(150n) Chirality-Driven Enhanced Uptake, Transport, and Drug Delivery of Graphene Quantum Dots (GQDs) into Cancerous Cellular Spheroid

Chirality plays a crucial role in understanding biological systems as the interactions of biomolecules often depend on their three-dimensional shape and spatial arrangement, which can be left-handed or right-handed. While many biomolecules exhibit chirality, only one type of chiral molecule can be biologically active (i.e., enantiomer). This rule applies not only to natural tissues and organisms but also to foreign molecules such as drugs and biomaterials, emphasizing the importance of considering chirality when designing biomedical materials and applications to avoid undesired side effects from opposite chiral materials (i.e., distomer). For instance, the thalidomide disaster showed how the distomer of the chiral drug induces severe side effects (e.g., fetal abnormalities) while the eutomer was effective in treating cancer and skin conditions. Therefore, understanding the role of chirality in realistic biological systems is significant to predict drug effects more precisely and developing advanced drug design accordingly.

This study employs chiral-modified graphene quantum dots (GQDs) as a model molecule for chirality-driven enhanced transport and drug delivery. GQDs have emerged as a promising tool for biological studies due to their unique physicochemical properties. GQDs possess a high surface area-to-volume ratio, tunable optical properties, and outstanding biocompatibility, making them ideal for various biomedical applications. One of the significant advantages of using GQDs in biological studies is their ability to act as imaging probes. Also, GQDs can be easily functionalized with specific biomolecules, such as antibodies, peptides, or nucleic acids, to target specific cells or tissues. Moreover, GQDs emit strong and stable fluorescence signals, allowing for high-resolution imaging of biological structures in vitro and in vivo. In addition, GQDs have shown low toxicity, making them safe for living organisms. Furthermore, their carbon- and electron-rich planar chemical structure is advantageous in loading chemical drugs containing aromatic groups through π-π stacking. Overall, the unique properties of GQDs make them a versatile and promising tool for biological studies, with potential applications in drug delivery, biosensing, and imaging.

On the other hand, three-dimensional (3D) cancer cell spheroids serve as a model system for chiral GQD uptake and transport tests. Traditional in-vitro models sometimes show inconsistencies with in-vivo animal and clinical studies, leading to a gap in understanding the therapeutic/side effects of chiral materials. To fill the gap, 3D cell culture has emerged as a promising model that mimics similar conditions to in-vivo environments. By replicating the physical alignment and chemical composition of real tissues, 3D cell culture systems provide a biomimetic microenvironment for cells to behave as they would in actual tissue, showing morphological and compositional similarity. Among them, cellular spheroids/organoids are a promising 3D culture model that starts from suspended cells that aggregate and mature to express extracellular matrix structures, enabling them to mimic the in-vivo microenvironment. In addition, cell spheroids can be used to study interactions between different types of cells, showing a better representation of drug responses. Therefore, cell spheroids can be one of the promising approaches for advancing the understanding and prediction of human biology and disease, as well as for developing more effective therapies with fewer undesired side effects.

This study investigated the effects of chiral nanoscale drug carriers, chiral GQDs, on their uptake and transport in tumor-like tissues. GQDs were synthesized using a modified Hummers method, and surface modification with L/D-cysteines was performed using an EDC/NHS coupling reaction (e.g., L-GQDs and D-GQDs). The GQDs were characterized using TEM, FTIR spectroscopy, CD spectroscopy, and zeta potential analysis. As a result, the distribution of each GQD had a similar profile in terms of both means and standard deviations. The average size of L-GQD was 7.88 ± 2.11 nm, while D-GQD was 7.99 ± 2.03 nm. The FT-IR analysis verified the chemical modification of the L/D-GQDs. In detail, both L- and D-GQDs did not represent only the GQD-related bonds (i.e., 3400, 1409, and 1350 cm^-1 for -OH group; 1712 cm^-1 for -C=O group; 1602 cm^-1 for C=C group; 1119 cm^-1 for C-O group) but showed cysteine related peak at 1250 cm^-1 (i.e., C-N group). This result demonstrated the reliable modification of GQDs with L/D-cysteine. On the other hand, from the circular dichroism (CD) spectra, both GQDs had ellipticity peaks with the same wavelengths (i.e., 213 nm and 258 nm) but negative ellipticity without significant difference in absorbance under CD measurement. Combining all, L/D-GQDs do not show any significant difference in their characteristics except for their chiral properties (e.g., L-GQDs or D-GQDs), implying the successful superficial modification of chirality onto GQDs.

To test the chiral GQDs in an in vitro model mimicking the real 3D cell organisms, we prepared cellular spheroids derived from a human hepatoma cell line (i.e., HepG2 cell) by combining Hanging-drop and Suspension culture methods. In detail, the cell suspension was hung on the nonpyrogenic surfaces for three days, followed by suspension culture in a fresh cell culture medium for seven days. The size analysis of 3D cell spheroids over timelapse demonstrated their narrow size distribution and well-controlled proliferation/growth as a promising in vitro model. Moreover, 3-day-cultured and 10-day-cultured 3D cell spheroids demonstrated high cell viability under a confocal laser scanning microscope with Live/Dead staining. Furthermore, 10-day-cultured mature spheroids showed rich extracellular matrix (ECM) structure under scanning electron microscopy, demonstrating the similarity with real 3D cell organisms or tissues. Also, the mature cell spheroids showed a dose-dependent effect from a conventional cancer drug (e.g., Doxorubicin; DOX) as an effective tool for drug testing. Overall, the successful formation of 3D cell spheroids demonstrates the potential for more accurate and reliable drug testing in vitro models, in this study, for chiral GQDs.

With prepared chiral GQDs and 3D cell spheroids, chirality-driven enhanced uptake of nanoparticles was evaluated. Interestingly, L-GQD treatment to immature spheroids (e.g., 3-day-cultured spheroids created only with the Hanging-drop method) showed over 15% swelling in spheroid diameter, implying the interaction between ECM and L-GQDs. To perform chiral GQD uptake and transport studies in cellular spheroids, confocal laser scanning microscopic (CLSM) imaging is employed to monitor the transport and uptake of L/D-GQDs into the spheroids over 60 minutes. Qualitatively, L-GQD showed more efficient uptake into 3D cell spheroids in yield and rate, implying that chirality can induce enhanced nanoparticle uptake. Herein, various image processing was performed to qualify and quantify the different GQD uptake and transport to 3D cell spheroids (Figure 1A), including total intensity over 3D space, radial coordinate quantification, and time-dependent transport quantification (Figure 1B). Image data analysis based on molecular transport reveals a 1.7-fold higher apparent diffusion coefficient of L-GQD to 3D cell spheroids than that of D-GQDs, suggesting that the chirality of nanoparticles can improve their transport and uptake into 3D structures of the ECM (Figure 1C). Furthermore, Furthermore, we loaded the L-GQDs with the conventional cancer drug DOX through π-π stacking and tested their efficacy in cancerous cellular spheroids. The DOX-loaded L-GQDs showed more effective drug delivery and higher efficacy for cancerous cellular spheroids than D-GQDs (Figure 1D).

In summary, first, our study successfully developed reliable drug molecules (e.g., DOX-embedding GQDs) representing the specific chirality (e.g., L- or D-) on the surface and their similar characteristics with opposite chirality were appropriate to represent the transport of chiral drugs. Second, we successfully implemented the reliable in-vitro model mimicking the in-vivo circumstance both in physical alignment and chemical composition. In conclusion, by combining two successful models, we demonstrated that the chirality of nanocarriers such as GQDs plays a vital role in the transport and uptake by tumor-like tissue. Therefore, it raises the importance of considering chirality when designing drug carriers, as it can significantly impact their transport and uptake in 3D tissue-like environments.

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Figure 1. GQD uptake quantification via CLSM image processing and chirality-driven enhanced cellular uptake of DOX. (A) Workflow and model equations for GQD transport quantification. (B) Time-lapse mean GQD intensities at specific radiuses of the spheroids. (C) Apparent diffusion coefficients of chiral GQDs into 3D tumor-like cellular spheroids. (D) Quantitative analysis of dead cell area under DOX-load L/D-GQD treatment to cellular spheroids (ns: not significant, **: P<0.01, ***: P<0.001, ****: P<0.0001).