(W1230-01-06) Fibronectin-Targeted Dendron Micelles to Effectively Deliver BRD4-Inhibitors for Inflammation Reduction in an Idiopathic Pulmonary Fibrosis Model

Purpose: Idiopathic pulmonary fibrosis is a serious chronic disease with only two approved therapeutics, both with serious side effects. Bromodomain-containing protein 4 (BRD4), which plays a role in innate immune response, is a promising drug target[1]. The binding of BRD4-ibhibitors blocks inflammatory response activation and extracellular matrix remodeling[2,3]. These inhibitors are hydrophobic, requiring toxic surfactants for effective formulation. With their ability to encapsulate these small molecules, micelles are promising platforms for pulmonary delivery with significantly lower toxicities. While linear micelles are common platforms for encapsulating drugs, there are drawbacks. Dendrons offer unique ways to improve stability, targeting, and drug loading compared to their linear counterparts. Dendron micelles (DMs) are hyperbranched polymeric nanoparticles that have many terminal functional groups and the ability to encapsulate hydrophobic drugs. The building blocks are PEGylated dendron coils (PDCs), amphiphilic polymers that self-assemble into these DMs[4,5]. They are composed of hydrophobic poly(ε-caprolactone) (PCL) linked to hydrophilic polyethylene glycol (PEG) via a hyperbranched polyester dendron with eight terminal groups[4,5]. The high density of surface end groups from the hyperbranched structure gives flexibility in adding targeting moieties. Fibronectin is overexpressed in pulmonary fibrosis, serving as an optimal target, specifically by fibronectin-binding peptides[6]. Peptides by themselves are quickly cleared from the body, not allowing enough time to reach the disease site and bind with their receptor. Attachment to the surface of micelles allows the peptides to remain in the body for significantly longer. The strength of binding also increases due to multivalency, which is multiple peptides on a single micelle binding to multiple receptors at the same time versus the one-to-one of free peptides, thus resulting in higher binding avidity. In this study we aimed to optimize BRD4-inhibitor ZL0516[3] delivery and fibronectin targeting for enhanced inflammation reduction in pulmonary fibrosis. BRD4i-loaded DMs are expected to demonstrate dose-dependent anti-inflammatory effects, with potential time-delayed potency than free BRD4-inhibitors due to required release from the micelle. Fibronectin-binding peptide (pFN) conjugated DMs should have increased cellular specificity and enhanced binding kinetics when compared with free peptide due the multivalent effect of the hyperbranched DM.

Methods: PDCs were prepared then characterized using NMR spectroscopy. PCL_14k underwent bromination then azide functionalization before G3 polyester dendron conjugation via click chemistry. The dendron terminal groups were then activated and conjugated with methoxy-PEG_0.6k. DMs were formed via thin-film evaporation to evaluate drug loading and encapsulation. Size and morphology measurements were taken of empty, drug-loaded, and peptide-conjugated DMs using DLS and TEM. Drug release profiles were completed in PBS to represent intranasal delivery and analyzed with HPLC. Binding kinetics were measured via biolayer interferometry (BLI) with DMs that included different weight percentages of peptides. In vitro targeting was observed in hSAE cells with confocal microscopy. hSAE cells were also treated with BRD4i-loaded DMs and free BRD4i to evaluate the in vitro anti-inflammatory efficiency, specifically looking at IL-6, FN1, RelA, and Col1A1 RNA expression.

Results: Empty micelles were observed to have slightly larger diameters than drug-loaded ones, 44.10 and 40.78 nm, respectively. The diameter was also seen to increase with increasing weight percentages of peptide conjugated to the surface, ranging from 44.10 nm for a non-targeted DM up to 68.08 nm for a DM with 20% wt/wt peptide PDC. All DM formulations maintained negative surface charges. ZL0516, the BRD4i used in this study showed ~50% encapsulation efficiency when loaded with 20% wt/wt drug. This same drug showed moderately quick release, with nearly complete release at 4 hours. BLI showed stronger binding of DM-pFN than free pFN peptide, with a lower K_D of 5.320E-10 M versus 1.240E-06 M. It was also determined that DM-pFN 5% wt/wt peptide had the best binding efficiency. Targeted DM-pFN showed significantly increased cellular specificity. Finally, the in vitro efficiency assay showed a dose-dependent decrease in expression of all four inflammation/fibrosis markers when treated with both free drug and drug-loaded DM.

Conclusion: Hydrophobic-hydrophobic interactions between encapsulated drug and micelle interior, along with increased corona density from the hyperbranched structure, play a role in the smaller diameter, increased drug loading, slower release, and enhanced binding of DMs. The stronger multivalent binding of DM-pFN can increase residence time at the disease site, lowering the clearance rate from the lungs. Encapsulation of BRD4i-inhibitors within DMs does not alter the properties/potency of the drug, successfully reducing inflammation. This can mean increased doses with less dose-limiting toxicity. Given that the major issue in pulmonary fibrosis treatments is the serious adverse side effects from the solvents required for dissolution, this platform can provide a potential solution, with the overall goal to optimize this formulation for intranasal delivery via nebulizer. The modular nature and enhanced targeting, along with effective drug loading and controlled release allows DMs to serve as a promising functionalizable and tunable nanocarrier for combination therapeutics.

References: 1. Xu X, Mann M, Qiao D, Li Y, Zhou J, Brasier AR. Front. Mol. Biosci. 2021
2. Tian B, Liu Z, Litvinov J, Maroto R, Jamaluddin M, Rytting E, Patrikeev I, Ochoa L, Vargas G, Motamedi M, Ameredes BT, Zhou J, Brasier AR. Am. J. Respir. Cell Mol. Biol. 2019
3. Liu Z, Chen H, Wang P, Li Y, Wold EA, Leonard PG, Joseph S, Brasier AR, Tian B, Zhou J. J. Med. Chem. 2020
4. Hsu H, Han Y, Cheong M, Král P, Hong S. Nanomed.: Nanotechnol. Biol. Med. 2018
5. Bae JW, Pearson RM, Patra N, Sunoqrot S, Vukovic L, Král P, Hong S. Chem. Commun. 2011
6. Gao, X. Eur J Pharm Biopharm. 1998

Acknowledgements:This project was funded in part by the Dr. Ralph and Marian Falk Medical Research Trust Catalyst Award and Transformational Award.

DM-pFN binding efficiency values: overall dissociation constant (KD) and individual on and off rate constants (ka and kd).

Cell specificity of DM-pFN in hSAE cells after 6h incubation. (A) Confocal microscopy images of Cy5.5-labeled DM groups. (B) Pixel count quantification of A. Results are presented as average ± S.D. (n = 4), p < 0.05.

Non-targeted BRD4i-loaded DM in vitro efficiency after 24h treatment of fibrosis-induced hSAE cells. Relative RNA expression of (A) IL-6, (B) FN1, (C) RelA, and (D) Col1A1. Results are presented as average ± S.D. (n = 3).