Polymeric microspheres for local delivery of proteins by administration under the kidney capsule

Abstract

Acute kidney injury and chronic kidney disease are conditions characterized by the inability of the kidneys to adequately filter waste products from the blood. The most common treatment remains renal replacement therapy (dialysis and renal transplantation), which unfortunately is associated with increased mortality rates. This thesis investigates a relatively novel approach for the treatment of kidney related diseases, by using polymeric microspheres loaded with a therapeutic protein. These microspheres were injected under the kidney capsule as a novel administration method to deliver drugs locally to the kidneys. Microspheres were prepared with poly(D,L-lactic-co-hydroxymethyl glycolic acid) (PLHMGA) copolymer using the membrane emulsification method in order to achieve uniformly dispersed spheres. PLHMGA microspheres were first tested for their safety after subcutaneous and subcapsular renal injection. Next, near-infrared labelled albumin (NIR-BSA) was encapsulated into PLHMGA microspheres followed by an injection under the kidney capsule in order to determine the release rate and the redistribution of the protein. In the last step of this thesis, the effect of the formulation conditions was tested after encapsulation of a therapeutic protein, erythropoietin (EPO), into PLHMGA microspheres. These microspheres were prepared with solid-in-oil-in-water (s/o/w) and solid-in-oil-in-oil (s/o/o) methods. The release of EPO and subsequent stability was tested with ELISA and Western blot. PLHMGA microspheres showed good biocompatibility properties and can be safely used in vivo as a drug delivery system. Further, PLHMGA microspheres had an average diameter of 35 µm and were degraded within 28 days post injection. A continuous release of around 90% of NIR-BSA loading was observed from the injected depot within a period of 2 weeks. Mainly the intact protein was released from the injected depot. The released NIR-BSA was further metabolized in the liver and subsequently the degradation products were cleared by the kidneys. This shows that the protein from the depot is mainly located at the injection site in the kidney. The encapsulation of EPO into PLHMGA microspheres resulted in low amount of dimmers (2-11%). However, when subjected to in vitro release studies, only a burst release of EPO was observed and no further release was detected with ELISA, which could be explained that upon hydration of microspheres during in vitro release studies, EPO can undergo a moisture-induced covalent aggregation resulting in the formation of dimers, trimers and higher-order oligomers. However, there is evidence in the literature that these higher order structures of EPO are pharmacologically active. In conclusion, locally administered PLHMGA microspheres injected under the kidney capsule are characterized by a 2-week release period of an encapsulated protein, making them an attractive delivery system. The subcapsular renal injection of this delivery system has the potential to reduce side effects of therapeutic proteins by increasing their presence at the site of injection and decreasing it elsewhere in the body.