Measuring enzymatic degradation of degradable starch microspheres using confocal laser scanning microscopy

Degradable starch microspheres (DSM) have long been used for topical haemostasis, temporary vascular occlusion and as drug delivery systems. When used for the latter, exact degradation rates of DSM have high importance, as this ensures a controlled and timed drug delivery. Current methods of analysing degradation rates are based on whole batch measurements, which does not yield information regarding individual times of degradation nor does it provide direct correlation measurements between sphere diameter and specific degradation time. In this paper we present an alternative method for measuring degradation rates of biodegradable starch microspheres using confocal laser scanning microscopy (CLSM). We succeeded in visualizing the degradation by staining the DSM and then following the spheres over time in a confocal microscope, after the addition of α-amylase. Individual degradation rates of single spheres could be followed, allowing a precise correlation measure between sphere size and degradation time. Furthermore, physical abnormalities such as internal cavities were detected within some spheres. These physical differences also had a measurable effect on the rate of degradation. Finally, complete degradation rates could be determined very accurately. To our knowledge, this is the first paper in which DSM degradation is visualized and measured using CLSM. STATEMENT OF SIGNIFICANCE: Using degradable starch microspheres as a drug delivery system, is a continuously evolving field which shows promise in several different areas of illnesses. This paper presents a new method which visualizes enzymatic degradation of starch microspheres in real-time using confocal microscopy. The method is simple, yet the versatility of it suggests that it could be broadly applied within the field of biodegradation. Here, it illuminates a previously uninvestigated parameter: the effect of physical sphere deformities on the rate of degradation. It also provides precise correlation measures between initial sphere size and time of complete degradation.