Intracellular Fate and Impact on Gene Expression of Doxorubicin/Cyclodextrin-Graphene Nanomaterials at Sub-Toxic Concentration

Abstract

The graphene road in nanomedicine still seems very long and winding because the current knowledge about graphene/cell interactions and the safety issues are not yet sufficiently clarified. Specifically, the impact of graphene exposure on gene expression is a largely unexplored concern. Herein, we investigated the intracellular fate of graphene (G) decorated with cyclodextrins (CD) and loaded with doxorubicin (DOX) and the modulation of genes involved in cancer-associated canonical pathways. Intracellular fate of GCD@DOX, tracked by FLIM, Raman mapping and fluorescence microscopy, evidenced the efficient cellular uptake of GCD@DOX and the presence of DOX in the nucleus, without graphene carrier. The NanoString nCounter™ platform provided evidence for 34 (out of 700) differentially expressed cancer-related genes in HEp-2 cells treated with GCD@DOX (25 µg/mL) compared with untreated cells. Cells treated with GCD alone (25 µg/mL) showed modification for 16 genes. Overall, 14 common genes were differentially expressed in both GCD and GCD@DOX treated cells and 4 of these genes with an opposite trend. The modification of cancer related genes also at sub-cytotoxic G concentration should be taken in consideration for the rational design of safe and effective G-based drug/gene delivery systems. The reliable advantages provided by NanoString® technology, such as sensibility and the direct RNA measurements, could be the cornerstone in this field.

Mechanistic Insights into the Release of Doxorubicin from Graphene Oxide in Cancer Cells

“…We are prompted to ascribe these patches to sites of GO-DOX cell attachment and subsequent drug release. This observation is in agreement with recent findings obtained using graphene decorated with cyclodextrins and loaded with doxorubicin [30]. The authors, in fact, also due to lifetime analysis, evidenced the efficient cellular uptake of the whole adduct and the presence of DOX in the nucleus without the graphene carrier.…”

“…Additionally, our data do not contradict previous evidences showing that GO likely binds to integrins at the plasma membrane of cancer cells, activates the integrin-FAK-Rho-ROCK pathway and makes cancer cells more susceptible to chemotherapeutic agents [31]. From a methodological point of view, our results corroborate the idea that FLIM can represent a quantitative platform to analyze DOX cellular uptake and release from nanocarriers, in line with a growing body of evidences from the literature (see for instance References [29,30,32,33]).…”

Polymer-Based Graphene Derivatives and Microwave-Assisted Silver Nanoparticles Decoration as a Potential Antibacterial Agent

“…The size of nanosystems in water dispersion were investigated by Dynamic Light Scattering (DLS) analyses. Generally, DLS values are not indicative of the morphology and size of the graphene nanosystems, but provide information about their hydrodynamic size [ 63 , 64 ]. The functionalization of GO with PVA resulted in two hydrodynamic radius distributions ( Figure 6 , black line) centered at 5 nm and 70 nm: those are similar to that of the pure PVA (grey line), even while having different relative intensities.…”

Recent Advances of Graphene-Based Strategies for Arsenic Remediation

“…Specifically, major concerns such as safety, economic feasibility, and aggregation phenomena, especially in scaled up water purification systems, need to be reasonably addressed. To minimize the health risk, safety issues require a careful evaluation (Caccamo et al, 2020 ) with the implementation of in-vivo studies. The lack of standardized ways for G univocal characterization and the different fabrication methods make the replication of G published results difficult (Piperno et al, 2018 ).…”