Wenqiu Huang

Failure of chemotherapy may be caused by dose-dependent negative effects on normal cells and inefficiency caused by fast blood clearance, low intratumoral and intracellular accumulation and drug resistance. Nanoparticles (NPs), particularly liposomes with intrinsic amphiphilic properties, offer a potential solution for improving efficiency and efficacy while reducing side effects by modifying drug pharmacokinetics and delivery.

Liposomes are lipid vesicles with a stable bilayer membrane that can protect drugs from degradation in the bloodstream and changes drug pharmacokinetics mainly by prolonging circulation time. These advantages allow for drug accumulation at the tumor site and reduced or altered side-effects. In chapter 1, we describe strategies relating to smart drug delivery systems (SDDS) to overcome limitations of the chemotherapeutic doxorubicin (DXR) in the clinical setting. We show the use of liposomal DXR formulation as a basic model for optimizing SDDS and explore the characteristics of nano-drug-based delivery systems and intracellular behavior of the free drug.

In chapter 2, We explored various ways to enhance the effectiveness of antitumor treatment using nano-platforms, such as targeted delivery through ligand conjugation and responsive nanoparticles. Specifically, we underscore the potential of thermosensitive liposomes (TSLs) triggered by hyperthermia for achieving controlled release of their payload in a spatiotemporal manner. Combination of TSLs with hyperthermia enables a systemic treatment approach with emphasis on local delivery to obtain a systemic effect and local control.

In chapter 3, we addressed the issue of labeling instability of lipid-based NPs, which can affect reliability of the evaluation drug delivery efficiency. We provide insight for designing fluorescent NPs to address this issue resulting from external factors such as physiological compounds and the inherent factors of the lipophilic fluorescent probes. Importantly, we highlighted the advantages and necessity of using a double labeling strategy to accurately track NPs by high resolution microscopy.

In chapter 4, DXR-DNA interaction in the nucleus was identified in living tumor cells using fluorescent lifetime imaging (FLIM). We demonstrated that DXR has three statuses when in the nucleus, each with a unique sub-lifetime (τ): free DXR exhibited a value of 1 ns at concentration below 460 μM and biological pH conditions, DNA intercalated DXR had a short τ value of 0.35 ns and groove binding DXR exhibited a long value of 1.9 ns. The analysis of the amplitude weighted mean lifetime (τw) and amplitude contribution helps us to understand the DXR/DNA binding kinetics and the potential reasons for the dynamic changes in living nucleus. Moreover, a similar time-dependent drug-nucleus interaction was observed among three tumor cell lines which have similar IC 50 values: τw decreased before 5 h and then increased after adding DXR for 5 h. This recovery in lifetime may correspond to the reduced drug concentration in nucleus, indicating a potent drug resistance mechanism resulting from protective activities in both the nucleus and cytoplasm.

In chapter 5, we discussed how the distribution of DXR in the cytoplasm affects DXR interaction within the nucleus of tumor cells using FLIM. Our findings suggest that the drug lifetime in the cytoplasm can be tri-exponential fitted, with sub-lifetimes similar to those fitted in the nucleus in chapter 4: besides the free DXR below 460 μM showing a value of 1 ns, we observed distinct values for other two sub-lifetimes, indicating different conformations of DXR in cytoplasmic compartments.

When using Doxil as a model, the short τ (0.135 ns) and long τ (4.5 ns) indicated the concentrated drug in the core center and lipid-interacted drug, respectively. These values were consistent with those detected in cytoplasm. By evaluating the amplitude contributions relating to various drug dosing, tumor cell lines, as well as treatment duration with the drug, the influence of cytoplasmic drug behavior on nuclear behavior was underscored. Additionally, the fitted components in the images segmented DXR signals into three sub-statuses in cytoplasm and nucleus, allowing clear observation of the time-dependent intracellular distribution. We concluded that drug sequestration by lysosomes and interaction with cell membrane structures are primary factors contributing to drug redistribution from nucleus to cytoplasm, possibly related to drug resistance.

In chapter 6, we have deliberated on the benefits and practical applications of our work as outlined in this thesis. The insights we have gained may serve as a valuable guide for researchers seeking to optimize drug delivery systems, ultimately leading to improved clinical translation.