2.2.2 Characterization of THB@ANVs
THB@ANVs can be prepared by co-incubating THB nanoparticles (NPs) with 400 nm ANVs, followed by extruding the mixture through a 200 nm liposome membrane. The obtained THB@ANVs were compared with THB NPs and 200 nm ANVs without THB. As shown in Figure 1C, the characteristic absorption band of THB at 464 nm can be observed in the UV/Vis absorption spectra of THB@ANVs. The loaded THB NPs can be observed in the TEM morphology of THB@ANVs (Figure 1A, right, marked with triangles). Hydrodynamic particle size and Zeta potentials tested by DLS and NTA at optimal conditions before and after encapsulation were shown in Figure 1F~1H. The hydration effect leads to a larger particle size measurement by DLS as compared to NTA. The isolated ANVs and THB@ANVs mainly match the TEM results, with diameters ranging from 30 to 300 nm. The particle sizes of THB@ANVs prepared with different initial concentrations of THB NPs were evaluated at a fixed ANVs concentrations of 100 μg/mL (Figure S7). An optimal initial concentration of 50 μM THB NPs was chosen to obtained a hydrodynamic diameter of 204.1 nm for THB@ANVs, which is close to that of ANVs (230.6 nm). When the concentration of THB NPs is further increased, they can also distribute onto the outer surface of the membranes. This might result in electrostatic attraction between ANVs, leading to an increase of hydrodynamic diameter. Calculated by the standard curve (Figures S8), a final concentration of 35 nmol THB/100 μg ANVs was obtained, corresponding to a loading capacity of approximately 19.6%. As shown in Figure 1J, the red fluorescence of THB has also been utilized to co-localized with the membrane labeled with 3,3-dioctadecyloxacarbocyanine perchlorate (DiO), further confirming the successful coupling of THB into ANVs.
2.3 In vitro wound repair promoting experiment of ANVs and THB@ANVs