(G-274) Novel Liposome Charged Grafts (LCGs) for localized Delivery of Sodium Nitroprusside to Augment Regenerative Repair of Abdominal Aortic Aneurysms (AAAs)

Introduction:: Abdominal Aortic Aneurysms (AAAs) are localized, rupture-prone aortal expansions resulting from progressive degradation of the wall elastic matrix by overexpressed matrix metalloproteases (MMPs). Mitogen-activated protein kinases (MAPKs), which are upstream regulators of MMPs, are highly expressed in AAA wall. Their activation triggers chronic degradation and loss of elastic matrix in the aorta wall^1,2. Reversing AAA pathophysiology is a challenge due to the intrinsically poor ability of adult or diseased vascular smooth muscle cells (vSMCs) to repair and regenerate elastic fibers. Our previous work showed that the NO donor drug, sodium nitroprusside (SNP) effectively downregulates proteolytic enzymes (specifically MMP 2) by MAPK inhibition, in aneurysmal SMC cultures to stimulate elastic fiber neo-assembly and facilitate fiber crosslinking and maturation towards restoring ECM homeostasis³. Since oral drug administration has complex pharmacokinetics, is not efficient in site-specific (i.e, AAA) tissue repair, the goal of this project is to develop liposome charged grafts (LCGs) with biotinylated lipid nanoparticles (bLNPs) charged onto biotinylated polycaprolactone (bPCL) graft using neutravidin bridge towards enabling efficient local delivery of SNP to the AAA wall. We hypothesize that this will ensure strong bLNP retention on site despite the dynamic blood flow burden and provide sustained stimulus for elastic matrix regeneration and anti-proteolytic effect. This body of work presents the formation and characterization of bLNPs, bPCL grafts, LCGs (by conjugating bLNPs to bPCL graft), and in-vitro matrix regenerative efficiency of the novel LCGs.


Materials and Methods:: Lipid nanoparticles (LNPs) of different formulations were prepared using 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP), dioleoylphosphatidylethanolamine (DOPE), and cholesterol in the molar ratio of 1:1:1 using thin film hydration technique followed by extrusion using 100 nm polycarbonate filter⁴. PEGylation was done using both pre-insertion and post-insertion techniques to compare and determine the optimal size, charge and PDI for cardiovascular application⁴. Biotinylated LNPS (bLNPs) were formulated similarly using pre-insertion with DSPE-PEG2000-(Biotin). LNP size, charge and PDI were measured by Dynamic Light Scattering (DLS). Successful biotinylation on the LNPs were verified using QuantTag^TM biotin quantification kit. Encapsulation of SNP was achieved by hydrating the thin film with 100 mM of SNP in water followed by extrusion. SNP encapsulation efficiency was determined using Greiss test⁵. Furthermore, the PCL graft was 3D-printed, and the biotin was conjugated using ethylenediamine as a linker following a normal carbodiimide reaction chemistry to form biotinylated PCL graft (bPCL). On going experiments seek to conjugate bLNPs on bPCL using neutravidin bridge, determine the cytotoxicity of bLNPs and LCGs on human aneurysmal SMCs (hASMCs), and assess matrix regenerative benefits of LCGs using RT-PCR and WB for ECM homeostasis genes and proteins, biochemical assays for elastin and collagen, TEM for elastin ultrastructure etc.


Results, Conclusions, and Discussions:: Results: The average size of non-PEGylated LNPs, post-PEGylated LNPs (with 2.5 mol % of PEG), pre-PEGylated LNPs (with 40 mol % of PEG) were 124.0 ± 16.7 nm, 126.3 ± 12.6 nm, 124.9 ± 5.4 nm (Fig. 1a), the average charges were 42.9 ± 2.2 mV, 43.2 ± 2.0 mV, and 34.6 ± 3.1 mV (Fig 1b), and the average polydispersity index were 0.07 ± 0.03, 0.10 ± 0.17, and 0.08 ± 0.02, respectively (Fig. 1c). LNPs that were formulated using a pre-PEGylation technique had significantly lower zeta potential compared to non-PEGylated or post PEGylated (p< 0.0005) LNPs. Therefore, pre-insertion techniques were used for further experiments and for the formation of bLNPs. The size, charge and PDIs of bLNPs were 128.2±12.9 nm, 40.09 ± 2.8 mV, and 0.04± 0.03 respectively which was significantly lower than both non-PEGylated and post-PEGylated LNPs (p< 0.05) (Fig 1). All LNPs were stable for 14 days at 4 °C (Fig. 2a, b). The standard curve for biotin on bLNPs had the r-squared value of 0.9875 suggesting successful PEGylation and biotinylation of LNPs. The encapsulation efficiency of SNP on bLNPs was 52.17%. These results are promising towards our planned development for localized delivery and release of SNP for onsite regenerative repair of the AAA wall.



Conclusion: Our studies to date have successfully optimized the LNP formulations suggesting pre-PEGylation (with 40 mol % of PEG (with or without biotin) had the optimal size, charge and PDI for our application. We have also achieved successful biotinylation and encapsulation of SNP on LNPs. Our ongoing experiments seek to evaluate matrix regenerative capability of bLNPs and LCGs. If successful, the proposed approach has the potential to delay the growth of small AAAs to a rupture stage.






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References (Optional): : References: (1) Farrell, K et al. Experimental Cell Research 2019;384 (1), 111589. (2) Camardo, A et al. Drug Deliv Transl Res 2018;8 (4), 964–984. (3) Bastola S et al. Tissue Eng Part A. 2023;(7-8):225-243. (4) Nosova AS et al. Medchemcomm. 2019;10(3):369-377. (5) Griess, P et al. Chem. Ber. 1879, 12, 426–8.