The results of this study indicate that CurcuEmulsomes can successfully entrap curcumin inside the inner solid matrix composed of tripalmitin surrounded by phospholipids. The stable formulations are spherical in shape and preserve the surface characteristics of the nanocarrier. Most important, the solubility of curcumin is increased up to 0.11 mg/ml by means of CurcuEmulsomes, corresponding to an improvement in solubility by 10,000 times. Thus CurcuEmulsomes can achieve the effective concentrations of curcumin (i.e. 10–50 μM) [25, 26], and facilitate the delivery of bioactive molecules into the cell in vitro.
In the literature, various encapsulation approaches like diblock copolymers [35, 36], hydrophobically modified starch , beta-casein micelles , lipid nanoemulsions , curcumin-rubusoside complexes , cyclodextrin assemblies [40, 41], liposomes , curcumin-nanodisk  and polymeric NanoCurc™ formulations  have been successfully applied to increase the solubility and thereby the delivery of curcumin. Encapsulation of curcumin in a pluronic block copolymer showed not only anti-cancer activity comparable with free curcumin, but also demonstrated a slow and sustained release of curcumin . Therefore, the aforementioned approaches, as well as CurcuEmulsomes, look promising to enable the effective use of curcumin in medical applications.
However, having partially the characteristics of both liposomes and emulsions, CurcuEmulsome approach possesses certain advantages over its alternatives. Like liposomes, emulsomes are stabilized by phospholipid (multi-)layers as outermost structure, and thus, there is no need for surfactants stabilizing the nanoformulation. This endows emulsomes high degree of biocompatibility at therapeutic applications. More detailed, in the absence of any synthetic surfactants such as poloxamers, polysorbates or doxycholate, the use of emulsomes as a drug delivery system has demonstrable advantages, particularly for parenteral administration of poorly water-soluble lipophilic drugs , such as curcumin. Alternatively, due to their colloidal nature, emulsomes can be passively taken up from the blood stream by macrophages of the liver and spleen after intravenous or intracardiac administration as demonstrated in early in vivo studies [44, 45].
On the other hand, unlike lipid emulsions having a fluid core, emulsomes with a solid fat core can prolong the release of incorporated drugs - a property similar to polymeric nanoparticles [28, 46]. As previously demonstrated, zidovudine-emulsome formulations displayed a slow drug release profile in vivo (12–15% after 24 h) and prolonged the action at comparatively low drug doses . Therefore, the developed CurcuEmulsomes would be expected not only to circumvent the problems of low solubility and rapid elimination, but also to modify the drug release profile thereafter, due to the presence of curcumin in the internal solid lipid core.
Finally, having an analogous surface as liposomes , CurcuEmulsomes can further be tailored to fulfill specific requirements such as longer blood circulation or to enable cell targeting and active drug delivery. For instance, Gill et al. (2011) coated emulsomes with O-palmitoyl amylopectin , whereas Pal et al. (2012) coated them with O-palmitoyl mannan both with the aim of developing macrophage targeted systems . In a recent study, we showed that crystalline bacterial cell surface layer (S-layer) proteins are capable to coat emulsomes and modify their entire surface characteristics , e.g. by altering zeta-potential.
The colloidal characteristics of the emulsome evidence its robust character and indicate its potential in versatile use for lipophilic therapeutic agents other than curcumin. As previously reported [28–30], the size of emulsomes (a mean diameter of 286 nm, Figure 3) is predominantly determined by the phospholipid to tripalmitin ratio, and evidently, incorporation of curcumin did not influence neither particle size nor zeta potential characteristics. Moreover, the particle sizes can be tuned by altering the phospholipid to solid lipid ratio .
Although curcumin, DMC and BDMC show only very small chemical modifications with respect to their number of methoxy groups, a decrease in hydrophobicity in the order of curcumin > DMC > BDMC is known . Therefore, a shift in the ratio of the analogues inside the lipophilic fat core should be expected, but not in terms of a relative decrease of curcumin compared to DMC and BDMC (Figure 5). Hence, this result contradicts with the relative hydrophobicity of the analogues, as well as the findings of Rungphanichkul et al. (2011), where encapsulation of curcuminoids in non-ionic surfactant based liposomes, so-called niosomes, favored the incorporation of curcumin rather than its analogues . Although some thermodynamic parameters such as the polarity, as well as the molecular electrostatic interactions of curcuminoids with charged groups of lipid compounds, such as hexadecylamine, are thought to play a role in this selective incorporation process, the complete clarification of this finding merits further study.
Biological efficacy of CurcuEmulsomes was studied in vitro on HepG2 cell line model. In line with earlier studies on emulsomes , the delay in cytotoxicity is attributed to the slow release of curcumin entrapped inside the solid core of emulsomes. Hence, on the short terms the cytotoxic effect of CurcuEmulsomes remains limited. Nevertheless, CurcuEmulsomes displayed prolonged biological activity and acted as efficiently as free curcumin on long terms (Figure 6).
Like free curcumin, CurcuEmulsomes caused morphological changes in HepG2 cells where treated cells distinguished from untreated ones by their round shape. Based on AFM studies, Jiang et al. (2012) demonstrated the effect of curcumin on cytoskeletal arrangement of HepG2 cells and, combined with flow cytometric analysis, correlated this morphological effect with the upregulated expression of tubulin . The latter caused disorganization of the well-organized, filamentous network of healthy cells as deduced from the adopted round shape. Therefore, delivering curcumin into the cell, CurcuEmulsomes must be initiating the same effect (Figure 7).
Indicating for an enhanced stability, fluorescence images demonstrated that incorporated curcumin preserve its fluorescence intensity for longer times compared to free curcumin (Figure 8A). Parallel to our previous cross-sectional analysis of cells treated with empty emulsomes , the fluorescence microscopic data verified the accumulation of CurcuEmulsomes inside the cytoplasm upon their uptake by the cell (Figure 8B). Accordingly, CurcuEmulsomes accumulate inside the cell before any sufficient release of the load could occur. This finding may explain why CurcuEmulsomes caused cytotoxicity only after 24 hours (Figure 6).
Cell cycle analysis demonstrated that CurcuEmulsomes cause a prolonged induction of G2/M cell cycle arrest where the peak of G2/M phase rose steadily from 6 to 48 hours (Figure 9). In the contrary, free curcumin results in a sharp increase after 24 hours which declined after 48 hours. These findings, in line with cytotoxicity data, corroborate the slow and sustained release of curcumin from CurcuEmulsomes into the cells. Cell cycle analyses were only performed for 48 hours because the low viability profiles of treated HepG2 cells (Figure 6) did not allow longer investigations. However, speculatively, a further increase in G2/M phase arrest might be predicted due to the slow release profile of emulsomes.