Study of insulin-loaded PLLA porous microspheres for pulmonary drug delivery based on compressed CO2 antisolvent process

摘要

Poly-L-lactide porous microspheres (PLLA PMs) and insulin-loaded PLLA PMs (INS-PLLA PMs) for pulmonary drug delivery were successfully developed in an emulsion-combined precipitation of compressed CO_2 antisolvent (PCA) process using ammonium bicarbonate (AB) as a porogen. The resulting INS-PLLA PMs exhibited a rough and porous shape, with a geometric mean diameter (Dg) of 15.6 μm, an aerodynamic diameter (Da) of 4.3 μm, a fine particle fraction (FPF) of 64.58% and good aerosolization characteristics. A slight change was found in the secondary structure of insulin, while the hypoglycemic activity of INS-PLLA PMs was retained. The fluorescent INS-PLLA PMs (Fig. 1 The fluorescent image of INS-PLLA (INS labeled with FITC)) demonstrated that insulin was homogeneously distributed in the matrix, and the INS-PLLA PMs displayed a sustained release profile for 24 h. For biocompatibility evaluation, acute lung injury test and immune inflammation were conducted to investigate the lung toxicity. The results demonstrated the movements of air and PLLA PMs could induce a certain degree of injury, which caused a leakage of lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and other protein. Furthermore, the inflammatory factors such as interleukin-6 (IL-6), interieukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) were gathered and a gentle immune inflammation was observed, which remained a natural immune defense. For the systemic toxicity, the results indicated no cytotoxicity on A549 cells, and the toxicity grade was 1. Hemolysis test showed that the hemolysis rate was lower than 5%, which meets the requirements in medical materials. The acute toxicity test showed no acute toxicity in KM mice after the injection of PLLA PMs. For the functional evaluation, the FITC labelled INS-PLLA PMs could effectively deposit in the bronchia and the upper lung of SD rat (Fig. 2 The lung deposition of INS-PLLA PMs in SD rat: the comparison of fluorescence intensity between untreated lung tissue and administrated lung tissue). For the treatment of type Ⅱ diabetes, as shown in Fig. 3 (The fasting blood glucose levels of rats in different groups), the PLLA PMs showed no hypoglycemic activity while INS-PLLA PMs did have a remarkable hypoglycemic activity. The level of fasting blood glucoses achieved the minimum in 2 h and get back to an unmoral level after 4 h, which displayed a comparable hypoglycemic activity with subcutaneous insulin injection. The INS-PLLA PMs developed would have potential to be applied in the diabetes treatment by pulmonary drug delivery.