First-in-human clinical trial of allogeneic, platelet-derived extracellular vesicles as a potential therapeutic for delayed wound healing

Abstract

The release of growth factors, cytokines and extracellular matrix modifiers by activated platelets is an important step in the process of healthy wound healing. Extracellular vesicles (EVs) released by activated platelets carry this bioactive cargo in an enriched form, and may therefore represent a potential therapeutic for the treatment of delayed wound healing, such as chronic wounds. While EVs show great promise in regenerative medicine, their production at clinical scale remains a critical challenge and their tolerability in humans is still to be fully established. In this work, we demonstrate that Ligand-based Exosome Affinity Purification (LEAP) chromatography can successfully isolate platelet EVs (pEVs) of clinical grade from activated platelets, which retain the regenerative properties of the parent cell. LEAP-isolated pEVs display the expected biophysical features of EV populations and transport essential proteins in wound healing processes, including insulin growth factor (IGF) and transforming growth factor beta (TGF-ß). In vitro studies show that pEVs induce proliferation and migration of dermal fibroblasts and increase dermal endothelial cells' angiogenic potential, demonstrating their wound healing potential. pEV treatment activates the ERK and Akt signalling pathways within recipient cells. In a first-in-human, double-blind, placebo-controlled, phase I clinical trial of healthy volunteer adults, designed primarily to assess safety in the context of wound healing, we demonstrate that injections of LEAP-purified pEVs in formulation buffer are safe and well tolerated (Plexoval II study, ACTRN12620000944932). As a secondary objective, biological activity in the context of wound healing rate was assessed. In this cohort of healthy participants, in which the wound bed would not be expected to be deficient in the bioactive cargo that pEVs carry, all wounds healed rapidly and completely and no difference in time to wound closure of the treated and untreated wounds was observed at the single dose tested. The outcomes of this study evidence that pEVs manufactured through the LEAP process can be injected safely in humans as a potential wound healing treatment, and warrant further study in clinical trials designed expressly to assess therapeutic efficacy in patients with delayed or disrupted wound healing.

Keywords: extracellular vesicles; first-in-human platelet EV therapy; platelet EVs; wound healing.

FIGURE 1

Characterization of LEAP‐purified pEVs. (a) Size distribution of clinical pEVs measured by Spectradyne, detecting pEVs from 65 to 400 nm. (b) Percentage of pEVs in clinical pEVs present within the size ranges of 65–100, 100–200, and 200–400 nm. (c) Cryo‐TEM images (left zoomed out, right zoomed in) of clinical pEVs, confirming the size distribution and innate morphology of the pEVs. Scale bar: 500 nm (left), 200 nm (right). (d) Western blot analysis of clinical pEVs showed enrichment of known EV markers CD9, CD63, and syntenin relative to platelet lysate and showed the absence of the negative EV marker, calnexin.

FIGURE 2

Mass spectrometric analysis revealed the abundance of proteins present in clinical pEVs based on whether they are involved in: (a) molecular function, (b) biological processes and (c) subcellular localisation.

FIGURE 3

Confocal microscopy images demonstrate (a) pEV association with NHDFs, which increases over time of incubation, as opposed to (b) Exoria control, where no signal is detected. Red: Exoria‐labelled pEVs, blue: DAPI (nucleus). Scale bar: 10 μm.

FIGURE 4

(a) Growth curve of NHDF cells in minimal growth media over 70 h treated with 15 μg/mL of three batches of clinical pEVs (red) and without any treatment (blue). (b) Cell index of clinical pEV‐treated NHDF cells versus no treated cells after 48 h of incubation, demonstrating that clinical pEVs significantly improved cell proliferation (n = 3, *p < 0.05, one‐way ANOVA). (c) Representative images of scratched area displaying NHDF cells migration capacity when untreated (top) or treated with clinical pEVs (bottom). Blue: DAPI (nucleus). (d) Quantification of migrated cells in treated and untreated cells after 24 h using ImageJ (*p < 0.05, one‐way ANOVA). (e) Microscope images of HDMEC cells with clinical pEVs (bottom) and without (top) clinical pEVs treatment at different time points: day 1, day 2 and day 5. After approximately day 3, clinical pEV‐treated HDMEC cells show tubular formation due to enhanced angiogenesis capacity. Scale bar: 100 μm.

FIGURE 5

Measurement of (a) pAkt/Akt and (b) pERK/ERK ratios over time when untreated (black) or treated with pEVs (red). Comparison of (c) Akt and pAkt levels, and (d) ERK and pERK levels in cells after 5 min incubation with clinical pEVs (red) as opposed to untreated cells (white). n = 6, *p < 0.05, one‐way ANOVA.

FIGURE 6

(a) Plot of wound healing rate, showing the wound diameter (defined as the sum of the vertical and horizontal diameter) of treated (red) and untreated (blue) 4 mm punch biopsy induced wounds over time. (b) Plot of wound healing rate, showing the number of treated (red) and untreated (blue) wounds to have completely healed, as recorded during participant physical examination.