Initially, an established method involving protein post-loading approach was employed for the construction of HYAL-Psomes and MMPsomes, envisioned as potential ECM digestive nanocontainers (details in SI, Figure ). On the other hand, for the fabrication of CU(βCD)2-Psomes, the same post-loading protocol was used, with a preliminary step of using βCD molecules as a CU solubility enhancer (details in SI, Figure ). Moreover, it is intuited that the even more water-soluble CU(βCD)2 complex can be more effectively encapsulated within the vesicles besides the membrane. This enhances control over its release, pH dependent. Firstly, size information, pH responsiveness and stability for loaded-Psomes and Empty-Psomes were studied by Dynamic Light Scattering (DLS) (details in SI, chapter 3). Cyclic pH switches of Empty-Psomes were performed at pH 8.0 (collapsed state) and 5.0 (swollen state) proving no disassembly and membrane rupture as well as no aggregation (Figure ). To evaluate the difference of pH range therapeutic windows of both Psomes (empty and loaded) which feature membranes with different hydrophobic balances, a pH titration was carried out to determine the pH* value (semi-open state) and pH value for fully open state (Figure , Figure , Figure ). Based on these pH values, the pursued permeability was obtained in 1 mM PBS, where enzyme-Psomes A were semi-open at around 6.7, but fully open at pH 6.5 (Figure ). However, for the CU(βCD)2-Psomes D, it was necessary to reach pH 5 to obtain a fully open state (Figure ). The morphology (vesicular structure), diameter and membrane thickness were confirmed by cryo-TEM (Figure ). The resulting mean diameters (⌀) for Psomes A (⌀HYAL-Psomes 82.6 + 4.9 nm; ⌀MMPsomes 72.9 + 7.0 nm; ⌀Empty-Psomes 71 + 7 nm) and membrane thickness (MT) (MT~ 14 nm in all cases) are in the range of previously published results. Just an increase in diameter is observed in the case of HYAL-Psomes A, probably due to the higher loading efficiency (45%), which makes the system a little larger in the collapsed state at pH 8. The enzymes cargo seems not to affect membrane thickness even anchored into the membranes. The resulting ⌀ for Psomes D (⌀CU(βCD)2-Psomes 87.3 + 15.0 nm) and MT (MT~ 29 nm in all cases) show no difference between the empty and loaded samples, however, the effect of the incorporation of a hydrophobic monomer is seen, which results in a much thicker membrane in comparison to Psomes A (details in SI, Figure , Table ). RhB labelled enzymes were used to estimate the loading efficiency and compared with the reported data (Figure , loading efficiency of HYAL= 45%, ~0.1 mg mL -1 and MMP-1= 21%, 0.05 mg mL -1 per 0.5 mg mL -1 BCP, details in SI, section 3.3). HYAL is a smaller enzyme (60 kDa) than MMP-1 (100 kDa) which might allow better incorporation into the membrane giving higher loading efficiency, in contrast, MMP-1 size might be a limiting factor for the post loading capacity of Psomes. Zeta potential measurements were carried out using naked cargoes, empty and loaded Psomes at pH 9 and pH 5 (Table -S4). For enzyme-Psomes A at pH 9 (collapsed state), the values for the surface charge changed significantly after the post-loading (5.54 mV for Empty-Psomes, -1.66 mV for HYAL-Psomes, -9.15 mV for HYAL-Psomes) which confirms that the enzyme was loaded into the Psomes and present on the surface. The same trend was observed for CU(βCD)2-Psomes (-5.99 mV for Empty-Psomes D, -9.42 mV for CU(βCD)2-Psomes). In the case of CU(βCD)2-Psomes, the loading efficiency was calculated using their UV-VIS spectroscopic properties (A490 nm, loading efficiency of CU(βCD)2 = 17%, Figure , ~ 10 µg mL -1 ). Furthermore, it has been demonstrated that the solubility increases when utilizing βCD molecules, as evidenced by spectrometric analysis (Figure ) and biological measurements (MTT assay, Figure ), since higher cytotoxicity outcomes obtained with the CU(βCD)2 complex. This enhancement allows its incorporation into the lumen vesicle cavity of Psomes D and, thus, regulates its pH dependent release.