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Engineering Carbon Nanodots for (Nano)Technological and Biomedical Applications

Periodic Reporting for period 2 - e-DOTS (Engineering Carbon Nanodots for (Nano)Technological and Biomedical Applications)

Période du rapport: 2022-07-01 au 2023-12-31

Carbon nanodots (CNDs) are photoluminescent nanoparticles, with a characteristic size below 10 nm and a quasi-spherical morphology. They have attracted a lot of attention in the materials science field, mainly owing to their easy preparation and exciting properties. Though the determination of how they form would help in the control over the properties of CNDs, including luminescence, the mechanism of formation of these new materials is still elusive. Also, the superficial groups of CNDs control their interaction with the environment and are therefore critical, in particular for biological applications. However, there is little information on the functional groups present at the carbon nanodot surface. In particular, there is a lot of confusion on the structural characteristics of these nanoparticles.

During this project, we have studied the mechanism of formation of CNDs. Through a wide range of spectroscopic and analytical techniques, we have demonstrated that the formation of CNDs consists of 4 consecutive steps: (i) aggregation of small organic molecules, (ii) formation of a dense core with an extended shell, (iii) collapse of the shell and (iv) aromatization of the core. This information has been vital for the other tasks of the project.

We have also devised a general NMR methodology to establish the purity and the quality of CNDs. This technique should allow all the people working on CNDs to understand whether or not they have prepared CNDs.
Another objective of the project is the insertion of heavy transition metal atoms in the CND core. This resulted in the synthesis of CNDs with additional properties, e.g. suitable for practical applications, such as Magnetic Resonance Imaging (MRI). The preparation of contrast agents for MRI is a research hot topic, because the current compounds are not ideal and are often toxic. The preparation of safe MRI contrast agents is therefore an important topic also for the society.

The overall objectives of the present Advanced ERC project relate to the use of CNDs as new platforms for a variety of practical applications, ranging from (electrochemi)luminescent materials to nanothermometers, from circularly polarized luminescence to novel catalysts for organic reactions.
One major challenge of the project was the study of the mechanism of formation of carbon nanodots (CNDs). We have demonstrated that the formation of CNDs consists of four consecutive steps: (i) aggregation of small organic molecules, (ii) formation of a dense core with an extended shell, (iii) collapse of the shell and (iv) aromatization of the core. This information has been vital for the other tasks (task 2 and task 3). We have also devised a general NMR methodology to establish the purity and the quality of CNDs.

The synthesis and applications of novel CNDs include work in the field of Organocatalysis, Bioimaging, Circularly Polarized Luminescence (CPL), Electrochemiluminescence, Drug delivery, and Sensors, including:

a) Synthesis of CNDs to catalyze (asymmetric)-(photo-)organic transformations in water.
This work has resulted a series of excellent results, which have created a new research field. CNDs have proved to be very versatile catalysts in organic reactions, either ground state, photo-induced and asymmetric versions, including Michael additions, Knövenagel reactions and others.

b) Synthesis of CNDs to serve as a new platform for Magnetic Resonance Imaging (MRI).
New CNDs doped with gadolinium (Gd (III)), named Gd@CNDs, have been synthesized as multifunctional probes for Magnetic Resonance Imaging (MRI). These new amorphous Gd@CNDs display good homogeneity, they are free from emissive side products. and display suitable and stable longitudinal relaxivity (r1), with emissive behavior, therefore potentially useful for both MR and fluorescence imaging. MRI recording T1-weighted images on mice after intravenous injection of Gd@CNDs, show signal enhancement in the liver, spleen, and kidney 30 min post-injection.

c) Preparation of CNDs that display circularly polarized luminescence (CPL).
Chiral carbon nanodots starting from atropoisomeric precursors have been synthesized, purified and characterized. The obtained atropoisomeric carbon nanodots are soluble in organic solvents and have good thermal stability, which are desirable features for technological applications. Introducing axial chirality expands the strategies available to tailor the properties of carbon nanodots, paving the way for carbon nanoparticles that combine good processibility in organic solvents with engineered advanced chiroptical properties.

d) Biocompatibility studies of CNDs
CNDs have shown good biocompatibility in both cellular and in vivo studies, with enhanced cell permeability. This study has evaluated two basic biological phenomena, the protein adsorption and cell internalization. We also found that a careful concentration estimation enables the evaluation of the differences in biological effects related to chirality, namely the two CND enantiomers.

e) Synthesis of new CNDs for electrochemiluminescence applications.
In this work, we reported the synthesis of CNDs that display electrochemiluminescence (ECL) in water via coreactant ECL, which is the dominant mechanism in biosensing applications. The dependence of luminescence properties on the surface chemistry was also reported, by investigating the photoluminescence (PL) and ECL response of CNDs with surfaces rich in primary, methylated, or propylated amino groups. The ECL emission is influenced by surface states from the organic shell, but states of the core strongly interact with the surface, influencing the ECL efficiency.

f) Synthesis of CNDs for drug delivery of cancer-fighting drugs.
Irinotecan (CTP-11) is one of the standard therapies for colorectal cancer (CRC). CTP-11 is enzymatically converted to the hydrophobic 7-ethyl-10-hydroxycamptothecin (SN38), a one hundred-fold more active metabolite. Conjugation of hydrophobic anticancer drugs to nanomaterials is a strategy to improve their solubility, efficacy, and selectivity. In this work, we have used CNDs to improve drug vehiculation, stability, and chemotherapeutic efficiency of SN38 through a direct intracellular uptake in CRC. CND-SN38 successfully penetrates the CRC cells with a release in the nucleus affecting first the cell cycle and then the cytoskeleton. Moreover, CD-SN38 leads to a deregulation of the extracellular matrix (ECM), one of the major components of the cancer niche considered a possible target therapy for reducing the cancer progression. This work shows the combined therapeutic and imaging potential of CD-based hybrid materials for the treatment of CRC.

g) Synthesis of CNDs for sensing applications.
In the logic to support a green circular economy, CDs were prepared from a natural low cost precursor consisting in olive solid waste (OSW) by a simple pyrolysis process combined with chemical oxidation. The nanomaterial was used to fabricate and test a conductometric gas sensor (CDs-sensor) that was found to exhibit excellent performances in terms of high and selective response to sub-ppm concentration of NO2 at low temperature (150 °C), low limit of detection (LOD) of 50 ppb, good reproducibility and stability over use and aging.

h) Synthesis of CNDs with antibacterial activity.
Synthesized from D-glucose, negatively charged CNDs (ζ-potential = −32 mV) were obtained, well dispersed in water, with average diameter of 2.2 nm. The novel CNDs showed a broad spectrum of antibacterial activities toward different Gram-positive and Gram-negative bacteria strains. Both synthesized CNDs caused highly colony forming unit reduction (CFU), ranging from 98% to 99.99% for most of the tested bacterial strains. The elevated antibacterial activity of high-oxygen-containing carbon nanodots is directly correlated to their ROS formation ability.
All the work performed goes beyond the state of the art. In particular, there were no suggestions on the mechanism of formation of CNDs. The design of novel CNDs with ad hoc properties requires a comprehensive understanding of their formation mechanism, which is a complex task considering the number of variables involved, such as reaction time, structure of precursors or synthetic protocol employed. Therefore, shedding light on the mechanism of formation help formulate novel CNDs with improved properties. Our successive results emphasize the importance of this acquired knowledge in the development of CNDs.
Also, all the work reported under the previous section has been published in high impact Journals, which clearly shows notable advances with respect to the state of the art.
Overview of the e-DOTS project