N32 - A lipid-polymeric nanoplatform for collagen fiber depletion and enhanced drug penetration in liver fibrosis

Introduction: : Liver fibrosis develops due to elevated synthesis and deposition of extracellular matrix (ECM), coupled with inadequate remodeling processes. Exploring therapeutic methods that directly degrade and dissolve fibrotic scar tissue and enable deep tissue drug delivery 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 with collagenase encapsulated and further decorated with INT-777, a bile acid derivative that acts as an agonist of G- protein-coupled bile acid receptor (Gpbar1) expressed by Kupffer cells. Collagenase enzyme helps in the degradation of the deposited ECM, thereby promoting nanoparticle (NP) penetration and therapeutic drug delivery in liver fibrosis.

Materials and

Methods: : PLGA polymer NPs 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 NPs using the freeze-thaw method. INT-777 was conjugated to the lipids using carbodiimide chemistry (Fig. 1a). All-trans-retinoic acid (ATRA), an anti-fibrotic drug, was encapsulated within the PLGA core during NP synthesis. The size and surface charge of the NPs were confirmed using dynamic light scattering. 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. The cytocompatibility of final NPs was determined by WST assays. The mechanism of uptake was analyzed by assessing cellular uptake of NPs after treatment with clathrin and caveolae inhibitors. To confirm targeting efficiency of the final NPs in vitro, a competition assay was performed, where cells pre-treated with INT were then treated with the NPs. For in vivo studies, a mouse model of alcohol-associated liver fibrosis was developed using a combination of ethanol liquid diet and carbon tetrachloride injections. The targeting efficiency and biodistribution of indocyanine green-containing final NPs was determined by harvesting organs in mice following 24 and 72h of treatment, and IVIS imaging of the organs.

Results, Conclusions, and Discussions:: NP abbreviations used in the abstract and figures: LP - Lipid-PLGA NPs; LPC - lipid-PLGA NPs containing collagenase; ILP - INT-777-tagged lipid-PLGA NPs; ILPA - INT-777-tagged PLGA NPs with ATRA (no collagenase); ILPAC - INT-777 tagged lipid-PLGA NPs with ATRA and collagenase

The ILPAC formulations demonstrated stability, characterized by an average hydrodynamic size of 134.23 ±8.58 nm and a zeta potential of -20.36±0.87 mV, with a PDI of 0.33 ± 0.016 (Figure 1b). The conjugation efficiency of lipid and INT resulted in 20.8% efficiency. Collagenase encapsulation efficiency was approximately 30%. The phospholipid concentration, determined by the Stewart assays, was 320 µg of lipid per 600 µg of LPC. Compatibility of ILPAC was confirmed through WST assay (Figure 1d). The cellular uptake of ILPs by differentiated Thp1 (model Kupffer cells), HepG2, and LX2 hepatic stellate cells showed an increased uptake in Thp1 cells (Figure 1e). The competition assay validated that the ILPs showed an inhibited uptake when pretreated with INT, confirming the uptake via Gpbar 1 receptor (Figure 1f). The in vivo data also confirmed the targeting efficiency of INT by showing greater accumulation and retention of ILPs in the liver at 24h and 72h post-injection than seen in animals given NPs without INT (Figure 2a). The fibrosis marker TIMP 1 shows an elevated level in the fibrosis group compared to the control (Figure 2b). The mouse serum ALT and AST data showed elevated levels, confirming the development of fibrosis in vivo.

Conclusions: We have successfully developed INT conjugated lipid-PLGA NPs loaded with collagenase for targeting liver fibrosis. A significantly higher uptake of ILPs was observed in THP1 cells, indicating that INT can target the Kupffer cells in the liver. The mechanistic study showed the inhibition of NP uptake by INT, indicating that the uptake is through the Gpbar 1 receptor. Our next goal is to investigate the combinatorial effects of NP-delivered collagenase and anti-fibrotic therapies in 3D spheroids and in vivo. This strategy aims to improve ECM penetration by collagenase further and deliver the anti-fibrotic agent effectively to sites of liver fibrosis.

Acknowledgements and/or References (Optional):: This work was supported by the National Institute of Health grant numbers R21 AA029750 and R37 CA283937 to J.U.M.