(T1230-04-20) Theranostics Peptide Functionalized Graphene Oxide Quantum Dots for Targeted Detection and Treatment of Colorectal Cancer

Purpose: Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide (1). Recently, increasing evidence has revealed that the antigen Placenta-specific protein 1 (PLAC-1) is overexpressed in CRC (2). The aim of this work is to develop a theranostics agent for targeted detection and treatment of CRC using graphene oxide quantum dots (QD) labeled with a specific peptide (GILGFVFTL) able to target CRC cells bearing PLAC-1.

Methods: Peptide functionalized QD (QD-P) were obtained by covalent coupling between QD and peptide using 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride. The physicochemical and morphological characteristics of the prepared QD-P were determined using Fourier Transform infrared spectroscopy (FT-IR), Differential scanning calorimetry (DSC), Raman spectroscopy, Energy-dispersive X-ray spectroscopy (EDS), Dynamic light scattering, and transmission electron microscopy (TEM). Confocal microscopy was used to evaluate the uptake of QD and QD-P in HCT-116, HT-29 and LS-180 CRC cell lines. Immunofluorescence assay was performed to evaluate the selective targeting of antigen PLAC-1 overexpressed on HT-29 and HCT-116 cells. Cell proliferation and IC₅₀ of QD and QD-P were measured using 3-(4, 5-dimethyldiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) cytotoxicity assay kit. Invasiveness of CRC cell lines after treatment with QD-P was evaluated using Transwell chambers of 8 μm pores pre-coated with extracellular matrix proteins. PLAC-1 expression in HCT-116 and HT-29 cells after treatment with QD and QD-P was measured using flow cytometry.

Results: FT-IR analysis showed characteristic bands for O-H stretch, C=O stretch, N-H stretch, amide II and aromatic ring of phenylalanine in QD-P structure (Figure 1). EDS spectrum of QD-P showed the presence of carbon, oxygen and nitrogen elements. In DSC thermograms, QD-P and peptide presented similar melting point (26.01 and 27.21 °C, respectively). Raman spectra of QD-P showed a shift at 1002-1004 cm^-1 corresponding to phenylalanine in the peptide chain. Zeta potential measurement showed that the charge on the QD changed from -10.3 mV to 27.8 mV after coupling with the peptide. TEM micrographs showed that QD were spherical in shape with an average size of 10 nm, which remarkably increased after coupling with the peptide. Confocal imaging showed that cellular uptake of QD (100 µg/mL) in HT-29, HCT-116 and LS-180 cells is time-dependent (Figure 2). The internalization of QD-P in HT-29 and HCT-116 significantly increased compared to QD because of the expression of the antigen PLAC-1 on cell membrane. On the other hand, the internalization of QD and QD-P by LS-180 cells lacking the antigen on their cell surface was comparable. These results showed the ability of the peptide to specifically recognize the antigen PLAC-1 expressed on HCT-116 and HT-29 cells, increasing the specific uptake of QD-P in those cells. Immunofluorescence analysis using HCT-116 and HT-29 cells showed that QD (100 µg/mL) did not interact with the antigen PLAC-1 after 2 h of treatment while QD-P co-localized with the antigen PLAC-1 which confirm selective targeting due to the presence of the functionalization of the QD surface with the peptide. MTT assay showed that treatment with 100 µg/mL of QD and QD-P did not significantly affect the cell viability except for HCT-116, which exhibited more sensitivity to the treatment (Figure 3). On the other hand, treatment with 300 µg/mL of QD showed a significant decrease in cell viability for all cells. However, treatment with QD-P (300 µg/mL) showed a further decrease in cell viability for HCT-116 and HT-29 cells only. The time-dependent decrease in cell viability after 24 h of treatment was more significant in HT-29 cells. The results were in correlation with the IC₅₀ measurements which showed that HCT-116 were more sensitive after 2 h of treatment with QD-P while HT-29 cells showed more significant time-dependent reduction in cell proliferation when treated with QD-P for longer time. In addition, treatment with QD-P resulted in a significant reduction in cell invasiveness in HCT-116 and HT-29 cells when compared to non-functionalized QD. For LS-180 cell, there was no significant reduction in cell migration showing the specificity of the QD-P in targeting cells bearing the surface antigen. Flow cytometry analysis of cells treated with 200 µg/mL QD-P for 24 h showed a downregulation in PLAC-1 expression of 14.4 % and 12.4% in HCT-116 and in HT-29, respectively.

Conclusion: QD were functionalized with a specific peptide (GILGFVFTL) and characterized with different analytical techniques to confirm the covalent coupling between the QD and the peptide. The prepared QD-P showed a significant increase in targeting and specific uptake in cells expressing the antigen PLAC-1 compared to non-functionalized QD. Treatment with QD-P also increased the cell cytotoxicity and downregulated the expression of PLAC-1, reducing in this way, the migration and the invasiveness of HT-29 and HCT-116 cells. These results highlight the potential use of QD-P as a theranostics agent for the detection and treatment of CRC cells expressing the antigen PLAC-1.

References: 1. P. Rawla, T. Sunkara, A. Barsouk, Prz. Gastroenterol. 2019, 14, 89.
2. Ji. Ma, S. Zhang, D. Liang, L. Li, J. Du, C. Pan, C. Zhang, Y. Chen. European Journal of Cancer Prevention. 202, 30, 220.

Acknowledgements: This research was supported by University of Sharjah (UOS) targeted research project funds, 2101110345 and the UOS College of Graduate Studies research project funds.


Figure 1. Physicochemical characterization of synthesized QD-P showing FTIR spectra A, DSC thermograms (B) and Raman Spectra of QD, peptide and QD-P


Figure 2. Cellular uptake studies in HT-29, HCT-116, LS-180 cells (A). Immunofluorescence studies in HT-29 and HCT-116 cells showing QD and QD-P cellular interaction with the antigen PLAC-1 (B) (magnification 60×). In blue the nuclei are stained with DAPI, in green the QD and QD-P, in red the antigen PLAC-1. Scale bar 5 µm.


Figure 3. Invasion assay on HT-29 (A), HCT-116 (B), LS-180 (C) cells treated with QD and QD-P (300 µg/mL) for 24 h (magnification 10x). The bars represent the SEM. *p < 0.05 **p < 0.005.