Nanoscopic and functional characterization of keratinocyte-originating exosomes in the wound fluid of non-diabetic and diabetic chronic wound patients

Exosomes, a class of extracellular vesicles of endocytic origin, play a critical role in paracrine signaling for successful cell-cell crosstalk in vivo. However, limitations in our current understanding of these circulating nanoparticles hinder efficient isolation, characterization, and downstream functional analysis of cell-specific exosomes. In this work, we sought to develop a method to isolate and characterize keratinocyte-originated exosomes (hExoκ) from human chronic wound fluid. Furthermore, we studied the significance of hExoκ in diabetic wounds. LC-MS-MS detection of KRT14 in hExoκ and subsequent validation by Vesiclepedia and Exocarta databases identified surface KRT14 as a reliable marker of hExoκ. dSTORM nanoimaging identified KRT14+ extracellular vesicles (EVκ) in human chronic wound fluid, 23% of which were of exosomal origin. An immunomagnetic two-step separation method using KRT14 and tetraspanin antibodies successfully isolated hExoκ from the heterogeneous pool of EV in chronic wound fluid of 15 non-diabetic and 22 diabetic patients. Isolated hExoκ (Ø 75–150 nm) were characterized per EV-track guidelines. dSTORM images, analyzed using online CODI platform followed by independent validation using Nanometrix, revealed hExoκ Ø as 80–145 nm. The abundance of hExoκ was low in diabetic wound fluids and negatively correlated with patient HbA1c levels. The hExoκ isolated from diabetic wound fluid showed a low abundance of small bp RNA (<200 bp). Raman spectroscopy underscored differences in surface lipids between non-diabetic and diabetic hExoκ. Uptake of hExoκ by monocyte-derived macrophages (MDM) was low for diabetics versus non-diabetics. Unlike hExoκ from non-diabetics, the addition of diabetic hExoκ to MDM polarized with LPS and INFγ resulted in sustained expression of iNOS and pro-inflammatory chemokines known to recruit macrophages (mϕ).This work provides maiden insight into the structure, composition, and function of hExoκ from chronic wound fluid thus providing a foundation for the study of exosomal malfunction under conditions of diabetic complications such as wound chronicity.