Development Of Novel Fluorescent Carbon Dots And Their Application In Optical Imaging Of Brain Tumor

Brain tumors are the most life-threatening diseases due to their aggressive pathological characteristics and lack of advanced diagnostic techniques. Even traditional imaging techniques are able to visualize brain tumors, their capability to diagnose tumors are normally at advanced stages and/or with metastasis, leading to missing the golden chance for tumor therapy.A variety of medical imaging techniques, including magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), and positron emission tomography (PET), have been applied for the diagnosis of brain tumors. MRI possesses high spatial resolution but relatively low sensitivity, while SPECT and PET display high sensitivity but with low spatial resolution. Optical imaging has been emerging as a novel technique and has attracted increasing attention due to its high spatial resolution and sensitivity. The development of nano-based probes intensively broadens the application of optical imaging in the diagnosis of brain tumors.Nano techno logy has been extensively applied in the field of biomedicine. Fluorescent carbon dot emerges as a novel fluorescent nanomaterial and possesses many advantages, including resistance to photobleaching, high photostability and biocompatibility, and low toxicity and ease of functionalization. However, the emission wavelength of carbon dots ranges from 400 nm to 500 nm, which ususlly has low tissue penetration depth and may not be differentiated from autofluorescence of the tissues. Systemic administrated carbon dots display ubiquitous distribution and non-selectivity leads to low contrast between normal and tumor tissues. In addition, carbon dot possesses short half life in vivo due to small particle size and surface properties. In present study, we attempted to design a series of brain tumor-targeted carbon dots for in vivo imaging. We screened a variety of origins of carbon and identified carbon dots with red light fluorescence, which was further modified with PEG to prolong blood circulation. Angiopep-2 (ANG) or c[RGDyK] (RGD) peptide was functionalized on the surface of PEGylated carbon dots to realize tumor-targeted imaging. ANG is a ligand of low density lipoprotein-related protein (LRP) and is capable of traversing the blood-brain barrier (BBB) by receptor-mediated transcytosis. Modified ANG was envisioned to inspire two-order brain tumor-targeted imaging of carbon dots since LRP is also overexpressed on brain tumor cells. RGD peptide was able to facilitate tumor targeting through specific binding with integrin αvβ3, which is overexpressed on both neovasculature and tumor cells. This dissertation is composed of three chapters:In the first chapter, spider silk was chosen as carbon origin to prepare carbon dots (termed CDs-1) with a mean diameter of 5 nm and emission peak wavelength at 447 nm. CDs-1 was taken up by glioblastoma U87MG cells with dose-dependent fashion, and the intracellular dots displayed colocalization with lysosome and mitochondria. In spite of high biocompatibility, short emission wavelength of CDs-1 limits further in vivo applications.In the second chapter, we prepared carbon dots (termed CDs-2) with a mean diameter of 5 nm and emission peak wavelength at 500 nm by pyrolyzing glycine at high temperature. CDs-2 was taken up by rat glioblastoma C6 cells with dose- and time-dependent fashions, and the intracellular dots displayed colocalization with lysosome and mitochondria and rapidly escaped from the organelles. In vivo, CDs-2 was able to target orthotopic glioblastoma xenograft and possessed high biocompatibility. However, CDs-2 still had to be optimized to extend the emission wavelength.In the third chapter, the emission wavelength of carbon dots was extended by optimizing the mixing ratio between glutamic acid and sucrose. A novel carbon dot (CDs-3) with a mean diameter of 5 nm and effective emission wavelength at 600 nm was developed by pyrolyzing glutamic acid/sucrose (10:1) at high temperature. CDs-3 was further modified with PEG and peptide to prolong blood circulation and to facilitate glioblastoma-targeting imaging. CDs-3 was taken up by glioma cells with dose- and time-dependent fashion, and the modification of ANG or RGD enhanced uptake capacity. When incubated with human plasma, PEG modified CDs-3 displayed significantly lower absorption capacity of plasma proteins, indicating the potential for prolonging blood circulation. Peptide modified CDs-3 demonstrated low cytotoxicity and excellent biocompatibility. In vivo, biodistribution of ANG (ANG-PEG-CDs-3), RGD (RGD-PEG-CDs-3) or PEG (PEG-CDs-3) modified CDs-3 was investigated in orthotopic glioblastoma-bearing nude mice and subcutaneous breast carcinoma-bearing nude mice respectively, and the imaging efficiency was evaluated. The results indicated that both ANG and RGD modification were capable of inspiring tumor-targeted accumulation of CDs-3. ANG-PEG-CDs-3 was superior to RGD-PEG-CDs-3 for glioblastoma-targeted imaging. RGD-PEG-CDs-3 was suitable for the imaging of subcutaneous breast carcinoma.In conclusion, we developed a novel carbon dots that possessed high biocompatibility and red-light fluorescence. When modified with tumor-targeted ligands, such as ANG and RGD, the carbon dots enabled tumor-targeted imaging with high target to background signal ratio. In view of the efficacy of tumor-targeted imaging, the present study provided promising tools for the diagnosis of early-stage tumors.