## 4 Applications of CDs in Cancer Theranosis
### Cancer Diagnosis via Fluorescence Imaging
#### 4.1.2 In Vivo Diagnosis  
CDs are becoming widely used for in vivo cancer imaging owing to their low toxicity and high quantum yields, showing promising results in cancer diagnosis. The in vivo cancer imaging is typically performed using mice or zebrafish models to examine the biodistribution, clearance, and uptake of CDs in cancer imaging and treatment. For in vivo imaging, longer excitation wavelengths are preferred as they can penetrate tissues more effectively, facilitating deep tissue imaging. However, most CDs reported so far are those emitting in the blue to green region. [PERSON] et al. used D-glucose and L-aspartic acid (Asp) as carbon sources in pyrolysis to synthesize CD-Asp, which exhibited targeting capability towards C6 brain cancer glioma without conjugation of any additional cancer-targeting ligands [134]. In vivo fluorescence imaging showed rapid accumulation of CD-Asp in the brain 5 min after tail vein administration, with the highest contrast of CD-Asp 15 min after administration (Figure 9a). Furthermore, the ex vivo imaging of the brain confirmed that the CD-Asp targeted the glioma site, showing great potential for the diagnosis of brain cancer cells (Figure 9b). [PERSON] et al. developed large amino acid-mimicking CDs via guanidinium modification (LAAM GUA-CDs) to efficiently load small interfering or silencing RNA (siRNA) to target cancer cells in vivo [153]. In vivo cancer targeting of the LAAM GUA-CDs/Cy5-siRNA was found to accumulate within 1 h and peak at 3 h after IV administration (Figure 9c), while free Cy5-siRNA showed minimal cancer localization due to rapid enzymatic degradation during circulation. Furthermore, the  
Figure 8: (**a**) Confocal laser scanning microscope images of three different cancer cells after incubation with FA-CDs for 4 h at 37 \({}^{\circ}\)C (1 mg CDs/mL). The fluorescence images of (**i**) PC-12, (**ii**) HepG-2, and (**iii**) MCF-7 cells incubated with FA-CDs. The bright field images of (**iv**) PC-12, (**v**) HepG-2, and (**vi**) MCF-7 cells and the overlay images of (**vii**) PC-12, (**vii**) HepG-2, and (**viii**) MCF-7 cells. Scale bar, 20 \(\upmu\)m [151]. (**b**) Confocal laser scanning microscope images of the cell mixture of NIH-3T3 and HeLa cells after incubation (**i**) with and (**ii**) without (as control) FA-CDs (50 \(\upmu\)g CDs/mL) for 6 h at 37 \({}^{\circ}\)C and (**iii**) NIH-3T3 cells incubated with FA-CDs (as another control). (**iv–vi**) The differential interference contrast images of the corresponding cells on the right panels: in the image (**iv**), the arrows indicate NIH-3T3 cells. Scale bar, 20 \(\upmu\)m [152].  
biodistribution analysis revealed that the administration of free Cy5-siRNA resulted in no signal at the cancer and weak fluorescence signals in major organs with the exception of the kidneys, indicating a short circulation time due to renal excretion of the free Cy5-siRNA whereas the LAAM GUA-CDs/Cy5-siRNA demonstrated significantly stronger fluorescence signals at the cancer site compared with other organs owing to their active cancer targeting (Figure 9d). [PERSON] et al. reported red-emissive CDs that could penetrate the nuclei of both cancer cells and cancer stem cells [154]. The DOX was loaded onto the surface of the CDs at a concentration of 30 \(\upmu\)g/mL. The bioimaging and biodistribution of DOX-loaded CDs (CSCNP-R-CDs/DOX) were evaluated in vivo after IV administration into the cancer-bearing Balb/c nude mice. At 6 h after administration, weak fluorescence at the cancer site indicated CSCNP-R-CQDs/DOX accumulation and reached its peak at 12 h after administration, confirming the effective targeting of the CDs at the cancer site. Ex vivo fluorescence imaging of isolated organs, including the cancer, liver, kidney, lung, and brain, further revealed that the cancer site exhibited the strongest fluorescence intensity at 12 h after administration, indicating that the CSCNP-R-CQDs/DOX were mainly concentrated at the cancer.