The Effect Of Phagocytes In The Degradation Process Of Poly-L-Lactide(PLLA)

The aim of this paper is to explore the role of macrophages on the degradation of poly L-lactic acid (PLLA). The process of degradation of PLLA microparticles in vitro was observed by scanning electron microscopy. The activities of AcPase and NADPH oxidase during being degraded of PLLA microparticles were checked by transmission electron microscopy and high- voltage electron microscopy and the variations of Ca2^, pH and membrane voltage of macrophages stimulated by PLLA were examined by confocal microscopy. 1, Observation of the degradation of PLLA in several stages (2,4,6.,8h) in vitro with scanning electron microscopy In PBS buffer particles become slightly swelling with appearing of wrinkles and pores (pore's diameter is about 0.12 It m and the pore will be larger in the course of incubation) after 2 hour's incubation at 370C. After 4h, there are more pores on surface of particles and pores penetrate inside the particles forming reticular tunnel connected with each other. At 8h, pores (diameter is about 0.8 1.?m ) are spreading all over the particle like honeycomb, but still maintaining globular structure. After 8h, the number of pore decreases and some of the pores ftise each other to form cavity. At last, the particles are degraded into fragile thin walls. There are three degradation ways of particles: honeycomb-like, channel-like and laminary structure. Honeycomb-like structure is often observed during degradation of particles. The speed of particle degradation is positive relation to temperature and time of incubation. Two kinds of pores appear in degraded particle. One is primary pore, emerging in early stage, which penetrates inside particle to form channel-like structure, so it may be also called entrance of tunnel; the other is secondary pore, emerging in later stage, which origins from corruded protrusion formed by extension of inter tunnel into the surface of particle. 2. Observation of phagocytosis of PLLA particles by macrophage with electron microscopy In PBS buffer involving macrophages from mouse abdominal cavity, a mount of PLLA particles are engulfed by rnacrophages after l0²0 mm incubation at 37G. After 20-30 mm, particles engulfed fuse with lysosome to form well-distributed phagocytosis lysosome and indicate clearly AcPase positive. When 1 It m thickness slice indicated AcPase action is observed by high-voltage electron microscopy, honeycomb-like and lami nary three- dimension structures are clearly displayed. The result is consistent with observation of scanning electron microsopy. 3. After macrophages engulf PLLA particles, there are several changes in. physical and chemical character as follows. (I) Free Ca²⁺ inside macrophage plasm increases 30%; (2) pH inside macrophage drops 70⁸0%; (3) mitochondrial membrane voltage declines 90%; (4) cell membrane voltage increase 100% in a short time; (5) activity of NADPH oxidase quickly enhances 150%. These results suggest that acidifying cytoplasm would help to activize enzymes of lysosome which accelerate macrophage engulfing PLLA particles, that the decreasing of mitochondrial membrane voltage is due to macrophage apoptosis which induced by engulfing a large mount of PLLA particles. At the same time, it also suggests that the raising of cell membrane voltage may be account for largely internal flowing of Ca2 into cytoplasm. Higher NADPH oxidase activity shows metabolism of macrophages reinforc...