D14 - Enhancing the Targeting Efficacy of Endothelial Colony Forming Cells for Renal Regeneration via Kidney-Targeted Liposomal Nanoparticles

Introduction: : Stem cell-based therapy is a cornerstone of regenerative medicine, aiming to enhance the body’s innate repair mechanisms by stimulating, modulating, and replenishing endogenous stem cell populations to restore tissue homeostasis and promote regeneration. Traditionally, stem cells are delivered either intravenously (systemic delivery) or via intra-arterial injection to target specific organs. However, both methods often result in poor cell perfusion, viability, and retention, largely due to disease-related factors such as impaired blood flow, hypoxia, and inflammation.

To overcome these challenges, targeted stem cell therapies are being developed to improve efficacy and safety. In this research, we propose an innovative strategy to address key safety concerns by utilizing Endothelial Colony Forming Cells (ECFCs). ECFCs, a subset of endothelial progenitor cells, have demonstrated the ability to promote angiogenesis and kidney regeneration, notably by secreting factors that preserve renal microvascular function after ischemic injury. Despite their promise, efficient delivery and retention of ECFCs in the kidney remain significant hurdles, as current intravascular approaches do not achieve detectable engraftment in post-ischemic tissue.

We hypothesize that enhancing ECFC retention within kidney tissue will markedly improve their therapeutic impact on renal microvascular repair. Our approach leverages liposome-based nanoparticles (LNPs) modified with kidney-targeting peptides (KTPs), which serve as “backpacks” to guide ECFCs specifically to the kidney. This targeted delivery system aims to optimize ECFC efficacy in treating acute kidney injury (AKI), addressing a critical unmet need in regenerative medicine.

Materials and

Methods: : Cord Blood Endothelial Colony Forming Cells (CB-ECFCs) were isolated from human umbilical cord blood and cultured in endothelial growth medium supplemented with 10% fetal bovine serum at 37°C with 5% CO₂. Liposome-based nanoparticles (LNPs) were synthesized via thin-film hydration and functionalized with kidney-targeting peptides (KTP) using maleimide-thiol chemistry.

Nanoparticle size distribution and concentration were analyzed by Nanoparticle Tracking Analysis (NTA) using a NanoSight NS300. Dynamic Light Scattering (DLS) measured hydrodynamic diameter and polydispersity index. Morphology and size of LNPs were confirmed by Transmission Electron Microscopy (TEM) after negative staining. Fourier Transform Infrared Spectroscopy (FTIR) was employed to verify successful peptide conjugation on LNP surfaces.

Peptide content of LNPs was quantified using the Bicinchoninic Acid (BCA) assay. Fluorescent KTP with a FITC modification on the peptide was used to quantify the conjugated peptide on the surface of the LNPS.

Flow cytometry characterized the binding interaction of the KTP towards the HRPTEpCs a Cytek Aurora cytometer. Confocal microscopy (Nikon AXR) was used to evaluate LNP uptake and co-localization with ECFCs. Cells were fixed, stained with DAPI, and imaged in z-stacks for detailed spatial analysis.

All experiments were performed in triplicate. Data are presented as mean ± standard deviation, with statistical significance determined by Student’s t-test or one-way ANOVA (p < 0.05).

Results, Conclusions, and Discussions:: We successfully validated the binding capability of the Kidney Targeting Peptide (KTP) using liposome-based nanoparticles (LNPs) as carriers under in vitro conditions. Particle size and surface charge were characterized by DLS, TEM, NTA, and zeta potential analysis, confirming uniform nanoparticle synthesis. The presence and loading efficiency of KTP on LNPs were demonstrated using BCA assay, KTP-FITC fluorescence labeling, and FTIR spectroscopy.

To assess targeting specificity, we evaluated KTP binding to human renal proximal tubular epithelial cells (HRPTEpCs) via flow cytometry, testing four different LNP@KTP formulations and comparing them to LNP@PEG controls. Results showed significantly enhanced binding of LNP@KTP to HRPTEpCs, while negative control experiments using human pulmonary fibroblasts confirmed minimal off-target interaction, underscoring the selectivity of KTP.

Furthermore, we demonstrated the feasibility of creating “backpack” molecules by conjugating LNP@KTP to ECFCs. Optimization experiments identified the ideal LNP@KTP-to-ECFC ratio for maximal retention without compromising cell viability. Preliminary data indicate that ECFCs can retain the nanoparticle “backpack” for extended periods, supporting their use in targeted delivery.

Future work will focus on evaluating the ability of KTP to direct ECFC migration in vitro and, ultimately, assessing the angiogenic potential and therapeutic efficacy of targeted ECFCs in ameliorating acute kidney injury (AKI) in vivo. Collectively, these findings establish a strong foundation for the development of targeted regenerative therapies for renal diseases.

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