Deformable nanovesicles have been extensively investigated due to their excellent ability to penetrate biological barriers. However, suffering from serious physical and chemical instabilities, the wide use of deformable nanovesicles in medical applications is still limited. Moreover, far less work has been done to pursue the lyophilization of deformable nanovesicles. Here, we aimed to obtain stable deformable nanovesicles via freeze-drying technology and to uncover the underlying protection mechanisms. Firstly, the density of nanovesicles before freeze-drying, the effect of different kinds of cryoprotectants, and the types of different reconstituted solvents after lyophilization were investigated in detail to obtain stable deformable nanovesicles based on insulin-phospholipid complex (IPC-DNVs). To further investigate the underlying protection mechanisms, we performed a variety of analyses. We found that deformable nanovesicles at a low density containing 8% lactose and trehalose in a ratio of 1:4 (8%-L-T) have a spherical shape, smooth surface morphology in the lyophilized state, a whorl-like structure, high entrapment efficiency, and deformability after reconstitution. Importantly, the integrity of IPC, as well as the secondary structure of insulin, were well protected. Accelerated stability studies demonstrated that 8%-L-T remained highly stable during storage for 6 months at 25 °C. Based on in vivo results, lyophilized IPC-DNVs retained their bioactivity and had good efficacy. Given the convenience of preparation and long term stability, the use of combined cryoprotectants in a proper ratio to protect stable nanovesicles indicates strong potential for industrial production.
Keywords: combination; cryoprotectant; deformable nanovesicles; insulin; mechanism.