modern biomedical sciences. [8] Several drugs obtained from natural sources are known to increase the healing of different types of wounds. [9,10] Though some of these drugs have been screened scientifically for evaluation of their wound healing activity in different pharmacological models and patients, the potential of many of the traditionally used natural agents remains unexplored. In few cases, active chemical constituents were identified [11][12][13] In the present investigation, non-metallic C-Dot

Characterization of C-Dots
Characterization studies such as UV spectrophotmeter (Elico BL165), Fluorescence spectrophotometer (Spectrofluorometer F-2700 Hitachi) were used to analyze the structure and the composition, surface morphology and size and shape respectively of synthesized carbon dots using chemical method.  hrs. Post incubation, plates were visually examined for turbidity and the optical densities at 630 nm will be estimated using a Spectrophotometer. The MIC will be defined as the lowest concentration of the test compound that prevents bacterial growth (lack of turbidity or the OD at 630 nm relative to no growth control [14].

Antibacterial activity of C-Dots
The assay was done in duplicates and the obtained results (test compounds versus control) were statistically analyzed using ANNOVA software.

Dose selection for Wound Healing Studies
For the Selection of dose and treatment period, After the exposure period, the residual test item was wiped gently from the skin using cotton soaked in water.
Limit test: Based on the results from the range finding experiment, limit test was chosen. In the limit test, 5 male and 5 female rats for each test item were exposed to dose 2000 ul/animal. One day before the treatment, around 10% dorsal skin area of each rat was clipped free of hair, without any abrasion. The appropriate amount (2ml) of the test item. SCD was applied uniformly over the clipped area of each rat. After the application, the test item was held in contact with the skin for a period of 24-hours, using a porous gauze dressing and bandaged with non-irritating adhesive tape.
After the exposure period, the residual test item was wiped gently from the skin using cotton soaked in water.
Animals were observed for 14 days for any abnormality or death.

2.8c. Dead space wound model
This type of wound was created by implanting subcutaneously a 2.5×0.5 cm polypropylene tube in the lumber region in anesthetized rats [20]. Animals were treated with SCD, GCD and FCD topically from 0th day to 9th post wounding day. On the 10th post wounding day, the animals were sacrificed and the granulation tissue harvested on the implanted tube was carefully dissected out along with the tube [21]. The tubular granulation tissue was cut lengthwise to obtain a sheet of granulation tissue. The pieces of granulation tissue were collected, dried at 60 o C for 24 hr to get a constant weight and weighed [20,22].

Data Compilation and Statistical Analysis
The micro dilution assay for determination of MIC

Characterization of Carbon dots
When UV-Visible light is passed through a sample, the transmittance of light by the sample is measured. From the transmittance (T), the absorbance can be calculated as A=-log (T). The absorbance spectrum is obtained which shows the absorbance of a sample at different wavelengths. An emission peak at around 500 nm was observed. It should be mentioned here that the EG solution itself is non-emissive in the visible region, confirming the bright fluorescence to be originating from the synthesized C-dots.
The emission spectra recorded at different excitation wavelengths (250 to 350 nm) showed the fluorescence emission has a red shift as the excitation wavelength is increasing (Fig.3).Interestingly, with increase in the excitation wavelength from 250 nm to 350 nm, the emission from C-dots gradually shifted to higher wavelengths accompanied with decreased fluorescence intensity. This bathochromic shift of emission from C-dots has also been reported previously [14]. The strong fluorescence exhibited by the C-dots is thought to be responsible for the quantum confinement of the passivity surface energy traps [11].

Antibacterial activity of C-Dots
All the four organisms were found susceptible to the C-dots from sucrose, Glucose and fructose. The   (Fig. 8). All the three C-dots have shown potent antibacterial activity when compared to the control

3.4a. Excision wound
Effect on excision and incision wound-Test compound SCD, GCD and FCD produced a significant decrease (P<0.05) in percent wound contraction on day 14 onwards, when compared to control. (Image 1, Table 3, Fig. 9 ). SCD and GCD showed antibacterial activity at a concentration of 12.5µl/ml at a lesser concentration compared to FCD at 50 µl/ml. All the three test compounds showed good activity when compared to vehicle control.   SCD and GCD showed antibacterial activity at a concentration of 12.5µl/ml at a lesser concentration compared to FCD at 50µl/ml. All the three test compounds showed good activity when compared to control.     is stimulated by various growth factors [27]. Growth hormone is also known to promote the proliferation of fibroblasts and fibroblast proliferation form the granulation tissue. In the dead space wound model, C-Dots treatment increased granuloma tissue weight. The exact mechanism by C-Dots, increased the granuloma tissue weight of granulation tissue is still not known.

Conclusion
The study reveals that Carbon dots possess