Nanoscopic and Functional Characterization of Keratinocyte-Originating Exosomes in the Wound Fluid of Non-Diabetic and Diabetic Chronic Wound Patients

Abstract

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 (${\text{hExo}}_{\text{κ}}$) from human chronic wound fluid. Furthermore, we studied the significance of ${\text{hExo}}_{\text{κ}}$ in diabetic wounds. LC-MS-MS detection of KRT14 in ${\text{hExo}}_{\text{κ}}$ and subsequent validation by Vesiclepedia and Exocarta databases identified surface KRT14 as a reliable marker of ${\text{hExo}}_{\text{κ}}$. dSTORM nanoimaging identified KRT14⁺ extracellular vesicles (${\text{EV}}_{\text{κ}}$) 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 ${\text{hExo}}_{\text{κ}}$ from the heterogeneous pool of EV in chronic wound fluid of 15 non-diabetic and 22 diabetic patients. Isolated ${\text{hExo}}_{\text{κ}}$ (Ø75-150nm) were characterized per EV-track guidelines. dSTORM images, analyzed using online CODI followed by independent validation using Nanometrix, revealed ${\text{hExo}}_{\text{κ}}$ Ø as 80-145nm. The abundance of ${\text{hExo}}_{\text{κ}}$ was low in diabetic wound fluids and negatively correlated with patient HbA1c levels. The ${\text{hExo}}_{\text{κ}}$ 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 ${\text{hExo}}_{\text{κ}}$ Uptake of ${\text{hExo}}_{\text{κ}}$ by monocyte-derived macrophages (MDM) was low for diabetics versus non-diabetics. Unlike ${\text{hExo}}_{\text{κ}}$ from non-diabetics, the addition of diabetic ${\text{hExo}}_{\text{κ}}$ to MDM polarized with LPS and INFγ resulted in sustained expression of iNOS and pro-inflammatory chemokines known to recruit macrophage (mϕ) This work provides maiden insight into the structure, composition, and function of ${\text{hExo}}_{\text{κ}}$ from chronic wound fluid thus providing a foundation for the study of exosomal malfunction under conditions of diabetic complications such as wound chronicity.

Keywords: Diaexosomes; Extracellular vesicles; Nanometrix; dSTORM microscopy; keratinocyte-originated exosomes; macrophage; resolution of inflammation; wound healing.