To perform a more comprehensive evaluation of structural parameters and stability in environments closer to biological ones, simulated biological fluids have been used, a valuable tool for research and development of medical technologies providing a safe, ethical, and efficient alternative to better understand the state of aggregation in more complex environments. Thus, the hydrodynamic diameter (Dh, mean peak) of the Psomes was measured by DLS in various simulated body fluids such as: phosphate-buffered saline (PBS), simulated blood plasma (SBP), simulated sperm medium (SSM), and simulated oviduct fluid (SOF) (Figure , i and iv). The Dh of loaded-Psomes A and D are stable in PBS, SBP and SSM, even after 24 h incubation. However, within a more complex fluid such as SOF, an increase in diameter is observed, indicating the presence of aggregation or agglomeration after 24 h. The colloidal stability was further studied using DLS following storages for different periods, showing stability after 8 weeks at -20℃ (Figure ). To complete the structural characterization of the different loaded-Psomes in presence of simulated body fluids, Asymmetric Flow Field Flow Fractionation coupled with light scattering (AF4-LS) was carried out (Figure , S15, Table ). AF4 as a separation method overcomes the limits of other techniques and the absence of a stationary phase minimizes sample adsorption and degradation ensuring a gentle separation and intact structure of the loaded-Psomes during the measurements. The molar masses and radii of gyration (Rg) distributions of the different samples were determined in 1 mM PBS and previously incubated in SBP (but 1 mM PBS is always the eluent). The conformation plot Rg versus Mw gives valuable information about the compactness and molecular shape of Psomes by using the scaling parameter (ʋ, slope of the plot, details in SI). Empty-Psomes possess a scaling exponent of 0.38 which is equal to the theoretical value of perfectly spherical objects. Post-loading of Psomes with both, MMP1 and HYAL, leads to slight deviation from the scaling parameters of 0.36 (PBS)-0.30 (SBP) for HYAL-Psomes and 0.46 (PBS)-0.26 (SBP) for MMPsomes. This proves the remaining spherical shape but with slight surface changes due to enzyme surface modification and microenvironment (Figure ) as also observed in previous studies. The apparent density of Empty-Psomes versus loaded-Psomes shows similar trends as found in a previous study with MMPsomes. As for Cu(βCD)2-Psomes (Figure ), ʋ moves further away from the spherical shape, 0.43 (PBS)-0.46 (SBP), which also proves a perhaps less smooth surface due to the incorporation of CU or even beta CD units that may interact with the PEG chains. We know from former studies that in situ enzyme loaded Psomes can improve the stability of the loaded enzyme located mainly into the lumen. However, the post-loading process (used for MMPsomes and HYAL-Psomes) promotes surface and membrane locations and it might compromise the stability of therapeutic enzymes in the presence of peptidases. Therefore, a comprehensive investigation of the stability of enzyme post-loaded Psomes in simulated body fluids and in the presence of a peptidase such as Trypsin (Tryp) has been done. First, the stability of the enzyme-Psomes in SBP and PBS after 0 h, 6 h and 24 h incubation at 37°C was validated (enzyme assays are described in detail in the SI). Enzyme-Psomes exhibit remarkable stability in SBP and even after 24 h (Figure ). In SBP, the calculated values after 24 h for both enzymes are high. Given its high salinity, SBP might offer optimal conditions for the enzyme functionality and preservation of its native form. The next step was to evaluate whether Psomes help to slow down the degradation of the therapeutic enzymes by proteases such as Tryp. This was attempted with both enzymes, but MMP-1 digestion assays with Tryp are not reproducible, therefore, just HYAL-Psomes were used (). Naked HYAL and HYAL-Psomes were incubated in SBP and in PBS in absence and in presence of Tryp (25 μg mL -1 ) (Figure ). Given the duration of the enzyme assay, measurements were carried out after 0 h and 2 h of incubation at 37°C for HYAL-Psomes. Three samples of naked enzymes in the presence of Tryp were tested resulting in a remaining normalized active HYAL concentration of 19 % after 2 h. In contrast, HYAL-Psomes show an average normalized active enzyme concentration of 90 % after 2 h at 37°C. The residual normalized active enzyme concentration of HYAL in HYAL-Psomes is at least three times higher than of naked one, another hint towards the protection of enzymes such as HYAL from degradation by peptidases.