It is worth noting that the majority of NPs are double-color labeled, indicating the high efficiency of sonication-induced hybridization

of PLGA NPs and liposomes. Figure 2 Confocal images of LPK NPs. The images illustrate that KLH was labeled with rhodamine B (red) and liposome was labeled with NBD (green), confirming that PK NPs were enclosed by liposome. Scale bars represent 10 μm. Stability of NPs in PBS, FBS, and human serum For vaccines, having a desirable stability could ensure prolonged circulation in blood and sustained induction of immune response. Size stability of NPs in various solutions, (a) 10 mM PBS, (b) 10% (v/v) FBS, and (c) 10% (v/v) human serum, was evaluated by DLS (Figure 3). All the NPs, especially LPK NPs, were highly click here stable during incubation in 10 mM PBS (Figure 3A): no significant size change of LPK NPs was detected over 8 days of test; the size of PK NPs did not increase until day 7. In both FBS

(Figure 3B) and human serum (Figure 3C), a marked size change was detected for PK NPs after 4 h of incubation. In contrast, all the LPK NPs stayed stable for at least 2 days in both FBS and human serum. Especially LPK++ NPs kept a constant size in FBS for 7 days and in human serum for 8 days. Interestingly, size stability of LPK NPs appears to be related to lipid compositions; NPs with more positive charges exhibited higher stability compared to those with less positive charges. Higher see more stability of positively charged hybrid NPs may have resulted from a strong electrostatic attraction between cationic lipid layer and anionic PLGA core [22, 23]. Figure 3 In vitro stability of NPs. Size stability of NPs in various solutions: (A)

10 mM PBS, (B) 10% (v/v) FBS, and (C) 10% (v/v) human serum. Sizes of all NPs, except PK NPs, were stable in Bacterial neuraminidase PBS over 9 days of incubation. LPK NPs demonstrated superior stability compared to PK NPs in the three solutions. In both FBS and human serum, sizes of all NPs increase more quickly compared to that in PBS. The inserts show antigen release from NPs within 10 h of incubation. Double asterisks indicate that the size of NPs at this point was significantly higher compared to that at 0 h (p value <0.05). In vitrorelease of antigen from NPs The evaluation of in vitro antigen release from NPs in human serum could simulate the antigen release in vivo. In agreement with other reports that a lipid shell could help retain molecules loaded inside PLGA cores [15], in this work, LPK NPs displayed more controlled and delayed release of the payload, KLH. As shown in Figure 4, a burst release was observed between 10 and 12 h for PK NPs, and more than 70% of KLH was released in the first 16 h.