Islet transplantation is a recognised treatment for type 1 diabetes: islets are transplanted into the liver and the procedure can stabilise blood glucose levels. A major limitation for successful clinical islet transplantation is the significant loss of islets post-transplantation due to an immune mediated inflammatory reaction. Etanercept, a TNF blocker that binds specifically to TNF-α, is currently provided as a systemic injection to patients on the first, third and fifth day post-transplantation. Whilst administration of etanercept has improved efficacy of clinical islet transplant, it is important to achieve targeted and controlled delivery to support islet engraftment locally. Encapsulation of etanercept within microparticles (MPs) may provide a suitable route for delivery. However, MPs are typically fabricated with commercially available polymers that are non-specific to the liver resulting in inefficient delivery.
Asialoglycoprotein receptor (ASGPR) exhibits high affinity as a galactose receptor and is the only liver-specific receptor identified on hepatocytes thus far. Herein, we demonstrate targeted delivery to the liver by synthesis of a novel poly(lactic-co-glycolic acid) (PLGA) polymer by covalent conjugation with galactose moieties. MPs fabricated from this polymer demonstrated sustained and controlled release of etanercept that reduced the inflammatory response suitable to promote islet survival.
PLGA 85:15 (55 kDa) was functionalised with amino-functionalised lactobionic acid through amide bond formation in dimethyl sulfoxide for 16 h. One gram of galactosylated PLGA (Gal-PLGA) MPs encapsulated with 10 mg etanercept was fabricated using standard water-in-oil-in-water double emulsion method. In vitro functional drug release was measured using ELISA assay from aliquots of MPs suspended in PBS and gently rocked at 37 ºC for up to 7 days. Immunomodulatory response of MPs was performed using THP-1 differentiated macrophages. MPs were assessed in vivo following delivery via the portal vein.
RESULTS AND DISCUSSION
Gal-PLGA was synthesised to provide specific binding site to hepatocytes; MPs fabricated from Gal-PLGA provided an enhanced MPs retention (>85%, compared to 40% with conventional PLGA) in vivo. MPs fabricated with a mean size of 13 µm resulted in less hepatic necrosis when compared to larger particles. MPs exhibited a mean controlled daily release of 0.3 µg of protein per 1 mg of MPs between 2 and 7 days of in vitro release. Immunomodulatory response of MPs performed using macrophages suggest functional release of etanercept in the in vitro setting binds with TNF-α and potentially reduces inflammation observed by down-regulated pro-inflammatory genes (CXCL2, IL-1β) as well as reduced release of soluble inflammatory cytokines (TNF-α, IL-6). Preliminary investigation in vivo has shown no adverse safety concerns in mice.
We demonstrated a novel synthesis route for galactosylation of commercial PLGA and the subsequent fabrication of Gal-PLGA MPs as a novel protein carrier for targeted liver delivery with controlled functional release kinetics of etanercept. Macrophages have shown reduced inflammation co-culture with etanercept MPs in vitro. Preliminary in vivo study has displayed no adverse safety concerns post-injection.
This project is funded by the UK Regenerative Medicine Platform 2 (UKRMP2) [MR/R015651/1].