For poorly water-soluble or lipophilic drugs, their clinical efficacy is often hampered by their poor aqueous solubility, which is one of the main factors to influence drug oral bioavailability. Excepting aqueous solubility, the needs to overcome the biological epithelial barriers such as intestinal epithelial and blood brain barrier etc. and to facilitate the drug absorption are other major challenges in drug delivery although the mechanism for several well developed drug delivery systems (DDS) such as nanocrystals (NCs) and inclusion complexes to overcome epithelial barrier and related transport mechanisms is not fully understood. In order to improve the solubility of poorly water-soluble drugs and address the related transport mechanisms for NCs and inclusion complex with Cucurbit[7]uril, a highly lipophilic florescent marker, coumarin 6 (C6), has been selected as the model compound. Madin-Darby canine kidney II (MDCKII) and larval zebrafish were selected as the in vitro epithelial barrier model and in vivo model, respectively, to evaluate the transport mechanisms of two DDS. The first developed DDS in this study is drug NCs, in which a stabilizer is absorbed on the surface of the drug NCs with high drug loading. NCs represent a widely commercialized approach to improve solubility and oral bioavailability of many poorly water-soluble drugs. Although NCs are often utilized in oral formulations of many commercial products to deliver lipophilic drug, their transport mechanisms are not fully understood. This section aimed to explore the particle size effect on the transport mechanism of NCs using in vitro MDCKII cells and in vivo larval zebrafish models. C6 was selected as the model compound and has been formulated into NCs with the particle sizes of 67.5±5.2 nm and 190±9.2 nm, iv respectively. TEM images revealed that both NCs were uniformly distributed with spherical morphology, which is in agreement with the DLS analysis result. Both batches of NCs maintained their crystalline state in the fabricated NCs as that of C6, although the crystallinity has been reduced to a certain extent, as confirmed by both DSC and PXRD. As few C6-NCs were dissolved in vitro dissolution study, they could be utilized as the NCs form in the whole intracellular tracking pathways among organelles. Our results demonstrated that lipid raft pathway mediated the endocytosis, whereas lipid raft, ER/Golgi and Golgi/plasma membrane pathways were involved in exocytosis and transcytosis process for 70 nm NCs, and the NCs accumulated in lysosome and endoplasmic reticulum (ER) as the major destinations. However, 200 nm NCs accumulated more in lysosome, where lipid raft pathways were also involved in endocytosis process. In order to investigate the nanocrystal integrity in the MDCKII cell and larval zebrafish during uptake and transport studies, C6-DiI hybrid NCs with the particle size of ~140 nm has been fabricated (C6 and DiI were co-precipitated as NCs) and the integrity of the nanoparticles was observed by Fluorescence Resonance Energy Transfer (FRET) imaging. It was found that the C6-DiI hybrid NCs remained stable for about 30 min during the particle internalization process (i.e. endocytosis and exocytosis), when incubated with MDCKII cells. With extended uptake time, the added C6-DiI hybrid NCs incubated with the cells was gradually dissolved. When the incubated C6-DiI hybrid NCs were withdrawn after 30 mins incubation, the C6- DiI hybrid NCs will maintain stable for the another 30 mins in the cell line, indicating that the C6-DiI hybrid NCs will be complete and stable for about 1 h in MDCKII cells. When C6-DiI hybrid NCs incubated in larval zebrafish, they were maintained stable for about 2 h. v The second DDS we have investigated is the inclusion complexes with macrocyclic CB[7]. Due to its superior water-solubility and compatible size with various organic and organometallic drug molecules, CB[7] has received the greatest attention as a potential drug delivery vehicle although few studies have been performed to elucidate their transport mechanisms. In this section, the 1:1 guest–host complexes between C6 and CB[7] have been prepared and confirmed by fluorescence spectroscopy. The fluorescence intensity within the MDCKII cells gradually decreased from 5 min to 60 min, which is likely attributed to the initial CB[7] assisted C6 uptake in the complex formation and subsequent release of free C6 in the cytoplasm. In contrast, free C6 (either solubilized by tween-80 or C6 suspension) exhibited little uptake by the MDCKII cells regardless of incubation time. In the larval zebrafish, strong fluorescence was observed in the C6@CB[7] complex treated groups, and no obvious or weak fluorescence was observed in zebrafish treated with free C6 suspension or tween-80 solubilized C6. Through the inhibition study by MβCD, filipin, nystatin and EIPA, it was found that clathrin and lipid raft, as well as macropinocytosis were mediated endocytosis and exocytosis of the complex in the absorption process. In summary, two DDS including C6-NCs and C6 inclusion complexes with CB[7] were employed to investigate the transport mechanism using both in vitro and in vivo models. Different transport pathways and cellular destinations have been observed in the transport of two difference sized batches of NCs. Moreover, formulation of lipophilic compounds as the nanosized DDS would enhance their cellular uptake as well as the transport. This study also provides solid evidence to support the use of CB[7] as a carrier for lipophilic drugs in order to enhance their cellular uptake and in vivo absorption. Moreover, as a complicated tool, this study vi has developed larval zebrafish as a dynamic and transparent in vivo model to study transport mechanism as well as the bio-fate of the NCs and inclusion complexes of lipophilic marker C6, which may provide a useful in vivo fast screening model in future work.