O2151: Treatment of pediatric intestinal ischemia reperfusion injury with engineered extracellular vesicles

Background: Intestinal ischemia reperfusion injury (IRI) causes intestinal injury in multiple pediatric diseases. Extracellular vesicles (EVs) derived from placental mesenchymal stem cells (PMSCs) may represent a potent therapy for the treatment of IRI via known pro-angiogenic, anti-inflammatory, and neuroprotective mechanisms. PMSC-EVs labeled with a collagen binding peptide (SILY) may demonstrate increased therapeutic efficacy due to tissue retention of SILY-PMSC- EVs. We aim to determine the optimal route of administration of SILY-PMSC-EVs for local tissue delivery and retention. Furthermore we aim to determine the therapeutic effects of native PMSC-EVs and SILY-PMSC-EVs in a pediatric rat IRI model.

Methods: EVs were isolated from PMSC culture media and analyzed with Nanoparticle Tracking Analysis (NTA) and western blot. TAMRA-labeled SILY peptide was conjugated to PMSC-EVs via amine crosslinking click chemistry with conjugation efficiency determined by ExoView co-labeling of EV particles with antibodies against CD9 and CD63 (n = 2). IRI was induced in postnatal day 14 rats by clamping of the superior mesenteric artery for 30 minutes, followed by intraperitoneal (IP) or per oral (PO) gavage administration of PBS, DiD-labeled native PMSC-EVs, or DiD-SILY-PMSC-EVs, with in vivo fluorescent imaging every 48-72 hours (n = 5-6). Statistical analyses included two-way ANOVA with Sídak’s multiple comparisons.

Results: PMSC-EVs were isolated and successfully validated by NTA for size (~100 nm) and by western blot with expression of EV-specific membrane proteins (ALIX, TS101, CD9, CD63, CD81) and no expression of calnexin as a negative control. ExoView analysis demonstrated a SILY conjugation efficiency of 77% (n = 2). Animal groups included sham laparotomy with administration of PBS, IRI with PBS, IRI with native PMSC-EVs, or IRI with SILY-PMSC-EVs. To determine the optimal route of administration treatments were administered by IP injection or PO gavage following IRI. In vivo imaging at post-operative days (POD) 3, 5, and 7 demonstrated intra-abdominal PMSC-EVs and SILY-PMSC-EVs by IP injection, with significant retention of SILY-PMSC-EVs at 7 days vs. PBS control (p < 0.0001) (Fig. 1). Furthermore, SILY conjugation increased retention over that of native PMSC-EVs (p = 0.005). In contrast, PO administration did not result in retention of PMSC-EVs or SILY-PMSC-EVs and was not significant (p > 0.4 among all comparisons)(Fig. 2).

Conclusion: We demonstrate successful PMSC-EV collection with high SILY-PMSC-EV conjugation efficiency. SILY conjugation prevented EV clearance and promoted local retention of EVs up to 7 days post-treatment with IP administration, while EVs were cleared with PO administration. Ongoing investigations aim to determine the therapeutic effects of PMSC-EV and SILY-PMSC-EV administration for the treatment of IRI, including photoacoustic imaging of intestinal oxygenation and perfusion, multiplex analysis of intestinal inflammatory markers and markers of proliferation, bowel architecture, and immunohistochemistry to investigate angiogenesis and enteric neuroprotection.