Cancer is the second leading cause of death and is responsible for approximately 9.6 million deaths worldwide in 2018. Among all oncological diseases, lung cancer claims the highest mortality (male: 23.5%; female: 22%) and the second most new cases (male: 13%; female: 12%) in the US. Approximately 40% of newly diagnosed lung cancer patients are in the advanced stage IV, for which platinum-based chemotherapy is the first-line treatment, either by itself or in combination with surgery or radiotherapy. Cisplatin, the first-generation platinum-based anticancer chemotherapeutic agent, has the highest potency against lung cancer but carries many severe adverse effects. Cisplatin also induces drug resistance during long-term chemotherapy. Many more platinum complexes have been investigated as better alternatives, which led to the approval of carboplatin and oxaliplatin by Food and Drug Administration (FDA). In addition, miriplatin suspended in iodolipds (lipiodolization) was approved in Japan for the treatment of hepatocellular carcinoma (HCC) in 2009. Miriplatin has the same non-leaving group as oxaliplatin but different leaving groups of two myristate chains, which make it highly lipophilic. Several characteristics of solid tumors in lung cancer constitute a physiochemical barrier to the homogenous distribution and deep penetration of chemotherapy agents. Nanocarriers provide a promising platform to overcome the physiochemical barrier and to reduce the systemic toxicity of anticancer chemotherapy. In this study, miriplatin is formulated with various lipid-based nanocarriers including micelles and solid lipid nanoparticles (SLNs) thanks to its highly lipophilic structure. The goal of this thesis is to develop and evaluate miriplatin-loaded nano formulations against lung cancer. Miriplatin-loaded formulations were prepared by different methods, including thin film hydration and several scale-up methods including chloroform dripping, chloroform injection, chloroform evaporation, co-solvent evaporation, chloroform slow evaporation and co-solvent slow evaporation. Between the two types of nano formulations under this study, micelles were much smaller (~10 nm in diameter) and more homogeneous (PDI < 0.3), while SLNs were bigger (~ 100 nm in diameter) and more heterogeneous (PDI ~0.8). A quantification method of miriplatin was established using inductively coupled plasma-optical emission spectrometry (ICP-OES). The quantification of platinum recovery from different miriplatin-loaded nano formulations was facilitated by digestion with 70% nitric acid and heating. The co-solvent slow evaporation method to prepare miriplatin-loaded nano formulations improved the platinum recovery prominently from 10% to 70%. Thus, co-solvent slow evaporation has been established as a pharmaceutically viable scale-up method to prepare nano formulations of miriplatin. Miriplatin-loaded nano formulations of different compositions were negatively stained with uranyl acetate and then imaged by transmission electron microscopy (TEM), which showed the formulations’ size and morphology that were consistent with the size and PDI data from dynamic light scattering studies by the Malvern Zetasizer. In the TEM studies, micelles showed a morphology of spherical dots at around 10 nm in diameter while SLNs showed both spherical and rod structures with a size distribution from 50 to 150 nm. A three-dimensional multicellular spheroid (3D MCS) model of A549-iRFP cells was used for in vitro evaluation of the nano formulations’ activity against lung cancer. A549-iRFP cells were engineered from the common lung cancer cell line A549 to stably express the near-infrared fluorescent protein (iRFP). The viability of A549-iRFP 3D MCS after exposure to cisplatin or nano formulations was similar to A549 3D MCS. The anticancer activity of miriplatin-loaded nano formulations against 3D MCS was positively associated with the platinum recovery as quantified by ICP-OES. The miriplatin-loaded nano formulations that had been prepared by the co-solvent slow evaporation method showed substantial anticancer activities against A549 3D MCS and A549-iRFP 3D MCS, which were comparable to cisplatin. Taken together, miriplatin-loaded nano formulations were successfully prepared by co-solvent slow evaporation. The formulations were developed to carry favorable physiochemical properties to enhance the activities of platinum drugs against lung cancer.

Lung cancer claims the highest mortality and the second-most estimated new cases among all oncological diseases [1]. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all newly diagnosed lung cancers [2]. Approximately 40% of newly diagnosed lung cancer patients are stage IV. For stage IIIB/IV NSCLC, cytotoxic combination chemotherapy is standard first-line chemotherapy. A regimen of platinum (cisplatin or carboplatin) plus paclitaxel, gemcitabine, docetaxel, vinorelbine, irinotecan, or pemetrexed is the recommended clinical treatment [3]. Cisplatin is the first-generation platinum-based anti-cancer drug. Although cisplatin is much more effective than other platinum drugs at the same dosage [4], accumulating reports have shown the failure of conventional platinum-based chemotherapy due to various side effects and drug resistance [5]. Miriplatin, a member of platinum drug family, has been approved in Japan in 2009 for transcatheter arterial chemoembolization treatment of hepatocellular carcinoma (HCC) [6]. Miriplatin is a lipophilic platinum drug that contains myristates (14-carbon chains) as leaving groups and diamino cyclohexane as the non-leaving carrier ligand. The application of miriplatin in clinic is limited because it has very poor solubilities both in water and in common organic solvents [7]. The structure of solid tumors and tumor microenvironment (TME) in lung cancer constitute a barrier to the deep penetration of chemotherapy agents, which limits the effectiveness of chemotherapy [8]. Nanoparticles with appropriate properties provide a promising delivery system to overcome the biological and physiochemical barriers that hinder anti-cancer activity [9]. Lipid-based nanoparticles such as liposomes, micelles, and solid lipid nanoparticles (SLNs) can delivery anti-cancer drugs to improve their anti-cancer activities. In this study, we formulated miriplatin into various micelles, liposomes, and SLNs by film-hydration and evaluated their physicochemical properties and anti-cancer activity against NSCLC cells in culture. Miriplatin-loaded formulations with different compositions were successfully prepared by the film-hydration method. Most miriplatin-loaded micelles were more homogeneous and much smaller than miriplatin-loaded liposomes and SLNs. The majority of miriplatin-loaded micelles were about 15 nm in diameter, while SLNs were around 120 nm, and liposomes were about 180 nm. Formulations with a higher molar ratio of PE-PEG2000 had smaller sizes. SLNs loaded with a higher molar ratio of miriplatin in the compositions showed smaller sizes. Inductively coupled plasma mass spectrometry (ICP-MS) and inductively coupled plasma optical emission spectrometry (ICP-OES) techniques were attempted to quantify the platinum element in the formulations. Formulations with a higher molar ratio of PE-PEG2000 had higher recovery of platinum element. Most miriplatin-loaded formulations had higher than 80% platinum recovery. The recovery of intact miriplatin was characterized by HPLC. Miriplatin-loaded micelles had much higher intact miriplatin recovery (about 100%) than SLNs (about 30%). By TEM imaging, the micelles showed the morphology of spherical dots of about 10 nm in diameter while SLNs showed both spherical and rodlike structures of about 120 nm in diameter. The TEM results were consistent with the size and PDI results by the Zetasizer. Three-dimensional multicellular spheroids (3D MCS) of A549 and A549-iRFP cell lines were successfully established as cell culture models to evaluate activity against non-small cell lung cancer. The viability of 3D MCS after 7-days treatment with miriplatin-loaded micelles was about 0%, which was similar to cisplatin. Miriplatin-loaded formulations with a higher molar ratio of PE-PEG2000 in the compositions had higher anti-cancer activity against 3D MCS. The anticancer activity of miriplatin-loaded formulations against 3D MCS was positively associated with the recovery of intact miriplatin from the formulations. The IC50 value of miriplatin-loaded micelles against A549-iRFP 3D MCS was around 25 µM, while that of cisplatin was 84.78 µM. In summary, the reported lipid-based nano-formulations represent a promising delivery system of miriplatin against NSCLC.