Biocompatible and cell traceable Graphene Quantum Dots (GQD) are used for targeted drug delivery of DNA intercalating drug Doxorubicin (DOX). Graphene Quantum Dots are highly dispersible and water soluble. Synthesized by acid oxidation and exfoliation of multi-walled carbon nanotubes (MWCNT). GQD loaded with DOX were covalently linked to tumor targeting module Biotin (BTN). Test performed on A549 cells reported a very low toxicity of the synthesized carrier (GQD and GQD-BTN). Cancer cells cytotoxicity was strongly dependent from cell uptake between cells. Treated with GQD-BTN-DOX system aka targeted drug. Treated with GQD-DOX system lacking targeting module biotin (BTN) aka free drug.

Graphene

Graphene can be described as a One-Atom thick layer of graphite. Graphene is the strongest thinnest material known to exist. Graphene and Graphene oxide (GO) are single atomic-layered structure. Graphene is 2D structure of carbon in honeycomb shape. Functionalized Graphene and Graphene Oxide have shown. It has improved drugs solubility, extended half-life of drug and reduced side effects to living cell.

Graphene Quantum Dots (GQD)

Graphene quantum dots are carbonaceous material prepared from graphene. Graphene quantum dots (GQDs) represent single-layer to tens of layers of graphene of a size less than 30 nm (1- 20 nm). It has properties such as low toxicity, stable photoluminescence, chemical stability. GQDs are considered as a novel material for biological drug delivery, opto-electronics, energy and environmental applications. GQD have molecule like character so it is easier to handle. GQD with different size can absorb light, when they release that energy it produces light of different color.

Targeted Drug Delivery System (DDS)

The major goal of any drug delivery system is to supply a therapeutic amount of drug to a target site in a body. Targeted drug delivery implies selective and effective localization of drug into the target at therapeutic concentrations with limited access to non-target sites. A targeted drug delivery system is preferred in drugs having instability, low solubility and short half-life. The design of effective DDS to treat cancer should include. A tumor targeting ligand unit that could specifically recognize cancer receptors on the cell surface. Induce receptor- mediated endocytosis. Targeting ligands such as arginine-glycine-aspartic acid (RGD), folic acid, biotin.

Materials & Methods

Solvents and reagents were obtained from commercial suppliers. MWCNTs were produced by catalytic chemical vapor deposition (CCVD) from isobutane on a Fe/Al2O3 catalyst. Purification of MWCNTS was done yielding pristine MWCNTs with >95% purity.

Methods

  1. Synthesis of GQD
  2. Synthesis of BTN
  3. Synthesis of GQD-BTN
  4. Synthesis of GQD-BTN-DOX and GQD-DOX

 

Synthesis of GQD:

Pristine MWCNT were treated with a HNO3/H2SO4 mixture in a 1:3 ratio. Mixture was refluxed and sonicated in ultrasonic water bath at 60 for 4 days. Mixture dilution and filtration under vacuum with 0.1μm Millipore membrane. Neutralization with NaOH and centrifugation of filtrate at 3000rpm. Washing with deionized water until no salts present in solution. Drying at 60 under vacuum of resulting material. Carboxylic groups amount present on the nanomaterial was evaluated by Boehm titration, using NaHCO3 as titrating agent.

Synthesis of BTN

1-ethyl-3-carbodiimide hydrochloride (EDC.HCl) and Triethylammine were added to a dispersion of biotin in Tetrahydrofuran and stirred for 1hour. 1-Hydroxybenzotriazole was added and the mixture was stirred for 1 hour more. Tert-butyl-2-ethylcarbamate was added and stirred at room temperature for 24h. After removal of the solvent, CH2Cl2 (Dichloromethane) was added and the organic phase was washed with water. Dried over sodium sulfate, filtered and evaporated for dryness. The residue was purified by MPLC on a silica gel column using a mixture of CH2Cl2/MeOH (9:1) to afford the BTN module protected at the amino functionality, in 95% yield. The BTN module was dissolved in CH2Cl2 and added with Trifluoroacetic acid (TFA) and stirred for 1h at room temperature. Toluene was added to form a TFA azeotrope and the solvent was removed under vacuum. The residue was purified by MPLC on a silica gel column using as eluent a mixture of CH2Cl2/MeOH (9:1) to afford BTN in 98% yield.

Synthesis of GQD-BTN

To a solution of GQD in CH2Cl2, EDC.HCl and 4-dimethylaminopyridine were added and the mixture was stirred at room temperature for 30  min. 1-Hydroxybenzotriazole  (HOBt,  0.134  mmol)  was  added and  the  mixture  was  stirred  for  30  min. Solution of BTN (0.134  mmol)  in  CH2Cl2 (10  mL)  was  added  and  the  suspension  was left  under  stirring  for  4  days  at  room  temperature. The resulting material was washed several times with CH2Cl2 and water. Centrifuged at 3000  rpm  until  no  organic  materials  were  present  in the  washing  solutions  and  dried  at  60   under  vacuum.

Synthesis of GQD-BTN-DOX and GQD-DOX

Solution of GQD-BTN or GQD  (30  mg) in deionized water  was stirred with a solution of doxorubicin  hydrochloride in 10  mL  of basic buffer solution  at  pH  7.4,  at room temperature for 48  h. Solution was then centrifuged at  3000rpm. Resulting material was washed several times with water  until  no drug was present  in the washing solutions and dried at  60 C  under vacuum. The amount  of  unbound  DOX was determined by measuring the absorbance  at  490  nm,  relative  to  a  calibration curve  recorded under the same  conditions. The drug loading for GQD-BTN-DOX  and  GQD-DOX  were  found  to  be  16.6%  and  17.8% respectively.

Biological Studies

Confluent monolayers of A549 cells were used to assess biocompatibility of the carriers and the drug uptake of GQD-DOX and GQD-BTN-DOX, by employing  FACS (fluorescence assisted cell sorting) technique.

Biocompatibility was detected by loading the carriers with Propidium iodine and the dead stained cells were recorded.

Drug uptake was evaluated by emission signals in cell suspension treated with GQD-DOX. Cells, maintained in the same medium with the addition of doxorubicin solutions, were included as negative and positive controls respectively.

Cell counts were detected Cytofluorimetrically to estimate mortality rate due to intercalating agent.

Physiochemical and Morphological characterization

This was done by XRD (X-ray Power diffraction), Electron Microscopy (GQD Morphology), DLS (Particle sizing Measurements of GQD), CONTIN analysis (correlation data treatment), Photoluminescence (PL) measurements, NMR spectra, Micro Raman scattering measurements, Thermo gravimetrical studies and FTIR.

Conclusion

In summary, from this study emerged the improved delivery of conventional chemotherapeutics whose activity is restricted by resistance mechanisms and dose-limiting side-effects, by using GQD  as  Nanocarrier.  These nanomaterials have shown a  great biocompatibility and ability to release drugs into cancer cells. Multimodal conjugation allows to incorporate both drugs and  targeting  ligands  in  the  same nanostructure  in  order  to  minimize  the  anticancer  drugs  systemic toxicity  and  undesirable  side  effects,  typically  associated  with conventional  chemotherapy

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