Angiogenic properties of poly(NAG-co-NAPA) nanoparticles: assessment via gray-level co-occurrence matrix-based image processing

Abstract

Angiogenesis is a complex physiological process that is involved in the formation of new blood vessels. Despite advancements in assessing angiogenesis, there is still a substantial opportunity for the development of more accessible, reliable, and automated processes for estimating this complex phenomenon. Although in vitro and in vivo studies are instrumental for identifying the molecular players in angiogenesis, computational and mathematical models are likewise crucial to understand and explain the formation of endothelial cell networks. Quantifying angiogenesis is difficult due to the non-availability of suitable methods. Manual analysis and quantification of such results with existing approaches is typically labour-intensive and affected by inter-experimental variability. With this in mind, the main aim of the present work is to develop a new method for assessing angiogenesis using a gray-level co-occurrence matrix (GLCM)-based textural-feature image processing tool, which could be co-related with more reliable parameters. To establish this method, amino acid-based model copolymer nanoparticles (NPs), i.e. poly[(N-acryloyl glycine)-co-(N-acryloyl-(L-phenylalanine methyl ester))], or p(NAG-co-NAPA), have been synthesized and tested for cell viability. These p(NAG-co-NAPA) NPs exhibited enhanced metabolic activity up to ∼115–120% with L929 (mouse fibroblast) cells, HUVECs (human umbilical endothelial cells) and RAW 264.7 macrophages. In the 2nd step, the pro-angiogenic properties of p(NAG-co-NAPA) NPs were investigated through an ‘in ovo’ model using the CAM assay. Approximately 1000 microscopic images of newly formed blood vessels were segmented using adaptive thresholding, and their angiogenic properties were analyzed using seven GLCM-based textural features. Furthermore, to predict their regeneration efficiency, the tube formation assay was performed using HUVEC cells and the results were cross-verified with the Angiotool method to establish the GLCM-based textural-feature method as a preliminary quantitative framework for the assessment of angiogenesis. In all, we have established a new approach for angiogenesis analysis, with the conclusion that p(NAG-co-NAPA) NPs could be paramount for various therapeutic applications.