Engineering bioactive nanoparticles to rejuvenate vascular progenitor cells

Cardiovascular disease is a leading cause of morbidity and mortality in diabetes, and fetal exposure to gestational diabetes mellitus (GDM) increases offspring risk of type 2 diabetes, hypertension, and cardiovascular disease. Hyperglycemia and oxidative stress drive early endothelial dysfunction, contributing to macro- and microvascular complications. Endothelial colony-forming cells (ECFCs), a subtype of endothelial progenitor cells enriched in cord blood, are promising for vascular repair due to high proliferative and vasculogenic potential. However, hyperglycemia and a diabetic intrauterine environment induce premature senescence and dysfunction in ECFCs, resulting in delayed colony formation, reduced migration, and impaired vasculogenesis. Prior strategies to rejuvenate ECFCs include systemic delivery of adjuvants, genetic modification, and pharmacologic preconditioning, but translation is limited by systemic side effects, immune responses, and regulatory barriers. The authors previously identified elevated transgelin (TAGLN/SM22α) in GDM-ECFCs, driven by TGF-β/Smad3 signaling, which disrupts actin cytoskeletal dynamics and reduces migration and vasculogenesis. The study hypothesizes that localized delivery of a TGF-β inhibitor (SB-431542) via cell-surface-conjugated liposomal nanoparticles to GDM-ECFCs would normalize TAGLN expression, enhance migration, and restore vasculogenesis in vitro and in vivo.

The paper situates its work within efforts to restore endothelial function using stem/progenitor cell therapies and adjuvant approaches (growth factors, gene therapy, pharmacologic preconditioning). ECFCs are highlighted for their in vivo vasculogenic capacity but are compromised by diabetic environments. Prior enhancements involved conjugating VEGF to microparticles and presenting proteins/glycomimetics on cell surfaces. However, growth factor and gene therapies face translational barriers such as immune responses, enzymatic degradation, and toxicity. TAGLN elevation in GDM-ECFCs has been linked to dysfunction; siRNA-mediated knockdown of TAGLN previously rescued migration and tube formation, supporting TGF-β pathway targeting. Liposomal nanoparticles have been used to deliver adjuvants to hematopoietic stem/progenitor cells with minimal immune response, indicating potential for clinical translation of surface-conjugated, drug-loaded nanoparticles to rejuvenate ECFCs.

Study population and cells: Human umbilical cord blood (40–60 mL) from normal/uncomplicated and GDM pregnancies (38–42 weeks) was collected with informed consent under IRB-approved protocols. Exclusions included pre-existing diabetes, disorders or medications affecting glucose metabolism, multiple gestations, preeclampsia, cardiovascular disease, or fetal chromosomal abnormalities. Mononuclear cells were isolated and cultured on collagen I in EGM-2 to derive ECFCs (passages 2–5). ECFC identity was confirmed by positive endothelial markers (CD31, CD141, CD105, CD144, vWF, Flk-1) and negative hematopoietic markers (CD41, CD14), and by colony-forming assays.

Nanoparticle fabrication: Multilamellar liposomal nanoparticles (NPs) were synthesized via thin-film hydration. Lipid components included MPB-PE (maleimide-functionalized), DOPC, DOPG, with Dil fluorescent tracer and SB-431542 (SB) as cargo. Dried lipid films were hydrated in PBS, vortexed, and extruded through 200 nm membranes, then purified by ultracentrifugation. Target particle size was ~150 nm, multilamellar structure confirmed by cryoTEM. Stability was evaluated by DLS and Nanosight at 4 °C and 37 °C over 30 days. Drug release of SB from NPs (10, 20, 40 μM initial loading) was measured daily up to 14 days and fit to the Korsmeyer-Peppas model.

Cell-surface conjugation: ECFCs were mixed with NPs (ratios 1:100 to 1:5,000 NPs per cell) at 37 °C for 30 min to couple maleimide groups on NPs with cell surface free thiols. Residual maleimide was quenched with 1 mg/mL thiol-terminated 2-kDa PEG (in EGM-2). Conjugation was confirmed by confocal microscopy and quantified by flow cytometry (MFI). Free thiol levels on ECFC surfaces were assessed and compared between normal ECFCs and GDM-ECFCs.

Phenotype assessment: Effects of NP conjugation on ECFC viability and proliferation were assessed (alamarBlue; monitoring over 3 days). Distribution of NP fluorescence with cell divisions was evaluated by flow cytometry (CFSE-labeled cells, Dil-labeled NPs) over 5 days. Expression of ECFC markers CD31, CD34, and CD144 was analyzed by flow cytometry in unconjugated and NP-conjugated cells.

Molecular assays: TAGLN mRNA levels were measured by qRT-PCR after soluble SB-431542 treatment (5 μM, 72 h) and after removal of SB at day 4 to assess persistence, and in ECFCs conjugated with vehicle-NPs (Vh-NPs) or SB-loaded NPs (SB-NPs) after 6 days. TAGLN protein expression was evaluated by Western blot at day 6, normalized to vinculin.

In vitro functional assays: Migration was assessed by transwell assays (4 h) and scratch wound-healing assays with time-lapse imaging (0–14 h), comparing Vh-NPs vs SB-NPs in normal ECFCs and GDM-ECFCs. Vasculogenesis was evaluated by (i) 2D Matrigel tube formation with kinetic analysis over 10 h, focusing on closed networks; and (ii) 3D collagen/fibronectin gels with encapsulated cells, assessing capillary network formation at 24–48 h. Quantification used the Kinetic Analysis of Vasculogenesis (KAV) plugin in FIJI.

In vivo vasculogenesis: Pre-vascularized collagen/fibronectin gels containing ECFCs (100,000 cells/gel) conjugated with Vh-NPs or SB-NPs were implanted subcutaneously into dorsal flanks of NOD/SCID mice (one gel per flank). After 14 days, perfusion labeling was performed via retro-orbital injection of rhodamine-conjugated UEA-I lectin (human vessels) and fluorescein-conjugated GS-IB4 isolectin (mouse vessels). Intravital imaging and multiphoton/confocal microscopy were used to visualize and quantify vessel interactions and percent area coverage by human and mouse vasculature; vessel diameter distributions were measured. Explanted grafts were fixed and analyzed by H&E and IHC for human CD31, mouse CD31, and mouse SMA; chimeric vessels were counted and normalized to graft area, with functional vessels defined by presence of at least one mouse erythrocyte.

Statistics: Experiments used ≥4 biological replicates (triplicate technical replicates). Data are mean ± SD. Statistical tests included Student’s t-test, ANOVA with Tukey post hoc, with significance at P<0.05, P<0.01, P<0.001, P<0.0001. Power analysis guided sample sizes.

• Nanoparticle engineering and conjugation:
  • Multilamellar liposomal nanoparticles ~147 ± 63 nm remained stable for ≥30 days at 4 °C and 37 °C.
  • Up to ~5,000 ± 100 NPs per cell could be conjugated via maleimide-thiol chemistry without affecting ECFC viability or proliferation; fluorescence intensity increased with NP:cell ratio and partitioned evenly to daughter cells with division.
  • SB-431542 loading (10–40 μM) produced sustained release over 14 days; Korsmeyer-Peppas fits yielded n=0.52–0.63 (non-Fickian diffusion).
  • NP conjugation did not alter ECFC marker expression (CD31, CD34, CD144) in normal or GDM-ECFCs.
• TAGLN modulation:
  • Soluble SB-431542 (5 μM, 72 h) transiently reduced TAGLN mRNA in GDM-ECFCs; TAGLN rebounded after SB removal.
  • SB-NPs conjugated to ECFCs stably decreased TAGLN mRNA over at least 6 days compared with Vh-NPs.
  • Western blot showed significant reductions in TAGLN protein in GDM-ECFCs with SB-NPs vs Vh-NPs; magnitude correlated with baseline TAGLN and GDM severity.
• Migration improvement (GDM-ECFC-specific):
  • Transwell migration increased significantly with SB-NPs in GDM-ECFCs (P=0.039) but not in normal ECFCs (P=0.955).
  • Wound healing at 8 h: SB-NPs enhanced wound closure in GDM-ECFCs (P=0.048) but not in normal ECFCs (P=0.988).
• In vitro vasculogenesis:
  • 2D Matrigel: SB-NPs increased closed network numbers at 5 h in GDM-ECFCs (P=0.010), with higher network counts maintained over 10 h; no significant effect in normal ECFCs (P=0.218).
  • 3D collagen/fibronectin gels (48 h): SB-NPs increased closed networks in GDM-ECFCs (P=0.0031) and showed no significant effect in normal ECFCs (P=0.051).
• In vivo vasculogenesis (NOD/SCID mice, 14 days):
  • Grafts with GDM-ECFCs + SB-NPs vascularized comparably to normal ECFCs and better than GDM-ECFCs + Vh-NPs, which sparsely vascularized periphery.
  • Human CD31 IHC: vessel density increased with SB-NPs vs Vh-NPs in GDM-ECFCs (44.7 ± 11.7 vs 102.5 ± 46.3 vessels/mm²; P=0.011); vessel area also increased (P=0.0058).
  • Intravital imaging: SB-NPs significantly increased percent area of human (UEA-I; P=0.021) and mouse (GS-IB4; P=0.006) vessels that were interconnected; mean vessel diameter shifted larger with SB-NPs (10.7 ± 3.6 vs 14.6 ± 5.8 μm; P=0.0018). Overall, localized, sustained delivery of SB-431542 via surface-conjugated liposomal nanoparticles normalized TAGLN expression, restored migration, and enhanced 2D/3D in vitro vasculogenesis and in vivo vessel formation in GDM-ECFCs without altering key progenitor phenotypes.

The study addressed the hypothesis that targeted inhibition of the TGF-β/SMAD3 pathway at the surface of GDM-ECFCs using SB-431542-loaded nanoparticles would normalize aberrant TAGLN expression and restore vasculogenic function. The engineered liposomal nanoparticles provided stable, controlled release and robust, benign conjugation to ECFC surfaces, enabling sustained pseudo-autocrine delivery. Findings demonstrated that SB-NPs specifically benefited dysfunctional GDM-ECFCs—reducing TAGLN levels, rescuing migration, and improving vasculogenesis in vitro and in vivo—while sparing normal ECFCs, consistent with selective correction of a dysregulated pathway. In vivo, enhanced vessel density, area, and integration with host vasculature (human-mouse interconnected networks) indicate functional restoration of vasculogenic capacity. These results validate TAGLN as a molecular marker linked to functional impairment and suggest that precision modulation of disease-perturbed signaling can rejuvenate progenitor cell function, potentially minimizing off-target effects compared to systemic or gene-based therapies. The platform aligns with translational workflows of ex vivo cell processing/banking, advancing therapeutic vasculogenesis for GDM-associated and broader cardiovascular complications.

This work introduces a simple, translatable strategy to rejuvenate vascular progenitor cells by conjugating bioactive, multilamellar liposomal nanoparticles carrying a TGF-β inhibitor (SB-431542) to the ECFC surface. The approach preserves ECFC viability and phenotype, provides sustained local drug delivery, normalizes elevated TAGLN in GDM-ECFCs, and restores migration and vasculogenic performance in vitro and in vivo to levels comparable to normal ECFCs. Clinically, this cell-surface engineering platform could be integrated into current ex vivo processing to enhance autologous cell therapies for vascular repair. Future research should define selection criteria (e.g., TAGLN threshold) for patient stratification, assess long-term safety/efficacy and immune interactions, optimize dosing and release kinetics, and explore applications to other dysfunctional vascular progenitor populations (e.g., T2DM, preeclampsia) and hPSC-derived vascular progenitors.

• Biological variability: TAGLN expression and responses varied among GDM-ECFC lines, correlating with patient GDM severity; generalizability across broader populations needs confirmation.
• Mechanistic specificity: While improvements correlate with reduced TAGLN, TGF-β inhibition may regulate additional targets; functional gains may not be exclusively TAGLN-mediated.
• Dosing/coverage limits: Conjugation was optimized up to ~5,000 NPs per cell; higher densities were not assessed and could alter membrane properties or NP fusion behavior.
• Model scope: In vivo validation used subcutaneous xenografts in immunodeficient mice over 14 days; long-term durability, systemic effects, and performance in disease-relevant models remain to be evaluated.
• Cell source and stage: Findings are based on cord blood–derived ECFCs (passages 2–5); effects in adult peripheral blood ECFCs or other progenitor subsets require testing.
• Immunogenicity/toxicity: Although similar formulations have shown minimal inflammatory responses in prior studies, comprehensive immune/toxicity profiling for this specific application was not performed here.