(T1430-01-04) Long-Term Storage of Targeted Lipid Nanoparticles for Vascular Gene Therapies

Purpose: Vascular disease is the leading cause for surgery in the United States, with over 50% of vascular surgeries requiring additional procedures within a year due to dysfunctional vessel remodeling and the development of intimal hyperplasia restenosis and loss of patency. Our lab seeks to develop a lipid nanoparticle (LNP) gene therapeutic to prevent restenosis development and reduce the need for secondary interventions. All vascular interventions denude the endothelial layer of the vessel wall, exposing underlying extracellular matrix proteins. We have previously established a collagen-targeting LNP (CT-PLP) that has specific binding affinity to the exposed collagen matrix of injured vascular tissue [1]. We have demonstrated CT-PLP binding under physiological flow conditions in human saphenous vein ex-vivo and in rodent carotid arteries in vivo following a surgical balloon angioplasty injury. While preliminary data shows that our base, non-targeting LNP formulations can be stored for up to a month at 4°C without degradation or aggregation, we have recently determined that the binding functionality of our vascular targeted CT-PLP formulation is significantly diminished by 24hr post-assembly. This not only prevents bulk assembly and storage of the CT-PLP needed for pre-clinical studies, but it also poses a significant hurdle when looking forward at the feasibility of clinical translation. Recent literature has shown alternative sample processing methods for storage of LNP gene therapies [2]. Here we aim to determine if the addition of sucrose as a cryoprotectant will aid to preserve the functionality CT-PLPs and permit long-term storage.

Methods: Collagen targeting lipids (CTL) were synthesized via standard click chemistry azide-alkyne cycloaddition reaction with equimolar DSPE-PEG-dibenzocyclooctyne plus azido-modified collagen targeting peptides. CT-PLPs were formed with bulk lipid DOPC and cholesterol at a 7:3 mole ratio plus 10mol% stearylated octaarginine, 5mol% DSPE-PEG, 5mol% CTL, and 1mol% Cy7-DOPE for fluorescent imaging. All lipids were dissolved in CHCl₃, mixed as stated, dried under N₂ gas, and resuspended in ethanol. An aqueous siRNA encapsulate was prepared in 10mM Tris-HCl+10mM CaCl­­₂. CT-PLPs were assembled via microfluidic mixing with a total flow rate of 12mL/min, an aqueous:ethanol flow rate ratio of 3:2, and a lipid:siRNA mass ratio of 100:1. CT-PLPs were purified by dialysis against PBS for 24 hours and extruded through 100nm polycarbonate membranes prior to characterization and storage. Total siRNA encapsulation was determined via RiboGreen assay. Nanoparticle size and polydispersity index (PDI) were measured with dynamic light scattering. CT-PLPs were prepared for long-term storage by diluting to a concentration of 10ug/mL siRNA with PBS plus 9.6% sucrose by volume, followed by 0.2um sterile filtration, and aliquoted at 1mL. One aliquot was used immediately to establish baseline binding while the remaining aliquots were flash frozen in liquid nitrogen and stored at -80°C. Stored CT-PLPs were used to assess efficacy for up to four months, and in each case were thawed overnight at 4°C prior to use. CT-PLP binding was assessed using a previously established rodent carotid balloon angioplasty injury model [3] followed by localized CT-PLP infusion of an isolated section of the injured vessel for 5min and systemic blood flow restored. After 5min of flow restoration the animals were euthanized and the carotid artery collected for fluorescent imaging. Images were quantified using VisionWorks software to determine fluorescent intensity per area.

Results: CT-PLP at assembly met the critical quality attributes (CQAs) considered acceptable for clinical translation (diameter < 100nm, PDI < 0.2). When stored in PBS at 4°C and measured 24hr later, size and PDI had both increased beyond the desired CQA parameters with a broad size distribution (Table 1). Following agitation of the CT-PLPs via vortexing two distinct species were observed, indicating irreversible LNP coalescence (Table 1). Furthermore, CT-PLP binding efficacy of these samples in vivo in the injured vessel was significantly diminished following 24hr storage at 4°C (Figure 1). For CT-PLPs stored in sucrose at -80°C, CQAs were not comparable given the light refraction interference of sucrose in the sample. However, when these CT-PLPs were thawed and used in our pre-clinical injury model, fluorescent intensity heat maps of carotid artery sections demonstrate CT-PLP binding functionality throughout the injured region of vessel after all storage time points (Figure 1). Quantification of fluorescent intensity per vessel area of bound CT-PLPs demonstrate binding functionality was not significantly different for up to 120 days of storage (Figure 2).

Conclusion: CT-PLPs are a promising platform for vascular targeted gene therapeutics but show aggregation and loss of functionality after as little as 24 hours. By using sucrose as a cryoprotectant, CT-PLPs can be frozen and stored for extended periods of time with no loss in binding functionality. Samples can therefore be made in larger batches and stored prior to use. Larger batches reduce material loss that occurs during the microfluidic process, and improved storage conditions will allow for easier translation into clinical applications.

References: [1] Grimsley, L. B., et al. (2021). "Liposomal nanocarriers designed for sub-endothelial matrix targeting under vascular flow conditions." Pharmaceutics 13(11): 1816.
[2] Kim, B., et al. (2023). "Optimization of storage conditions for lipid nanoparticle-formulated self-replicating RNA vaccines." Journal of Controlled Release 353: 241-253.
[3] Tummers, A. M., et al. (2010). "Serum levels of matrix metalloproteinase-2 as a marker of intimal hyperplasia." Journal of Surgical Research 160(1): 9-13.

Acknowledgements: All animal procedures conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication no. 85-23, revised 1996), and were approved by the Institutional Animal Care and Use Committee, certified by the American Association of Accreditation of Laboratory Animal Care. This research was funded in part by an STTR grant from the National Science Foundation (RFisher, DMountain). RFisher and DMountain are co-founders of Orion Therapeutics, which holds license rights to the technology herein. These interests did not influence the study design, data collection, or interpretation.

Table of DLS from CT-PLP at assembly and 24hr post

Fluorescent images of CT-PLP binding in rat carotid arteries

Quantification of CT-PLP binding after storage over time