Biodegradable microspheres for the sustained release of PDGF-receptor directed PPB-HSA targeted to the fibrotic kidney

Platelet Derived Growth Factor (PDGF) plays a key role in the development of fibrotic processes in several tissues. Accordingly, the PDGFβ receptor is abundantly present in these fibrotic tissues. Specific targeting to this receptor is established for a series of compounds in different animal models, all sharing the same targeting moiety, i.e. the cyclic peptide pPB. One of those compounds is pPB-HSA, which might function as carrier vehicle that binds to the PDGFβ-receptor without eliciting an intracellular response itself. When formulated as a solution for parenteral administration, targeting of fibrosis with this proteinaceous construct would involve long-term or even lifelong daily injections, which may jeopardize patient compliance. Therefore, the aim of this study was to develop a solid formulation for the sustained release of pPB-HSA and assess the delivery of the intact protein construct in vivo. pPB-HSA was encapsulated in biodegradable polymeric microspheres using a W/O/W emulsification process. Various mixtures of two semi-crystalline block copolymers consisting of poly ε-caprolactone, poly ethylene glycol and poly L-lactic acid of different block ratios were used to tailor the release characteristics. The delivery and targeting of the protein construct was evaluated in vivo using a mouse unilateral ureteral obstruction (UUO) kidney fibrosis model. By varying the ratio of the two polymers, the release of the model protein HSA from the microspheres could be tailored from 7 days to 2 months without burst release. To fit the design of the in vivo study, the microsphere formulation with a 50:50 ratio of the polymers, showing a sustained first order release of 14 days, was selected for further optimization. The optimized production process resulted in an encapsulation efficiency of 83% and an in vitro release of >90% after 14 days for the final pPB-HSA formulation. The median particle size of these microspheres was 25 μm, which makes them injectable with a 21G needle. All mice were sacrificed 7 days after ureteral obstruction and microsphere administration. The obstructed kidneys developed fibrosis and showed increased PDGFβ-receptor expression. The plasma concentration of pPB-HSA in the pPB-HSA treated mice (n=3) was 15 ± 4 ng/mL. Immunohistochemical staining and western blot analysis showed the presence of pPB-HSA in the fibrotic kidney of these mice, while the sham operated kidney was negative for pPB-HSA. In conclusion, the pPB-HSA construct was successfully formulated in polymeric microspheres produced by a W/O/W method, which showed a first order release profile in vitro. Furthermore, pPB-HSA was released from these microspheres in vivo. In addition, pPB- HSA was specifically localized in fibrotic tissue, where the target receptor, the PDGFβ receptor, is abundantly present. The delivery and site specific targeting of pPB-HSA for polymeric microspheres is thus confirmed in this study. Future studies include a pharmacokinetic in vivo study of the pPB-HSA microspheres in a mouse liver fibrosis model