Carbon dots based nanomaterials for biomedical applications

Abstract

Carbon dots (C-dots) are an emerging class of photoluminescent (PL) materials that are mainly composed of carbon, hydrogen, and oxygen, along with heteroatoms such as N, P, and S. C-dots are spherical in shape with a diameter between 1-20 nm, they demonstrate excitation wavelength-dependent emission with minimum photobleaching, while showing low toxicity levels for humans and the environment.

In this project C-dots were synthesized via hydrothermal decomposition of a mixture of CA and ethanolamine, and they were subjected to surface treatment with nitric acid followed by purification via dialysis against water. Moreover, the heptapeptide (lysine)7 (referred hereafter as K7) was synthesized by means of solid phase peptide synthesis and was subsequently purified and characterized via High-Performance Liquid Chromatography (HPLC) and by Liquid Chromatography–Mass Spectrometry (LC-MS). In addition, a quaternary ammonium salt (QAS) was synthesized and was covalently attached to polyethyleneimine (PEI) (Figure 1 A), giving rise to PEI/QAS (Figure 1B).

Finally, C-dots were conjugated with K7, Arginine-Glycine-Aspartic acid (RGD, a commercially available peptide), PEI/QAS, Jeffamine D-230 (Figure 2, a commercially available polymer) to generate a family of conjugates denoted as C-K7, C-RGD, C-PEI/QAS and C-J, respectively.

This project aims to explore the structure-property relationships within this family of novel nanomaterials. To that end, the conjugates were characterized by TEM, FTIR, elemental analysis, TGA, UV-vis, fluorescence and time-resolved fluorescence spectroscopy. TEM imaging indicated that all types of conjugates are spherical with an average diameter 11±2 nm. The quantum yield (QY) at excitation wavelength λex= 345 nm (using 4′,6-diamidino-2-phenylindole as the standard) was calculated 4.2%, 5.4%, 7.4%, 7.3% and 5.1% for C-dots, C-K7, C-RGD, C-PEI/QAS, and C-J respectively. Interestingly, the fluorescence spectra of the conjugates, while maintaining the characteristic excitation-wavelength emission mode, they showed pronounced blue shifts compared to C-dots. The average fluorescence lifetime decay (τavg) of the conjugates is close to 8 ns and 5.5 ns at λex= 375 nm and 450 nm, respectively, in line with the behavior observed for C-dots, with only C-J showing significant deviation from those τavg values possibly due to pronounced modifications of the local electronic environment.
MTT and PrestoBlue assays indicated that the viability of HeLa, U87, T96G and GC-1 SPG cells when incubated with the conjugates remain consistently at levels above 83% at 100 μg/mL loading, while haemolysis tests confirmed that the conjugates do not cause rupturing of the blood cells. Fluorescence microscope imaging showed that all conjugates are able to penetrate the HeLa cells, rendering the cytoplasm highly fluorescent, thus showing promise for in vivo
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bioimaging applications. However, only the C-K7 can effectively illuminate the Hela cells at concentrations as low as 10 μg/mL, while for the other conjugates much higher concentrations are required to induce similar effects.
Additionally, all conjugates show significant antibacterial activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) with the most effective being C-PEI/QAS that eliminates the population of E. coli and the S. aureus by 99.9% and 99.96%, respectively. For comparison, it was found that C-dots suppress the growth E. coli and the S. aureus by 94.4% and 96.7%, respectively. The release profiles of ibuprofen and temozolomide dispersed in hydrogels of copolymer [HO(CH2CH2O)20(CH2CH(CH3)O)70(CH2CH2O)20H] (otherwise known as Pluronic 123) in the presence of the conjugates were monitored at 25 °C. Incorporation of C-PEI/QAS, C-RGD and C-J to hydrogels facilitates the release of ibuprofen to 95% after 100 h, compared to only 60% release for the conjugate-free counterparts over the same period of time. Interestingly, introduction of C-RGD can facilitate an almost linear ibuprofen release as a function of time for 100 h, an effect that is highly desirable for controlled and sustained drug release applications. The C-dot and conjugate-enriched hydrogels showed a faster release of temozolomide compared to their nanoparticle-free counterparts.
In conclusion, this project contributes novel insights into the synthesis of non-toxic peptide/C-dots and polymer/C-dots conjugates that combine the supreme photoluminescent properties of C-dots with the cell-penetrating nature of K7 and RGD peptides and the biocompatible nature of PEI/QAS and Jeffamine, ultimately giving rise to a new generation of advanced materials that show great promise for a series of challenging applications related to bioimaging, controlled drug release and antimicrobial treatment.