(M1430-01-03) Enhanced Membrane Composition for Lipid Nanoparticle Vascular Drug Delivery

Purpose: Vascular disease is the most prevalent pathology in the United States and the most common cause of surgical intervention. Open and endovascular interventions create a mechanical injury to the vessel wall that results in acute inflammation and vascular remodeling.  Up to 60% of vascular interventions fail within the first 12 months due to dysfunctional vascular remodeling at the injured site, leading to intimal hyperplasia restenosis and loss of patency. Our research team is developing targeted lipid nanoparticles (LNPs) to deliver therapeutics to the vessel wall at the time of surgery, aiming to improve surgical outcomes.  In previous research, our team demonstrated the efficacy of these vascular targeted LNPs in the delivery of gene therapeutics, using PEGylated distearoyl-glycerol-phosphoethanolamine (DSPE-PEG) as a component of the LNP formulation to improve particle stability, circulation half-life, and drug retention [1,2]. However, current literature demonstrates that despite these pharmacokinetic improvements, PEGylated lipids with long hydro-carbon chains can reduce cellular uptake due to their barrier effect, ultimately attenuating LNP functionality [3].  Recently PEGylated lipids with shorter hydro-carbon tails have been investigated as alternative LNP components, and current literature suggests that a PEGylated dimyristoyl-glycerol (DMG-PEG) incorporation may provide greater LNP functionality [3,4]. Whether this enhanced functionality occurs via mechanisms that induce lipid polymorphisms influencing lipid desorption rates and membrane stability and fusion for enhanced cellular uptake or by enhancing endosomal destabilization and escape is a phenomenon that is still being investigated. Here, our objective is to determine if the substitution of DMG-PEG for DSPE-PEG in our previously established LNP formulation will improve in vitro vascular cell delivery, without sacrificing critical quality attributes (CQA) for potential clinical translation.

Methods: Our established base LNPs (R8-PLPs) are formulated with bulk lipid dioleoyl-glycero-phosphocholine (DOPC) and cholesterol at 7:3mol plus 10mol% sterylated octa-arginine (STR-R8) and 1 mol% DSPE-PEG to serve as the experimental control. For the purposes of these investigations, this base R8-PLP formulation is modified via the substitution of DMG-PEG for DSPE-PEG at variable mole percents 1, 5, and 10mol%. All LNPs are assembled using a previously described EtOH injection technique with a lipid:siRNA weight-to-weight ratio of 100:1, purified via 24hr dialysis, and extruded through 100nm polycarbonate membranes prior to characterization and storage at 4°C. CQAs are assessed using standard characterization techniques. RiboGreen Assay is used to measure siRNA drug loading and retention (encapsulation efficiency, EE%). Dynamic light scattering (DLS) and electrophoretic mobility is used to measure particle size and homogeneity (polydispersity index, PDI).  All characterization studies are performed at assembly (time 0) and repeated serially for up to 28 days of storage in order to determine particle stability over time. Cellular uptake is assayed using LNP formulations that incorporate 1mol% Rhodamine-PE for tracking.  Human aortic smooth muscle cells (HASMCs) in culture are treated with LNPs at 50uM total lipid for 24h and cellular uptake quantified by fluorometry.

Results: LNPs with DMG-PEG incorporation at 1-10mol% maintain stable particle size and homogeneity ( < 65nm, PDI < 0.2) for up to 28 days at 4°C storage and are not significantly different than control LNPs with 1mol% DSPE-PEG (Figure 1). Likewise, incorporation of DMG-PEG does not affect siRNA EE% at assembly or siRNA retention for up to 28 Days at 4°C compared to DSPE-PEG controls (Figure 2). Furthermore, vascular cellular association is significantly enhanced in LNPs with DMG-PEG at 1 and 5 mol% membrane incorporation compared to DSPE-PEG controls (P≤0.05, n=3, Figure 3).

Conclusion: In conclusion, substituting DMG-PEG for DSPE-PEG enhances the in vitro cellular association of our previously established LNP formulation without significantly affecting nanoparticle CQAs or nanoparticle stability. Future studies will investigate DMG-PEGs effect on LNP cytotoxicity and LNP potency for functional gene modulation in vitro and in vivo.

References: 1. Fisher, R.K., 3rd, et al., Advances in the Formulation and Assembly of Non-Cationic Lipid Nanoparticles for the Medical Application of Gene Therapeutics. Nanomaterials (Basel), 2021. 11(3).
2. Grimsley, L.B., et al., Liposomal Nanocarriers Designed for Sub-Endothelial Matrix Targeting under Vascular Flow Conditions. Pharmaceutics, 2021. 13(11).
3. Mui, B.L., et al., Influence of Polyethylene Glycol Lipid Desorption Rates on Pharmacokinetics and Pharmacodynamics of siRNA Lipid Nanoparticles. Mol Ther Nucleic Acids, 2013. 2(12): p. e139.
4. Hattori, Y., et al., Effects of PEG anchors in PEGylated siRNA lipoplexes on in-vitro gene‑silencing effects and siRNA biodistribution in mice. Molecular Medicine Reports, 2020.

Acknowledgements: This research was funded in part by a grant from the National Science Foundation (DMountain) and the UTMC Hospital Auxiliary (DMountain). The authors declare no conflicts of interest.

LNP Size Over Time

Drug Retention Over Time

LNP Cellular Association