HPLC/MS data obtained from interaction of SJG-136 (2) with Hairpin-5.

<p><b>A</b>, HPLC chromatogram showing the annealed Hairpin-5 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152303#pone.0152303.g002" target="_blank">Fig 2</a>) alone at RT 7.64 min; <b>B</b>, HPLC chromatogram after incubation of annealed Hairpin-5 with <b>2</b> for 24 hours, showing approximately 50% conversion to the adduct peak at RT 14.03 min; <b>C</b>, MALDI-TOF spectrum of the adduct at RT 14.03 min from Chromatogram B above. Observed mass of 1:1 <b>2</b>/Hairpin-5 adduct: 6305.1 m/z (theoretical mass: 6305.41 m/z), observed mass of DNA Hairpin-5 alone from Chromatogram <b>A</b>: 5748.3 m/z (theoretical mass: 5748.8 m/z).</p

Structures of Hairpins 1–9 used in this study.

<p>Hairpin-1, the AP-1 transcription factor recognition sequence; Hairpins 2–4, the same AP-1 sequence but with two of the four guanines replaced with inosines in each case to study cross-linking; Hairpins 5–8, the same AP-1 sequence but with three of the four guanines replaced with inosines in each case to study mono-alkylation; Hairpin 9, the same AP-1 sequence but with three of the four guanine bases (except the 5’-terminal-guanine) mutated to an A.</p

AP-1 consensus sequence.

<p>Schematic diagram of the various possible mono-alkylated (blue) and interstrand cross-linked (red) adducts that could potentially form between SJG-136 (<b>2</b>) and the parent AP-1 sequence (Hairpin-1).</p

Summary of HPLC/MALDI-TOF results obtained on the interaction of SJG-136 with Hairpins 1–8.

<p>Summary of HPLC/MALDI-TOF results obtained on the interaction of SJG-136 with Hairpins 1–8.</p

Schematic and molecular models of anthramycin (1), SJG-136 (2) and GWL-78 (3) covalently bound to the terminal guanine of Hairpin-5.

<p><b>A</b>, Schematic model of <b>1</b> (green) covalently bound to G1 (magenta) of Hairpin-5 with the A-ring pointing toward the TTT-loop (A-ring-3’ orientation); <b>B</b>, Low energy snapshot of a 10 ns molecular dynamics simulation illustrating the C2-tail of <b>1</b> orienting outside of the DNA structure, suggesting that a DNA triplet is necessary for full accommodation of the molecule; <b>C</b>, Schematic model of <b>2</b> bound covalently to G1 of Hairpin-5, with the bulk of the molecule positioned within the minor groove (without cross-link formation) pointing toward the TTT-loop (<i>i</i>.<i>e</i>., the forward direction); <b>D</b>, Low energy snapshot of a molecular dynamics simulation (10 ns) of <b>2</b> covalently bound to the 5’-terminal G1 of Hairpin-5, illustrating the good accommodation of <b>2</b> (green sticks) within the minor groove and the non-covalent interactions between the central methylene linker of the ligand and the A2:T18 and C3:I17 base-pairs. <b>E</b>, Schematic model of <b>3</b> covalently bound to G1 of Hairpin-5, with the heterocyclic C8-sidechain pointing towards the TTT-loop; <b>F</b>, Low energy snapshot of a 10 ns molecular dynamics simulation of <b>3</b> (blue) covalently bound to G1 (magenta) of Hairpin-5, illustrating the comfortable accommodation of the C8-poly-pyrrole tail of <b>3</b> in the minor groove. It is likely that the poly-pyrrole tail forms non-covalent interactions with 5’-ACAT-3’ at 5’-positions 2–4 (yellow), guiding the PBD core to the G1 base.</p

Probing milk extracellular vesicles for intestinal delivery of RNA therapies

Background Oral delivery remains unattainable for nucleic acid therapies. Many nanoparticle-based drug delivery systems have been investigated for this, but most suffer from poor gut stability, poor mucus diffusion and/or inefficient epithelial uptake. Extracellular vesicles from bovine milk (mEVs) possess desirable characteristics for oral delivery of nucleic acid therapies since they both survive digestion and traverse the intestinal mucosa. Results Using novel tools, we comprehensively examine the intestinal delivery of mEVs, probing whether they could be used as, or inform the design of, nanoparticles for oral nucleic acid therapies. We show that mEVs efficiently translocate across the Caco-2 intestinal model, which is not compromised by treatment with simulated intestinal fluids. For the first time, we also demonstrate transport of mEVs in novel 3D ‘apical-out’ and monolayer-based human intestinal epithelial organoids (IEOs). Importantly, mEVs loaded with small interfering RNA (siRNA) induced (glyceraldehyde 3-phosphate dehydrogenase, GAPDH) gene silencing in macrophages. Using inflammatory bowel disease (IBD) as an example application, we show that administration of anti-tumour necrosis factor alpha (TNFα) siRNA-loaded mEVs reduced inflammation in a IBD rat model. Conclusions Together, this work demonstrates that mEVs could either act as natural and safe systems for oral delivery or nucleic acid therapies, or inform the design of synthetic systems for such application. Graphical Abstract</p