Journal of Biotechnology & Biomaterials
Open Access

C-dot nanoparticles (SCD, GCD, FCD); IC₅₀; Cytotoxicity

Cancer is one of the first causes of mortality worldwide. As a result, the principle challenges faced in anticancer therapy using nanotechnology have been the design and development of nanomaterials with multiple functions [1]. The anticancer activity of metal nanoparticles like zinc oxide and are much explored. The silver nanoparticles are well known for their antimicrobial properties and studies have shown that they possess anticancer properties; hence researches are going on to extract these nanoparticles via Green Synthesis. The Pharmaceutical activities of non-metals like carbon dots are not much known. In one of the recent studies carbon dot supported atomically dispersed gold can be used as a mitochondrial oxidative stress amplifier for cancer treatment. Anticancer therapy using multifunctional nanomaterials can target a cancer or tumour, deliver therapeutic drugs, and monitor the tumour tissues. In recent years, the use of various types of biological labelling probes such as gold nanoshells semiconductor quantum dots, iron oxide nanoparticles carbon nanotubes and polymer nanocarriers has resulted in the development of sensitive and specific targeted cell imaging, sensing, and therapy for invitro and in vivo applications. However, many of those probes possess properties that considerably decrease their therapeutic efficacy, including poor photostability and solubility in aqueous media, poor bio distribution, and a lack of target selectivity. The Pharmaceutical activities of non-metals like carbon dots are not much known. C-Dots are small carbon nanoparticles (less than 10 nm in size) with various surface passivation schemes in which chemical functionalization with organic molecules have been most effective.

Alternatively, carbon-based multifunctional water-soluble nanomaterials can be fabricated from bulk biological sources into fluorescent probes, thus reducing ecological concerns and economic problems. Recently, our group reported green carbon dots (G-dots) from food waste-derived sources as novel fluorescent probes for cell imaging. Although G-dots do not possess any targeting ligands on their surfaces, they are biocompatible and stable in aqueous systems, permitting their use in biomedical applications [2]. Compared to previous metallic or inorganic nanoparticles, carbon dot delivery systems can be used to minimise cytotoxicity and improve clinical outcomes and comprise the next generation of multifunctional nano medicine. Nevertheless, there are several critical issues involved in the design of carbon nano materials, such as surface functionalization.

Past attempts have involved attachment of gold nanoparticles to carbon nanotubes, resulting in the production of large clusters, which were unsuitable for biomedical applications. While these various imaging and therapeutic nanoparticles represent an exciting improvement in the field of bio sensing and cell imaging, it would be ideal to engineer ‘advanced’ versatile carbon dots that are capable of performing biological functions (e.g. sensing, imaging, and drug delivery) by a very simple manufacturing method. Nano biotechnology has introduced a new perspective for using nano sized elements against cancer diseases. Nanoparticles, because of their size (<100 nm), have unique physiochemical features including a large surfaceto- mass ratio, easy surface functionalization, quantum characteristics and consequently, novel biological properties. The cellular transport of nanoparticles is also significantly different from that of chemical compounds or drugs because they are able to move across cells, reach nuclear membranes, and target specific structures such as proteins or gene sequences.

In the present investigation, non-metallic C-dots were synthesized by wet chemical method from three different compounds namely glucose, sucrose and fructose. The synthesized nanomaterials were characterized with UV-Visible and fluorescence spectroscopic techniques. The cytotoxicity of the synthesized nanoparticles was tested on theHepG2and MCF-7 cell lines by the MTT method. MTT assay is a colorimetric assay used for the determination of cell proliferation and cytotoxicity, based on reduction of the yellow colour watersoluble tetrazolium dye MTT to formazan crystals. Mitochondrial lactate dehydrogenase produced by live cells reduces MTT to insoluble formazan crystals, which upon dissolution into an appropriate solvent exhibits purple color, the intensity of which is proportional to the number of viable cells and can be measured spectrophotometric ally at 570 nm [3].

Materials and Methods

Cell lines and medium

HepG2 and MCF-7 cell lines were from (NCCS, Pune). Foetal Bovine Serum [#RM10685], MTT Reagent [5 mg/ml, # 4060], D-PBS [#TL1006] were from Himedia.

Chemicals used

Sucrose, Glucose, Fructose, Camptothecin (# C9911, Sigma) for HepG2, Oxaliplatin (# O9512, Sigma) for MCF7.

Synthesis of C-Dots

C-dots were synthesized by acidic oxidation of sucrose, glucose and fructose in separate glass beaker with H₃PO₄at 60^OC till color changes to the formation of yellow C-dots. In this method 0.698g of sucrose, 0.1396g of glucose and fructose were dissolved in 150ml distilled water in three different glass beakers, then 0.106ml of phosphoric acid was added to each beaker and kept on the heating mantle at 60oC till the colour changed to pale yellow. The synthesized nanoparticle solutions were then transferred to dialysis bag and subjected for 24 hours of dialysis treatment. Detailed synthesis and characterization of C-dots published in our earlier article [4, 5].

Cell Culture

The cell lines MCF-7 and HepG2 were obtained from the NCCS Pune and maintained in Dulbecco’s modified Eagle’s culture medium supplemented with 10% Fetal bovine serum (Sigma-Aldrich), 1% penicillin, and streptomycin (Sigma-Aldrich) at 37 ⁰ C in a humidified atmosphere of 5% CO2/95% air in a CO₂ Air-Jacketed Incubator until confluent before used for cytotoxicity assay.

Cytotoxic activity by MTT assay

Cytotoxicity studies with normal cell culture systems of C-dots have not been studied extensively and this is vital for the safety evaluation of these nanoparticles. Therefore, the objective of this study was to evaluate the potential cytotoxic activity of C-dots against HepG2 and MCF-7 cell lines.

The cytotoxicity study was carried out for C-dots (Test Compound) from Sucrose (SCD), Fructose (FCD) and Glucose (GCD). They were screened for their cytotoxicity against HepG2 and MCF-7 cell lines at different concentrations to determine the IC50 (50% growth inhibition) by MTT assay. In-Vitro assay C-dots formulation was carried out for their confirmation of cytotoxic effect on HepG2 and MCF-7 cell lines. Percentage of viable cell can be obtained by performing try pan blue dye exclusion technique. The cytotoxicity activity is carried out by using MTT assay. Cell lines derived from NCCS, Pune were free from any kind of bacterial and fungal contamination.

Percentage cell viability of cell line was carried out by using try pan blue dye exclusion technique. 200μl cell suspension was Seeded in a 96-well plate at required cell density (20,000 cells per well), without the test agent. The cells were allowed to grow for about 12 hours. Final concentrations of C-dots (Test Compound) from Sucrose (SCD) Fructose (FCD) and Glucose (GCD) 50, 150, 250, 350 and 450 μg/ml were added. The plate was incubated for 24hrs at 37°C in a 5% CO2 atmosphere. After the incubation period, the plate was removed from the incubator; spent media was removed followed by addition of MTT reagent to a final concentration of 0.5 mg/ml. The plate was wrapped with aluminum foil and placed in the incubator for 3 hours, after which MTT was removed and 100 μL of DMSO was added. Absorbance was measured in an ELISA reader at 570 nm.

The Absorbance of medium control was deducted from Absorbance in treatment wells before estimation of viability. The mean and SD % viability was estimated for each concentration .

Statistical Analysis

All the invitro experiments were performed in duplicates using two replicate samples for each formulation. The cytotoxicity of each formulation was expressed in terms of it IC₅₀ (concentration causing 50% if death of cell population) calculated from concentration – response curves. The IC₅₀ value was determined by using linear regression equation i.e. y= mx+c. Here, y = 50, m and c values were derived from the viability graph.

The percent viability of cells was estimated using the following equation:

% Viability = [Absorbance of treated cells/Absorbance of untreated cells] * 100

Results and Discussion

Cytotoxicity activity of C- Dots

The present study carried out for C-dots from Sucrose (SCD), Fructose (FCD) and Glucose (GCD) showed that the % viability of HepG2 and MCF-7 cell line is 90.85%, which is most suitable to perform cytotoxicity study. These C-dots were screened for its cytotoxicity against HepG2 and MCF-7 cell lines at different concentrations to determine the IC₅₀ (50% growth inhibition) by MTT assay. IC₅₀ of the C-Dot nanoparticles (SCD, GCD, FCD) on HepG2 cells were 67.24, <50 and and<50 μg/ml respectively. Results of % Viability of HepG2 cell line of GCD, FCD & SCD were presented in and graphically represented.

The IC₅₀ of the C-Dot nanoparticles (SCD, GCD, FCD) onMCF-7 cells were 105, <50 and <50μg/ml respectively. Results of %Viability of MCF-7 cell line of untreated/control, GCD,FCD and SCDwere presented in and graphically represented. The percentage viability was found to be increasing with decreasing concentration of test compounds.

Conclusion

A Wet chemical method adopted for the synthesis of Carbon Dots has been reported. The method is very simple, cost effective and environmental benign and can be used for various applications. These nanoparticles are advantageous with their well-known intrinsically nontoxic in vitro and in vivo, eco-friendly and can be used in the field of medicine for their efficacy. The present work reveals the potential application of C-dots for in vitro cytotoxic activity against two cancer cell lines human liver carcinoma cells (HepG2) and human breast carcinoma cells (MCF -7) and further it recommends advanced studies of anticancer drug discovery using the Carbon dot nanoparticles as its pharmaceutical applications are less explored.

Acknowledgements

Authors wish to acknowledge the management of M.S Ramaiah Institute of Technology for the constant support and encouragement.

Conflicts of Interest

There are no conflicts of interest among the authors.

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