(B) Assessment of the intracellular uptake of liposomes by A549 tumor cells using fluorescence microscopy. PEI-1, PEI-2, PEI-3, and PEI-4 represent PEI contents of 10%, 40%, 70%, and 100% (w/w total lipid) in liposomal formulations, respectively. Error bar represents mean ± SD (n = 3); *p < 0.001. HIF inhibitor Cytotoxicity assay Prior to assessing the in vivo localization of DSPE-PEI-2 liposomes,

the in vitro cytotoxicity of free DOX (positive control), control liposomes (negative control), and DSPE-PEI-2 liposomes was measured in A549 cells using an MTT assay (Figure 4). Free DOX was found to be more cytotoxic to A549 cells than liposomal DOX due to the higher cellular uptake of free DOX by tumor cells via diffusion mechanisms [26, 27]. Furthermore, DSPE-PEI-2 (cationic liposomes) also showed significantly higher cytotoxicity compared to control liposomes (p < 0.01). The lower cytotoxicity of control

liposomes may be a result of their low intracellular uptake. Cellular uptake of negatively charged control liposomes was inhibited as demonstrated by the measured zeta potential (Figure 2C) and Hydroxychloroquine molecular weight by the flow cytometric study (Figure 3A). DSPE-PEI-2 liposomes, on the other hand, do interact electrostatically with A549 cell membranes, resulting in increased cytotoxicity of DOX-loaded DSPE-PEI liposomes. Figure 4 Cytotoxicity after liposomal DOX uptake in A549 cells. Error bar represents mean ± SD (n = 3); *p < 0.05. Tumor tissue localization of liposomes The possible role of cationic charge in enhancing the accumulation of liposomes in tumor tissue was assessed by fluorescence microscopy. Figure 5 shows the localization of free calcein, control liposomes (negative charge), and DSPE-PEI-2 liposomes (positive charge) in tumor-bearing mice after intratumoral injection. As shown in Figure 5, the image of DSPE-PEI-2 liposomes exhibits prominent fluorescence 10 min after

injection, and DSPE-PEI-2 liposomes at the tumor site show a longer retention time (240 min) than either control liposomes or free calcein. This result implies that the interaction of tumor vessels Immune system with cationic liposomes, specifically with DSPE-PEI-2 liposomes, may occur electrostatically between the negative cell surfaces and positive DSPE-PEI-2 liposomes. The observed effect is likely a result of the surface charge of the cationic liposomes that were not taken up by the tumor tissue, resulting in an enhancement of the localization efficiency of the cationic liposomes. Toward increasing the localization of payloads, extensive research investigation has been carried out into methods of modifying various carriers including ligand-labeled liposomes [28], hydrogel-based intratumoral injections [7], and magnetic-based carriers [29]. Although these investigations have yielded promising results, the additional formulations of such carrier systems require optimization.