Targeting the gut barrier: identification of a homing peptide sequence for delivery into the injured intestinal epithelial cell

Abstract

Background: Severe injury results in intestinal barrier dysfunction that may be responsible for significant morbidity and mortality. We postulated that mining a peptide library that was displayed on phage would identify peptide sequences that bind and internalize into the gut epithelium following injury.

Methods: We utilized a severe full thickness burn in mice as a model of severe injury. Candidate peptides were identified by screening 10(12) phage displaying unique peptide sequences. In vivo assessment was performed by injecting targeted phage into the lumen of a segment of distal ileum following burn injury, then analyzed for uptake of peptide sequence using quantitative polymerase chain reaction (PCR), DNA sequencing, and confocal microscopy of the peptide bound to quantum dots (Qdots).

Results: Phage screening identified the peptide sequence T18 (LTHPQDSPPASA) as an optimal candidate for in vivo testing. PCR of intestinal cells following injury showed a higher level of T18 sequence when compared to untargeted phage. Confocal microscopy of the peptide sequence bound to Qdots showed internalization into gut mucosa following injury.

Conclusion: We have identified a peptide sequence that targets the injured intestinal epithelium and may allow for the development of targeted therapies to attenuate inflammation, or other pathologic conditions of the small bowel.

Fig 1

Candidate peptide sequences from screened phage clones. A total of 32 unique candidate 12-amino acid sequences were identified.

Fig 2

Ex vivo staining of intestinal sections to screen candidate peptide sequences. Candidate peptide sequences were incubated with fixed intestinal specimens from burned animals. Sections were then stained with anti-M13, which recognizes phage and DAPI. Positive staining was seen in candidate sequences T18 (LTHPQDSPPASA) and AS8 (NPNLNTRVLVTG). Empty phage bearing no peptide sequence was used as a control.

Fig 3

Quantitative PCR of intestinal extracts from in vivo phage screening. Quantitative PCR of the T18 sequence (LTHPQDSPPASA) was compared to empty phage bearing no peptide sequence, PBS, and the previously published 7-peptide TMT sequence (YPRLLTP). (A) Quantitative PCR of intestinal mucosa from animals 2 h following severe cutaneous burn. (B) Quantitative PCR of intestinal mucosa from sham animals.

Fig 4

DNA sequencing of PCR product obtained from in vivo phage screening. (A) DNA sequence of the peptide-phage conjugate recovered from intestinal mucosa of animals 2 h following severe burn. (B) Translation of the DNA sequence obtained yields an mRNA sequence which would yield the T18 peptide sequence (LTHPQDSPPASA) confirming that the T18-phage was internalized into the intestinal mucosa and recovered.

Fig 5

Confocal microscopy of candidate peptide sequence conjugated to Quantum Dots (Qdots). Sections of distal small intestine obtained from animals 2 h following severe cutaneous burn after in vivo exposure to intraluminal Qdot-T18 conjugates or Qdots alone. Fluorescence from Qdots viewed using a laser scanning confocal microscope. Intestinal sections also stained for LYVE-1, an antibody specific for lymphatic endothelium. The T18-Qdot conjugate allows for visualization of internalization of the T18 sequence into burn-injured intestinal mucosa. The T18 sequence co-localizes with the LYVE-1 antibody suggesting affinity for the intestinal lymphatics. Size bar = 50μm.