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  • PSB 1115 Liposomes that are less than nm in diameter

    2021-11-25

    Liposomes that are less than 200nm in diameter are necessary to optimize uptake of liposomes by PSB 1115 (Oesterling et al., 2014). The liposomes constructed in these experiments were found to be sufficiently small in diameter for use in in vivo and in vitro experiments. Even smaller diameters are associated with more effective cell interactions (Oesterling et al., 2014). A negative zeta potential of the liposomes indicates resistance to degradation, or increased shelf life. This value can be changed based on the ratio of PEG-2000 DSPE or cholesterol used in liposomal formulation (Haeri et al., 2014). The PDI is a measurement of the uniformity of size of the liposomes. A PDI of less than 0.2 indicates sufficiently uniform liposomes (Fig. 3). Results from the current experiment indicate liposomes that are of sufficient diameter, loading efficiency, PDI, and zeta potential for experimental use. The prepared liposomes were also found to have stable particle size and zeta potential for almost a year at -20°C(data not shown). All the in vitro studies listed here were performed on PC-12 cells. PC-12 cells line is derived from rat pheochromocytoma of the rat adrenal medulla. PC-12 cells are regarded as a reliable and widely used model of neuronal differentiation. PC-12 cells differentiate terminally into neuronal cells after treatment with NGF which promotes the growth and division of new neuronal cells. NGF causes undifferentiated, adhered PC-12 cells to differentiate into neuronal cells, which can be used as a model for human nerve cells. After differentiation, the cells are dependent upon survival factors like NGF to prevent apoptosis (Vaudry et al., 2002). The removal of NGF without the addition of other growth factors causes apoptosis in neuronal PC-12 cells. Depletion of NGF was observed in previous studies by other groups in the striatum and cortex of the ischemic hemisphere of t-MCAO rats 24h after reperfusion (Lee et al., 1998, Yang et al., 2008). Along the same lines, we observed that after differentiation using NGF, neuronal cells were formed and after withdrawal of NGF the cells underwent apoptosis as indicated by decreased cell viability (Fig. 4, Fig. 5) in the negative control groups compared to the positive control groups in all our in vitro experiments. However we observed prevention of cell apoptosis (cytoprotection) after addition of IRL-1620 (Fig. 4, Fig. 5). This is in agreement with our previous study that showed IRL-1620, in addition to acting as an ETB receptor agonist, has the ability to promote neurogenesis and angiogenesis although the exact mechanism is not completely known (Leonard and Gulati, 2013). We also studied whether IRL-1620 encapsulated in PEGylated liposomes was more efficient than free IRL-1620 in protecting serum-deprived differentiated PC-12 cells when NGF was withdrawn. IRL-1620 is known to promote proliferation and prevent apoptosis; however the concentration of IRL-1620 is crucial factor to these effects. This study shows that different concentrations of IRL-1620 had different effects on the cell viability of differentiated and serum-deprived PC-12 cells. Saturation of ETB receptors seems to be unlikely because as depicted in Fig. 4, 100nM of IRL-1620 resulted in higher cell viability when compared with 50nM. However, this may be due to a nonlinear dose response of IRL-1620 as observed in dose–response PSB 1115 studies. The liposomal IRL-1620 formulation contained enough IRL-1620 to promote proliferation and prevent apoptosis. This explains why the effect of liposomal IRL-1620 on cell viability was at least comparable to the positive control, where the cells survived using NGF. The encapsulation of a number of IRL-1620 molecules in one liposome and internalization of these liposomes by differentiated PC-12 cells could be the reason for higher cell viability in the 1nM liposomal IRL-1620 group compared to the 10nM liposomal IRL-1620. Usually particulate drug carriers are take up better by cells compared to solubilized free drug which could also contribute toward enhanced efficacy of liposomal IRL-1620 (Torchilin, 2005, Immordino et al., 2006). The diminished response in the 10nM liposomal and free IRL-1620 groups is consistent with the dose–response bar graph at the 10nM concentration. To understand the mechanism of increased proliferation of serum-deprived NGF-differentiated neuronal PC-12 cells in the presence of IRL-1620, levels of NGF were checked using an ELISA assay. Results indicate that the increased cell viability in vitro is not due to an increased concentration of NGF. However, the kit used to detect NGF is non-reactive with the following other neural growth factors in the NGF family: BDNF, NT-3, or NT-4. While NGF was the most likely candidate for preventing cell death, the possibility of involvement of other neural growth factors or IRL-1620 itself acting directly on the cells to prevent cell death cannot be neglected. From our experiments it is clear that NGF is not being produced, suggesting further investigation on other growth factors to evaluate exact mechanism of action of IRL-1620 for stimulating proliferation of neuronal cells.