Cationic Nanocylinders Promote Angiogenic Activities of Endothelial Cells

Abstract

Polymers have been used extensively taking forms as scaffolds, patterned surface and nanoparticle for regenerative medicine applications. Angiogenesis is an essential process for successful tissue regeneration, and endothelial cell-cell interaction plays a pivotal role in regulating their tight junction formation, a hallmark of angiogenesis. Though continuous progress has been made, strategies to promote angiogenesis still rely on small molecule delivery or nuanced scaffold fabrication. As such, the recent paradigm shift from top-down to bottom-up approaches in tissue engineering necessitates development of polymer-based modular engineering tools to control angiogenesis. Here, we developed cationic nanocylinders (NCs) as inducers of cell-cell interaction and investigated their effect on angiogenic activities of human umbilical vein endothelial cells (HUVECs) in vitro. Electrospun poly (L-lactic acid) (PLLA) fibers were aminolyzed to generate positively charged NCs. The aninolyzation time was changed to produce two different aspect ratios of NCs. When HUVECs were treated with NCs, the electrostatic interaction of cationic NCs with negatively charged plasma membranes promoted migration, permeability and tubulogenesis of HUVECs compared to no treatment. This effect was more profound when the higher aspect ratio NC was used. The results indicate these NCs can be used as a new tool for the bottom-up approach to promote angiogenesis.

Keywords: cationic nanocylinder; cell–cell interaction; endothelial cells; migration; tubulogenesis.

Figure 1

Scanning electron microscope (SEM) images of (a) electrospun PLA nanofibers before and (b,c) after aminolyzation to different aspect ratios nanocylinders; (b) aspect ratio small (ARS) and (c) aspect ratio large (ARL); and (d) distribution of NC length.

Figure 2

Analysis of HUVECs migration by in vitro scratch assay. Images were acquired at 0, 6, and 12 h post scratch generation. The degree of migration was determined by quantifying the number of cells migrated to the center of scratch. (Red: RFP HUVECs; Green: Alexa Fluor 647 tagged NCs).

Figure 3

NCs induce endothelial cell permeability in vitro. Quantitative transwell assay to measure HUVEC permeability showed increased permeability after treating with NCs. (* p < 0.05, ** p < 0.01 relative to control).

Figure 4

Confocal images of human umbilical vein cells (HUVECs) cultured on matrigel substrates when NCs were treated for (a) 6 h; (b) 12 h; and (c) 24 h (red: RFP-HUVECs/green: Alexa 647-tagged NCs). Zoomed images of HUVECs cultured with (d) ARS and (e) ARL after 12 h. Scale bar = 200 μm.

Figure 5

The effect of NCs on capillary-like structure formation: (a) total tube length; (b) branching points; and (c) loop numbers of capillary-like structures of HUVECs on Matrigel without (control) or with NC treatment.

Figure 6

Quantitative real time PCR of angiogenic gene expression in HUVECs on Matrigel with NCs. Results are expressed as mean values ± S.D. * statistical significance at the level of p < 0.05.