PEG-PG-PCL, PEEP-PCL, PEG-PCL and PEG-DSPE micelles are commonly used for novel nano-preparations. As shown in Figure 1, PEG-PG-PCL has a unique structure of linear-hyperbranched blocks with many arms, whereas PEEP-PCL, PEG-PCL and PEG-DSPE are long chain polymers. Despite the structural differences among these nanomaterials, they all exhibit amphiphilic properties, making it possible for them to form micelles and load drugs. Among these four kinds of micelles, the size of the PEG-PG-PCL micelles was larger than the other three types of micelles; however, the zeta-potentials of these micelles in PBS (pH 7.4) did not differ. The CMCs were all below 3 μg/ml, and the micelle concentrations in our investigation were higher than 5 μg/ml (even in vivo, the micelle concentration was approximately 80 μg/ml), indicating the micelles could maintain their micellar state in the present study in vitro and in vivo.
Two cell lines, J774.A1 and Eahy.926, were used in this study. J774.A1 cells are mononuclear macrophages. The cell line is used as an immune cell model to study the immune responses after stimulation by the micelles. Eahy.926 cells are vascular endothelial cells and are used as a cellular model of the vascular wall. Eahy.926 cells are used to study the toxicity of nanomaterials on the vascular endothelium [32, 33]. The reason for the choice of these two cells lies in the application of these nanomaterials in drug delivery. After the micelles are administered intravenously, they (or their original materials and degradation products) typically persist in the blood circulation and have direct access to the blood cells (including immune cells, such as LYM, WBC and macrophages) and vascular endothelial cells.
Macrophages are very sensitive cells in the blood and could respond rapidly to acute nanoparticle toxicity. Normally, macrophages exist in a resting state. When they are stimulated to become active, macrophages grow in volume and are able to engulf foreign antigens and secrete cytokines . As we have demonstrated, J774.A1 cells treated with the micelles exhibited no significant change in shape (Figure 3); however, upon further investigation, we observed an increase in J774.A1 cell size in the PEG-PG-PCL and PEEP-PCL groups (Figure 4). Moreover, PEG-PG-PCL and PEEP-PCL micelles induced TNF, and all four kinds of micelles induced an increase in MCP-1 (Figure 5), indicating that the stimulation process was initiated by the contact between micelles and J774.A1 cells.
During (or after) the stimulation process, protein and ATP content in macrophages increases, oxygen consumption increases significantly, cellular enzymatic activity increases, and the generation of ROS increases . ROS levels significantly increased after treatment with the micelles; PEG-PG-PCL micelles induced the highest level of ROS, and PEEP-PCL micelles induced the second-highest levels (Figure 6). These results suggested that treatment with micelles might elicit an immunological response, resulting from the increasing of ROS levels.
Among all of the micelles, PEG-PG-PCL micelles, followed by PEEP-PCL micelles, most strongly stimulated J774.A1 cells. The reason for difference might lie in the size difference of the micelles and the structure difference of these nanomaterials; phagocytosis generally occurs when particle sizes are larger than 100 nm . The size of the PEG-PG-PCL micelles was 173 nm. Thus, J774.A1 cells may have recognized the PEG-PG-PCL micelles and activated. However, there is no clear reason to explain the phenomenon in PEEP-PCL micelles. We speculated that the interaction might be related to the negatively charge of PEEP-PCL micelles. PEEP, with many phosphoesters, deduced the micelles negatively charged in aqueous solution (-14.4 mV), although it was nearly neutral (-4.75 mV) when added to PBS due to buffer action. As it is reported, negatively charged nanoaprticles can show stronger interaction with cells through nonspecific binding and clustering of the particles on cationic sites on the plasma membrane (that are relatively scarcer than negatively charged domains) compared to nanoparticles with neutral surfaces . In another study on how hydrophilic and hydrophobic structures influence micelle transport in epithelial MDCK cells, PEEP-PCL micelles indeed exhibited unique behavior in terms of endocytosis, exocytosis, organelles colocalization and transcytosis. For example, PEEP-PCL micelles were easier to locate in lysosomes than endoplasmic reticulum in the first ten minutes, while PEG-PCL micelles were concentrated more in endoplasmic reticulum in the first 10 minutes .
In the study on Eahy.926 cells, cytotoxicity and apoptosis analyses were conducted. Cytotoxicity and apoptosis analyses are typically used to detect the direct damage of nanocarriers or their degradation products on vascular endothelial cells. The results of the cytotoxicity and apoptosis revealed that among these four kinds of micelles, PEG-DSPE micelles (83.3 μg/ml) significantly inhibited the growth of Eahy.926 cells (Figure 7) and increased the percentage of late apoptotic and necrotic cells (Figure 8). Apoptosis is a form of programmed cell death that occurs through the activation of cell-intrinsic suicide machinery . The increasing percentage of late apoptotic and necrotic cells indicates that PEG-DSPE micelles may trigger apoptosis, leading to the inhibition of cell growth that was observed. We considered that the higher cytotoxicity and apoptosis of PEG-DSPE micelles might result from the higher cellular uptake ability of PEG-DSPE micelles. In our previous study, it was shown that the uptake of the three micelles ranked as PEG-DSPE > PEG-PCL > PEEP-PCL . We considered that as a type of phospholipid with a similar structure to the cell membrane, DSPE had good affinity with cell membrane and deduced higher uptake.
Monitoring cell membrane fluidity is based on the principle that materials exhibit a fat-soluble structure that can insert into the cell membrane and affect its properties. However, in our research, there was no evidence demonstrating that these micelles had any effects on the membrane fluidity of Eahy.926 cells at the given concentration (Figure 9). It is possible that the contact process between the micelles and cells was too long, allowing the cell membrane sufficient time to recover to its origin state, and the interaction between the cell membrane and the micelle process could not be observed.
There were certain differences in the influence of micelles on J774.A1 cells and Eahy.926 cells. J774.A1 cells were stimulated largely by PEG-PG-PCL and PEEP-PCL micelles, but Eahy.926 cells were influenced mainly by PEG-DSPE micelles. We considered the reason for this difference might be associated with the different characteristics of J774.A1 and Eahy.926 cells. As Lewinski et al. reported, from the toxicity study on the effect of C60 exposure under various experimental conditions with different cell lines, the results indeed were related to cell type . J774.A1 cells are of macrophages that can rapidly respond to the environmental changes by secreting various factors. In contrast, Eahy.926 cells are human endothelial cells that exhibit different functions in the human body. As they have unique functions, different reactions to similar stimulations are reasonable.
As we have observed certain physical and chemical changes in the cell models above, it remains to be understood whether these cellular changes occur and cause pathophysiological changes in vivo? To answer this question, we conducted a toxicity study in KM mice. There were no obvious body weight and behavior changes post-exposure for all the micelle groups and control (data not shown). Generally, when micelles are injected into vessels of mice, they immediately contact the blood cells and may be delivered to every possible organ and enter cells . We therefore monitored the changes in complete blood cell counts, lymphocyte subset analysis, plasma inflammatory cytokines and changes in target organs, such as the heart, liver, spleen, lung, kidney and thymus. After multiple doses, the micelles of these nanomaterials caused certain changes in blood cells; for example, PEG-PCL micelles decreased the level of WBC, LYM and MID, which are all immune cells that may influence the levels of inflammatory cytokines. PEG-DSPE and PEEP-PCL increased the level of GRN, another type of immune cells, whereas PEG-PG-PCL increased the number of PLT (Figure 10), which may induce hemorrhage, thrombosis or splenomegaly. In the lymphocyte subpopulation analysis, PEEP-PCL induce some increase in the CD4+ lymphocyte subpopulation (Figure 11), indicating that PEEP-PCL micelles could stimulate the immune system.
As a result of changes in the circulatory system, other changes in inflammatory factors and organs could follow. However, in our research, rapid changes in inflammatory factors and pathological target organs changes were not observed. One possible explanation might be the time point at which we detected inflammation factors, 24 h after injection, which was long enough for the micelles and inflammation factors to be cleared by the circulation system of mice. Another reason might be that the micelles did stimulate the blood cells and immune cells; however, the stimulation was not strong enough to cause obvious changes in our detection. Alternatively, the micelles of these nanomaterials in circulatory system may have stimulated lymphocytes of blood, which led to cellular stress and subsequent differentiation; this defense system to avoid a further damage on target organs, and the short-term secretion effect of inflammatory cell stress was eliminated after 24 h.
When these in vitro (on cells) and in vivo (on mice) results were compared comprehensively, we found that the toxicity in vivo was not as significant as that in vitro. There are several possible reasons: (1) The micelles in vivo exist mainly in the blood system, which is a dynamic environment, whereas the in vitro studies are performed in a relatively static environment, thereby providing more chances for micelles to contact cells. (2) The body’s innate ability to self-regulate is much more prevalent than regulation in cultured cells.