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Drug Delivery

Drug Delivery - Poster Session D

Poster X10 - A lipid-polymeric nanoplatform for collagen breakdown and improved drug penetration in liver fibrosis

Friday, October 25, 2024
3:30 PM - 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Liver fibrosis develops due to elevated synthesis and deposition of extracellular matrix (ECM), coupled with inadequate remodeling processes. Exploring therapeutic methods that break down fibrotic scar tissue for greater drug penetration could offer a promising approach to treating advanced fibrosis. We report the development of a targeted lipid- poly lactic co glycolic acid (PLGA), polymeric nanoplatform containing collagenase and further decorated with INT-777, a semi-synthetic bile acid derivative that acts as an agonist of G- protein-coupled bile acid receptor (Gpbar1) expressed on Kupffer cells, for liver fibrosis treatment. Collagenase enzyme helps in the degradation of the deposited ECM, thereby promoting nanoparticle penetration and therapeutic drug delivery in liver fibrosis.

Materials and

Methods:  PLGA polymer nanoparticles were fabricated using a standard single emulsion process, followed by passive lipid coating through thin film hydration. Collagenase was subsequently loaded into these lipid-coated PLGA nanoparticles using the freeze-thaw method. INT-777 was conjugated to the NH2 end groups of the lipids using carbodiimide chemistry (Fig 1a). The size and surface charge of the nanoparticles were confirmed using dynamic light scattering (DLS). The phospholipid concentration was analyzed using the Stewart assay with UV-visible spectrometry. The encapsulation efficiency of collagenase was validated using a collagenase assay kit. Collagen degradation and nanoparticle penetration were assessed in 3D spheroids and confirmed via Sirius red staining for collagen fibers. The penetration of nanoparticles was examined using Z-stacks with a confocal microscope. Spheroid viability was determined using a Live/dead assay.

Results, Conclusions, and Discussions: Stable lipid-PLGA with collagenase (LPC) formulations were synthesized with an average hydrodynamic diameter of 198.2±8.86 nm, a zeta potential of -12.20±1.6 mV, and a PDI of 0.26 ± 0.016 (Figures 1b and c). Collagenase encapsulation efficiency was approximately 30%. The phospholipid concentration, determined by Stewart assay, was 320 µg of lipid per 600 µg of LPC. Cytocompatibility of LPC was confirmed through live/dead staining (Figure 2), and fluorescent microscope images reveal the disintegration of spheroid structures under LPC treatment, supporting the hypothesis that collagenase aids in ECM collagen fiber degradation. Sirius red staining further validated Type 1 collagen fiber breakdown. LPC penetration was visualized using z-stacks with a confocal microscope at 10x magnification, showing nanoparticle fluorescence at various depths within the spheroids, indicating successful penetration of LPC compared to LP (Fig 3).

Conclusions: We have successfully developed lipid-PLGA nanoparticles loaded with collagenase and conjugated with INT-777 for targeting liver fibrosis. We observed significant disintegration of spheroids treated with LPC, indicating successful delivery of collagenase to the ECM, which promotes collagen fiber degradation and enhances nanoparticle penetration into spheroids. Future studies will focus on synthesizing INT-decorated LPC incorporating anti-fibrotic agents. This strategy aims to further improve ECM penetration by collagenase and deliver the anti-fibrotic agent effectively to sites of liver fibrosis.

Acknowledgements (Optional):  We acknowledge funding support from the National Institutes of Health [grant numbers R21 AA029750 and R37 CA283937] to J.U.M.