# Improved Anticancer Activity of the Malloapelta B-Nanoliposomal Complex against Lung Carcinoma
## 4 Discussion
The investigation of liposomal stability in this study showed that malB-nanoliposomes were stable at 4 \({}^{\circ}\)C in PBS for approximately 30 days, as the average size of the liposomes was still \(<\)200 nm and they had a slight negative charge. However, the PDI of liposomes was significantly increased at around 30 days to almost twice that on day 1 (data not shown). The PDI parameter is an indicator of whether the formed liposomes are monodispersed and have an average size distribution. The PDI should be as low as possible, as higher PDI indicates a broader size distribution of nanoparticles. Thus, the results of the present study suggested that malB-liposomes exhibited an appropriate PDI and this characteristic seems to change after 30 days of storage at 4 \({}^{\circ}\)C in PBS. With regard to assessment of bioactivity, malB encapsulated in nanoliposomes still showed strong anticancer activities in vitro that were comparable to those of the free form. Indeed, the nanoliposomal formulation inhibited cancer cell survival after 48 h with IC\({}_{50}\) values ranging from 11.86 to 13.12 uM, which was slightly higher than that of free malB (8.67 - 10.35 uM). However, the anti-clonogenic activity of malB-nanoliposomes was more obvious than that of the free compound in the A549 colony-forming assay. In addition, malB and malB-nanoliposomes affected the activity of caspase 3, a crucial protease that mediates apoptosis. This caspase catalyzes the specific cleavage of many key cellular proteins, and plays an important role in apoptotic chromatin condensation and DNA fragmentation [22; 23]. The significant caspase 3 induction and activation by malB and malB-nanoliposomes, which contributed to both intrinsic and extrinsic apoptotic pathways, were reported here for the first time. Furthermore, the improved cytotoxic and antimurigenic effects of malB-nanoliposomes were also verified in tumor-bearing mice. The malB-nanoliposomes showed the ability to effectively inhibit tumor growth in comparison with negative controls as well as with free malB at the same concentration of 5 mg/kg. There have been many studies on loading of potential anticancer agents into nanoliposomes to improve their activities. Those studies showed that the encapsulation of drugs into liposomes reduces toxicity, improves bioactivity, and increases the circulation time of drugs in the body, leading to increased effectiveness of these drugs in clinical treatment [24]. The intraperitoneal (i.p.) administration of liposomes also has advantages, such as reduction of local toxicity [25]. In addition, when administered by the i.p. route, the drug is absorbed into the organs through the peritoneum or the lymphatic system [26]. Drugs packaged in liposomes could be stable for longer period in the abdominal cavity or in the lymph vessels, thus improving the effectiveness of treatment for peritoneal carcinomas, such as ovarian cancer and liver cancer [27]. However, the uptake of the drug into the blood via the abdominal cavity also depends on the size of the liposomes. [PERSON] reported that liposomes approximately 100 nm in diameter show a high concentration in blood equivalent to intravenous (i.v.) administration, and could therefore be used to treat cancer far from the peritoneum [28]. On the other hand, PEGylation of liposomes plays a central role in prolonging their circulation in the blood. PEGylation helps protect drug-nanoliposomes from mononuclear phagocytes by impeding the absorption of opsonin protein in the circulation on the liposome surface, which would minimize clearance of the drug by these cells [29; 30]. The characteristics of PEGylated liposomes allow them to circulate longer in the bloodstream than free drugs, leading to increased effectiveness in cancer treatment. In the present study, the PEG-cholesterol components in the malB-nanoliposome complex may have enhanced the antitumor activity in comparison with the free drug.