Engineering Extracellular Vesicles to Target Pancreatic Tissue In Vivo

Abstract

Extracellular vesicles (EVs) are naturally released, cell-derived vesicles that mediate intracellular communication, in part, by transferring genetic information and, thus, have the potential to be modified for use as a therapeutic gene or drug delivery vehicle. Advances in EV engineering suggest that directed delivery can be accomplished via surface alterations. Here we assess enriched delivery of engineered EVs displaying an organ targeting peptide specific to the pancreas. We first characterized the size, morphology, and surface markers of engineered EVs that were decorated with a recombinant protein specific to pancreatic β-cells. This β-cell-specific recombinant protein consists of the peptide p88 fused to the EV-binding domain of lactadherin (C1C2). These engineered EVs, p88-EVs, specifically bound to pancreatic β-cells in culture and transferred encapsulated plasmid DNA (pDNA) as early as in 10 min suggesting that the internalization of peptide-bearing EVs is a rapid process. Biodistribution of p88-EVs administrated intravenously into mice showed an altered pattern of EV localization and improved DNA delivery to the pancreas relative to control EVs, as well as an accumulation of targeting EVs to the pancreas using luciferase activity as a readout. These findings demonstrate that systemic administration of engineered EVs can efficiently deliver their cargo as gene carriers to targeted organs in live animals.

Keywords: EVs engineering; Extracellular Vesicles; Imaging; Pancreatic β-cells; Targeted delivery..

Figure 1

Design and schematic presentation of a C1C2-peptide fusion protein (A) Peptide-C1C2 fusion protein expression driven by a CMV promoter in pcDNA6.0 derived pcS vector. The recombinant protein comprising a lactadherin signal peptide (SP), Hemagglutinin tag (HA), peptide sequence, (GGGGS)₃ Linker, EV anchor region of lactadherin (C1C2), and polyhistidine tag (HA). (B) Depiction of EV with engineered EV displaying peptide on its surface and encapsulated pDNA, and predicted protein structure of peptide-hC1C2 chimeric protein. (C) Schematic flow of EV isolation process.

Figure 2

Successful isolation and characterization of engineered EVs displaying peptides of interest. (A) Representative size distribution of the naïve, pep1- and p88-display EVs determined by Nanoparticle Tracking Analysis. (B) Transmission electron microscopy images of naïve, pep1-, and p88-EVs showing gold labeled HA and CD63 surface markers. (C) Western blot analysis of engineered EVs (p88 fusion peptide-44Kda; pep1 fusion peptide-42KDa) for the presence of EV biomarkers CD63(30-60KDa) and TSG101(44KDa), and peptide HA-tag. Additionally, analysis of cell lysate and engineered EVs for cellular biomarkers Calnexin (67KDa) and β-actin (42KDa) (D) Summary of particle number and pDNA copy numbers determined by NTA and qPCR of pep1- and p88-display EVs. (E) pDNA quantification before and after DNase I treatment of EVs. Naïve EVs mixed with pDNA and p88 EVs were treated with DNase I. pDNA were quantified by qPCR following pDNA isolation. *pDNA undetected

Figure 3

Specific binding of targeting EVs to pancreatic β-cell line in vitro. (A) NIT-1 cells and 4T1 cells received non-tarting (NP)- or β-cell-targeting (p88)-EV treatment after PFA fixation. Representative image of EV (gLuc) binding to NIT-1 or 4T1 cells. The total photon flu (p/s) from EVs bound to the cells was quantified using IVS. The value represents the means ± SD (n=3) in the graph. (B) NIT-1 and 4T1 cells were co-cultured and treated either with mCherry-EVs (upper row), or p88-mCherry-EVs (lower row) for 1 hr. The cells were fixed, and the binding was assessed by confocal imaging of EVs (red), anti-insulin antibody (FITC-conjugated) and nuclear staining with DAPI. microscopic images of cocultured NIT1 (FITC insulin labeled; Green), 4T1 cells treated with p88-mCherry-EVs, and mCherry-EVs (red). DAPI stain (blue) Scale bars, 20 μm.

Figure 4

Altered EV biodistribution by peptide-display, and pancreas enriched pDNA delivery by β-cell-targeting EVs. (A) Representative images of the organs from the Balb/cJ mice received intravenous injections of PBS, NP-gLuc- or p88-gLuc-EVs. (B) Balb/cJ mice received intravenous injection of NP- or p88-labeled EVs. The organs were removed from the mice post-mortem and homogenized for pDNA isolation. qPCR assay was used to determine the copy number from heart, lungs, liver, kidneys, pancreas and spleen. The amounts of recovered pDNA were normalized by mitochondrial DNA. The fold change values represent the average fold change of samples (N).