# Bioavailability of Liposomal Vitamin C in Powder Form: A Randomized, Double-Blind, Cross-Over Trial
## 3 Results and Discussion  
### Properties of liposomal Vitamin C in Powder Form  
The hydrolysis of the ester bonds and/or the oxidation of the unsaturated acyl residues of the phospholipids, as well as changes in the average particle size and size distribution due to aggregation or carrier fusion--occurring in liquid liposomal suspensions--not infrequently lead to leakage of the entrapped active ingredient [36; 41], translating into short shelf-lives of the product and a reduction in its in vivo effectiveness [36]. An effective way to improve the stability, bioavailability, and bioactivity of liquid liposomal suspensions is to subject them to additional \"post-processing techniques\", including drying [48]. Dehydration of the liquid intermediate product significantly extends the shelf life of the product, makes it easier to transport, and reduces distribution costs, as the cold chain is not mandatory [40]. Spray drying, used in liposomal vitamin C powder technology, is more convenient and economical compared to popular freeze drying [40; 48]. Spray drying, unlike lyophilization, allows for direct transformation of a liquid feed into powder without the need for grinding, and importantly, it does not generate the risk of damaging the integrity of the lipid bilayer by ice crystals formed during freezing of the semi-finished product with a liquid consistency [49]. The process parameters we chose allowed for rapid dehydration of the liposomal suspension. The effectiveness of the performed process of the microencapsulation of liposomal particles was confirmed in the analysis of the morphology of the powder obtained under a scanning electron microscope (SEM) (Figure 2A,B). This analysis highlighted the intact surface of the microcapsules formed with carbohydrate material and the liposomal particles entrapped inside. Their mean diameter (median distribution), determined using the laser light diffraction method, was 23.4 \(\pm\) 1.2 \(\upmu\)m (Figure 2C). The intact/undamaged surface of liposomal vitamin C powder particles obtained by nanofiber weaving (Zeal) technology was visualized under the SEM in a study described by [PERSON] et al. [50]. However, the authors did not determine the value of the median particle distribution. The microcapsules obtained in this study, composed of carbohydrate material, further protected the vitamin C encapsulated in the liposomes from the adverse effects of various environmental factors. They also minimized the risk of the destructive effects of high temperature and high shear forces on the integrity of the lipid vesicle structure (see ref. [40]). The latter was highlighted in cryogenic electron microscope (cryo-TEM) images, allowing visualization of the shape and internal structure (lamellarity) of the liposomes, with sizes ranging from 5 to about 500 nm [51]. The images obtained by cryogenic transmission electron microscopy (cryo-TEM) yielded liposomes, mostly large unilamellar vesicles (LUVs), with a spherical shape and smooth surface (Figure 3). The continuity of the lipid vesicle walls indicates that the liquid liposomal intermediate was carried out in powder form without violating the continuity of the phospholipid bilayer. The presence of multivesicular vesicles (MVVs) next to the LUVs confirms earlier reports of a larger population of unilamellar liposomes with fractions of bi- or multiwalled vesicles in cryo-TEM imaging [51; 53]. This reflects the fact that the technological process does not achieve a uniform, monodisperse formulation of liposomal carriers [54]. In the discussed case, it cannot be excluded that changes in the structure of liposomes occurring during spray drying led to the formation of bi- and multilamellar vesicles [55]. Finally, in some cases, the bilayer carriers seen in cryo-TEM may actually be invaginated single-walled structures formed during sample preparation for microscopic analysis [51; 53].