DISCUSSION
diabetic ulcers, obesity, Théry, shock, diabetic ulcer, diabetes, cardiovascular disease
DIABETIC ULCERS, OBESITY, DELAYED WOUND HEALING, DELAYED HEALING, HEAT, SHOCK, PROLIFERATION, DIABETIC ULCER, EVENTS, SECONDARY, BLOOD, PERIPHERAL VASCULAR DISEASE
Several preclinical studies have demonstrated that pEVs represent a promising potential wound healing therapeutic (Guo et al., Blood and its derivatives are complex biofluids from which to isolate EVs (Clayton et al., LEAP chromatography is an affinity‐like, ion‐exchange resin‐based biologic purification protocol highly amenable to manufacturing scale‐up of clinical‐grade material. Ion‐exchange resin‐based chromatography is well‐established in the commercial production of proteins and antibodies (Liu et al., LEAP resins are IEX resins based on cation exchange. Although EVs have a net‐negative charge, local areas of positive charge exist on the outer surface of their lipid bilayer structure. LEAP technology uses charged ligand groups spaced 5 Å apart along the resin backbone to interact with those positive charges to capture EVs (Joseph et al., To validate the use of LEAP to produce clinical‐grade EVs at scale and to explore the potential of LEAP‐purified pEVs as a wound healing therapeutic, human platelet packs donated for research were sourced from a clinical‐grade platelet supplier. Pooled platelet packs were activated by an in‐house protocol to stimulate pEV release, and the pEVs were then isolated from the releasate using LEAP chromatography. The LEAP unit operation proved to be time‐efficient, does not require manual operator intervention once set up, and can be performed by two operators working approximately 8 h to purify the EVs from each activated platelet pack.The pEVs purified through the LEAP protocol were characterised using a range of techniques and were found to display the expected EV biophysical characteristics. Proteomic analysis was performed to analyse the protein cargo of LEAP‐purified pEVs. The pEVs carried the expected EV protein markers, including CD9, annexins, and heat shock proteins (Théry et al., Fibroblasts and endothelial cells have previously been shown to increase their proliferation rate in response to pEV treatment (Guo et al., The migration and proliferation of several cell types into the wound bed are crucial steps in the healing of wounds (Baum & Arpey, Angiogenesis is a critical facet of wound healing. New capillary formation supplies the wound with the required oxygen and nutrients to support recovery. Impaired circulation and poor angiogenesis are known risk factors in, for example, non‐healing diabetic ulcers (Prompers et al., To probe the LEAP‐purified pEVs’ possible cellular mode of action, activation of the ERK and Akt signalling pathways in dermal fibroblasts was assessed. The ERK pathway drives the proliferation and migration of multiple cell types, including keratinocytes and epithelial cells (Lee et al., Studies conducted to date suggest that EV therapeutics have a strong safety profile. The thousands of blood and plasma transfusions conducted daily, in which non‐autologous EVs are safely transferred into patients in large numbers, is one positive indicator of EV safety. EVs have also been tested in a small but growing number of early‐stage human clinical trials, and shown to be generally well tolerated (Kordelas et al., The secondary objective of the clinical study was to conduct an exploratory assessment of the bioactivity of the pEV treatment. No difference was observed in the total time to wound closure between the treated and untreated arms. A mean healing time of 22.8 ± 8.7 days was recorded for both groups, and each wound had entirely healed within 30 days. There was no difference in the physician wound evaluation between treatment groups.As a phase I study focused on assessing safety, several aspects of the study design may explain the lack of observed bioactivity in this clinical trial. Based on previous studies and our in vitro assays, delayed wound healing is the target indication of the LEAP‐purified pEV product. However, the delayed healing patient cohort is typically elderly, and displays health complexities including multiple co‐morbidities such as diabetes, cardiovascular disease, obesity and peripheral vascular disease. Therefore, as a first‐in‐human study, a trial design based on assessing product safety in a small number of healthy participants was selected. The trial design was not powered to draw conclusions about therapeutic efficacy. As such, in depth analysis of product bioactivity, such as wound histology during healing, was not performed.Although an exploratory assessment of wound healing rate was conducted, a 4 mm skin punch biopsy wound in a healthy individual naturally heals rapidly, making any differences in healing time between the clinical pEV treatment and placebo challenging to observe. Furthermore, wound healing from a skin punch biopsy in a healthy individual differs significantly from healing in a chronic wound in a patient with an underlying health condition, such as a non‐healing diabetic ulcer. A healthy wound is not expected to be deficient in the biomolecules that pEVs carry, which may further explain the lack of bioactivity observed. Conversely, patients with chronic wounds suffer from inhibition of the biological events that lead to healing, which pEVs could be able to reverse. For example, in a previous animal study, treatment with pEVs leads to faster wound healing in a rat model of diabetic ulcer than in an untreated wound (Guo et al., In conclusion, we have shown proof of concept that LEAP chromatography is amenable to the scalable manufacture of clinical grade pEVs from platelet cell releasate as an experimental therapy for wound healing. The pEVs were shown to be active in a range of wound‐healing in vitro assays and safe when administered to healthy volunteers in a phase I clinical trial for safety in the context of wound healing. All wounds healed rapidly and completely and no difference in time to wound closure of the treated and untreated wounds was observed. Although the secondary endpoint of the trial, to assess efficacy, was not met in this cohort of healthy volunteers at this dose, the result was not unexpected in the context of a trial designed to assess safety involving only healthy volunteers with a normal swift wound healing response. Further exploration of the efficacy of the pEV therapeutic in future clinical trials for delayed wound healing would be warranted.