(G-260) Enhancing the Targeting Efficacy of Endothelial Colony Forming Cells for Renal Regeneration via Kidney-targeted Liposomal Nanoparticles

Introduction:: Endothelial Colony Forming Cells (ECFCs) are rare circulating endothelial progenitor cells that can be isolated from both peripheral and umbilical cord blood [1]. ECFCs are positive for endothelial markers CD31 and CD144, as well as negative for the immune cell markers CD45 and CD14 [2]. ECFCs have been used to treat different diseases and have shown that they can home to the site of injury in a model of stroke and a model of retinopathy. This improvement has been attributed to neo angiogenesis, direct integration of ECFCs into pre-existing vasculature [3]. Treatment of nephrotic syndrome with drugs often requires high drug concentrations in the kidney and those high concentrations are often associated with adverse effects. Furthermore, high renal drug concentrations may not translate into high concentrations in the target cell and pathological conditions [4]. Previous results in our group have shown that Liposome Nanoparticles (LNPs) can be attached to the surface of ECFCs via the free-thiol groups on the lipid bilayers [5]. A modification of the LNPs with a kidney-targeting peptide (KTP) held at its NH2-terminus encourages the delivery of LNPs and ECFCs directly to the kidneys [6]. This will allow us to achieve a high accumulation of LNPs@KTP@ECFCs in the proximal tubules and vascular endothelium of the kidney. In this way, we would be able to enhance the targeting efficacy of ECFCs for renal regeneration.

Materials and Methods:: A mixture of DOPC, DOPG, MBP-PE, and Dil dye lipid components were combined and subjected to vacuum drying for 30-45 minutes, resulting in a dry lipid film. The dry lipid film containing DOPC/DOPG/MPB-PE/Dil was then hydrated with 1 ml PBS solution. The resulting mixture was vortexed for 5-7 minutes and extruded 21 times using gas-tight syringes, through a 200 nm polycarbonate membrane sandwiched in the Mini-Extruder block. The resulting solution was incubated at room temperature for 1.5 hours, and then at 4°C overnight. The NPs were purified from free phospholipids by ultracentrifugation at 50,100 rpm at 4°C for 1 hour. To conjugate the LNPs with the KTP peptide, 2-iminothiolane, a cyclic thioimidate compound that reacts with primary amines (-NH2), was used to introduce sulfhydryl (-SH) groups while maintaining charge properties similar to the original amino group. The KTP-LNPs were conjugated on the surface of cells by mixing equal volumes of the Primary Human Glomerular Microvascular Endothelial Cells (HGMVEC) and KTP-LNPs in cell media, with nanoparticles-to-cell ratios ranging from 1:100 to 1:5,000. The cells were then incubated for 30 minutes at 37°C with gentle agitation to facilitate the conjugation of maleimide in liposomes to the free thiols on HGMVEC. The residual maleimide groups on cell-bound particles were quenched with 1 mg/mL thiol-terminated 2-kDa PEG for 30 minutes in cell media (Fig A). After nanoparticles were conjugated on the cell surface, their presence was confirmed by confocal microscopy. Mean fluorescence intensity was measured using FACS and FlowJo software.

Results, Conclusions, and Discussions:: In this study, we aimed to confirm the ability of the KTP peptide with LNPs to directly target our kidney cell line (HGMVEC) and guide the ECFCs as a therapeutic agent to that area (Fig. A). Our goal is to successfully conjugate the LNPs to the KTP peptide. We strive to create the “backpack” molecule (ECFCs@LNPs@KTP) and confirm its presence using confocal imaging and FACS. Furthermore, our objective is to be able to demonstrate that the “backpack” molecule effectively targeted the HGMVECs, indicating its potential as a therapeutic agent. We demonstrated that the KTP peptide conjugated with LNPs with a diameter < 200nm (Fig. B) can effectively target HGMVECs (Fig. C). The largest population (77.1) is shown in Q2, indicating the binding of the LNPs with the kidney cells. The next step is to create a “backpack” molecule capable of guiding ECFCs. Confocal imaging confirmed the nature of the HGMVEC cells due to their markers, specially plasmalemmal vesicular associated-protein (PLVAP), which is an endothelial cell-specific membrane protein marker for kidney cells, and VE-cadherin (CD144), which is the major component of endothelial cell junctions and is specific to endothelial cells (Fig. D). The multi-lamellar structure of LNPs also provides a potential platform for drug delivery, which could further enhance the therapeutic response of the backpack molecule. Overall, this study presents a promising approach for the targeted delivery of ECFCs to the kidneys for potential therapeutic applications in kidney diseases. Further studies are needed to optimize the backpack molecule and investigate its therapeutic efficacy in vivo.

Acknowledgements (Optional): : We acknowledge support from the University of Notre Dame through “Advancing Our Vision” Initiative in Stem Cell Research, American Heart Association through Career Development Award (19-CDA-34630012 to D.H.-P.), National Science Foundation (2047903 to D.H.-P.) and from National Institutes of Health (1R35-GM-143055-01 to D.H.-P.).

References (Optional): :

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