Faculty Bibliography

Carbon nanomaterials, including 2D graphene-based materials, have shown promising applicability to drug delivery, tissue engineering, diagnostics, and various other biomedical areas. However, to exploit the benefits of these materials in some of the areas mentioned, it is necessary to understand their possible toxicological implications and long-term fate in vivo. We previously demonstrated that following intravenous administration, 2D graphene oxide (GO) nanosheets were largely excreted via the kidneys; however, a small but significant portion of the material was sequestered in the spleen. Herein, we interrogate the potential consequences of this accumulation and the fate of the spleen-residing GO over a period of nine months. We show that our thoroughly characterized GO materials are not associated with any detectable pathological consequences in the spleen. Using confocal Raman mapping of tissue sections, we determine the sub-organ biodistribution of GO at various time points after administration. The cells largely responsible for taking up the material are confirmed using immunohistochemistry coupled with Raman spectroscopy, and transmission electron microscopy (TEM). This combination of techniques identified cells of the splenic marginal zone as the main site of GO bioaccumulation. In addition, through analyses using both bright-field TEM coupled with electron diffraction and Raman spectroscopy, we reveal direct evidence of in vivo intracellular biodegradation of GO sheets with ultrastructural precision. This work offers critical information about biological processing and degradation of thin GO sheets by normal mammalian tissue, indicating that further development and exploitation of GO in biomedicine would be possible.

Studies unraveling the interactions between graphene oxide (GO) and the biological milieu, including cells and tissues, are multiplying quickly as the biomedical applications of this and other 2D materials continue to be explored. Many of such studies rely on real-time RT-qPCR as a powerful yet simple technique to assess gene expression. However, a systematic investigation of potential GO-induced changes in the expression of reference genes, crucial for appropriate qPCR data normalization, is still lacking. We aimed to cover this gap investigating the stability of the expression of ten candidate reference genes upon exposure to increasing, but subtoxic, GO concentrations, with two established algorithms (Bestkeeper and NormFinder). The study was performed in a human cancer cell line (MCF7) and in mouse, non-cancerous, primary cells (mouse embryonic fibroblasts, MEFs), to assess different behaviors between cell types. Both algorithms evidenced significant deviations in the expression of various reference genes. Ribosomal proteins scored among the most significantly dysregulated in both cell types. ACTB and GAPDH, the most frequent calibrators in real-time RT-qPCR, were also affected, although differences existed between cell lines. This study illustrates the need to validate reference genes for appropriate real-time RT-qPCR normalization, according to specific experimental conditions, when GO-cell interactions occur.

The development of efficient and safe nucleic acid delivery vectors remains an unmet need holding back translation of gene therapy approaches to the bedside. Graphene oxide (GO) could help bypass such bottlenecks, thanks to its large surface area, versatile chemistry and biocompatibility, which could overall enhance transfection efficiency while abolishing some of the limitations linked to the use of viral vectors. Here, we aimed to assess the capacity of bare GO, without any further surface modification, to complex a short double-stranded nucleic acid of biological relevance (siRNA) and mediate its intracellular delivery. GO formed stable complexes with siRNA at 10 : 1, 20 : 1 and 50 : 1 GO : siRNA mass ratios. Complexation was further corroborated by atomistic molecular dynamics simulations. GO : siRNA complexes were promptly internalized in a primary mouse cell culture, as early as 4 h after exposure. At this time point, intracellular siRNA levels were comparable to those provided by a lipid-based transfection reagent that achieved significant gene silencing. The time-lapse tracking of internalized GO and siRNA evidenced a sharp decrease of intracellular siRNA from 4 to 12 h, while GO was sequestered in large vesicles, which may explain the lack of biological effects (i.e. gene silencing) achieved by GO : siRNA complexes. This study underlines the potential of non-surface modified GO flakes to act as 2D siRNA delivery platforms, without the need for cationic functionalization, but warrants further vector optimization to allow the effective release of the nucleic acid and achieve efficient gene silencing.

Overexpression of Oct3/4, Klf4, Sox2, and c-Myc (OKSM) transcription factors can de-differentiate adult cells in vivo. While sustained OKSM expression triggers tumorigenesis through uncontrolled proliferation of toti- and pluripotent cells, transient reprogramming induces pluripotency-like features and proliferation only temporarily, without teratomas. We sought to transiently reprogram cells within mouse skeletal muscle with a localized injection of plasmid DNA encoding OKSM (pOKSM), and we hypothesized that the generation of proliferative intermediates would enhance tissue regeneration after injury. Intramuscular pOKSM administration rapidly upregulated pluripotency (Nanog, Ecat1, and Rex1) and early myogenesis genes (Pax3) in the healthy gastrocnemius of various strains. Mononucleated cells expressing such markers appeared in clusters among myofibers, proliferated only transiently, and did not lead to dysplasia or tumorigenesis for at least 120 days. Nanog was also upregulated in the gastrocnemius when pOKSM was administered 7 days after surgically sectioning its medial head. Enhanced tissue regeneration after reprogramming was manifested by the accelerated appearance of centronucleated myofibers and reduced fibrosis. These results suggest that transient in vivo reprogramming could develop into a novel strategy toward the acceleration of tissue regeneration after injury, based on the induction of transiently proliferative, pluripotent-like cells in situ. Further research to achieve clinically meaningful functional regeneration is warranted.

The control of temperature during photothermal therapy is key to preventing unwanted damage in surrounding tissue or post-treatment inflammatory responses. Lack of accurate thermal control is indeed one of the main limitations that hyperthermia techniques present to allow their translation into therapeutic applications. We developed a nanoprobe that allows controlled local heating, combined with in situ nanothermometry. The design of the probe follows a practical rationale that aims at simplifying experimental requirements and exploits exclusively optical wavelengths matching the first and second biological windows in the near-infrared. Methods: Hybrid nanostructures were chemically synthesized, and combine gold nanostars (photothermal agents) with CaF₂:Nd³⁺,Y³⁺ nanoparticles (luminescent nanothermometers). Both components were simultaneously excited in the near-infrared range, at 808 nm. Following the goal of simplifying the thermal monitoring technique, the luminescent signal was recorded with a portable near-infrared detector. The performance of the probes was tested in 3D tumor spheroids from a human glioblastoma (U87MG) cell line. The location of the beads within the spheroids was determined measuring Nd³⁺ emission in a commercial Lightsheet microscope, modified in-house to be able to select the required near-infrared wavelengths. The temperature achieved inside the tumor spheroids was deduced from the luminescence of Nd³⁺, following a protocol that we developed to provide reliable thermal readings. Results: The choice of materials was shown to work as an optically excited hybrid probe. Depending on the illumination parameters, temperature can be controlled in a range between 37 ºC and 100 ºC. The near-infrared emission of nanothermometers also allows microscopic tracking of the hybrid nanostructures, confirming that the probes can penetrate deeper into the spheroid mass. We observed that, application of optical thermometry in biological environments requires often neglected considerations, since the optical signal changes along the optical path. Accordingly, we developed data analysis protocols that guarantee reliable thermal readings. Conclusions: The prepared hybrid probes are internalized in 3D tumor spheroids and can be used to induce cell death through photothermal effects, while simultaneously measuring the local temperature in situ. We show that luminescent thermometry in biomedical applications requires the development of protocols that guarantee accurate readings. Regarding photothermal treatments, we observe a sharp thermal threshold at around 55 ºC (for 10 min treatments) that separates high survival ratio from complete cell death.

Reprogramming adult, fully differentiated cells to pluripotency in vivo via Oct3/4, Sox2, Klf4 and c-Myc (OSKM) overexpression has proved feasible in various independent studies and could be used to induce tissue regeneration owing to the proliferative capacity and differentiation potential of the reprogrammed cells. However, a number of these reports have described the generation of teratomas caused by sustained reprogramming, which precludes the therapeutic translation of this technology. A recent study by the Izpisúa-Belmonte laboratory described a cyclic regime for short-term OSKM expression in vivo that prevents complete reprogramming to the pluripotent state as well as tumorigenesis. We comment here on this and other studies that provide evidence that in vivo OSKM induction can enhance tissue regeneration, while avoiding the feared formation of teratomas. These results could inspire more research to explore the potential of in vivo reprogramming in regenerative medicine.

Advances in genomics and gene therapy could offer solutions to many diseases that remain incurable today, however, one of the critical reasons halting clinical progress is due to the difficulty in designing efficient and safe delivery vectors for the appropriate genetic cargo. Safety and large-scale production concerns counter-balance the high gene transfer efficiency achieved with viral vectors, while non-viral strategies have yet to become sufficiently efficient. The extraordinary physicochemical, optical and photothermal properties of graphene-based materials (GBMs) could offer two-dimensional components for the design of nucleic acid carrier systems. We discuss here such properties and their implications for the optimization of gene delivery. While the design of such vectors is still in its infancy, we provide here an exhaustive and up-to-date analysis of the studies that have explored GBMs as gene transfer vectors, focusing on the functionalization strategies followed to improve vector performance and on the biological effects attained.