Enhanced Translocation of Nanoparticles with Low Surface Coverage of Phage Display Peptide into Rat Intestinal Mucosa
Authors: Myers Daniel , Nelson Antoinette , Holloway Jennifer , Szekely Zoltan , Zhang Xiaoping , Sinko Patrick J 
Published: November 2023

IntroductionLittle is known about exploitable mechanisms for nanoparticle (NP) translocation across the intestinal mucosal barrier. Using T7 phage display, a unique peptide (P19) was identified that could function as a ligand that promotes intestinal uptake of NPs. Our objective is to demonstrate P19's potential for NP intestinal translocation and to learn more about its functional conformation.Materials and MethodsThe P19 sequence was optimized (P19opt) by replacing oxidation‐sensitive residues and functionalized with a short PEG‐based linker containing an azido group and fluorescein for chemical ligation and imaging, respectively. A negative control peptide (P19NC) was synthesized by replacing an aspartic acid with a neutral asparagine residue. P19opt and P19NC were conjugated to an amine‐functionalized polycaprolactone‐poly(enthylene glycol) (PCL‐PEG) block copolymer via click chemistry using dibenzylcyclooctyne reagent. Polymer‐peptide conjugates were mixed with PCL‐PEG polymer chains at 2 ratios (0.5% and 10%) to form NPs with a vitamin E core using flash nanoprecipitation. In vitro: Caco‐2 cell monolayers were grown in DMEM media for 21 days on transwell inserts and then treated with NPs displaying low (0.5%) and high (10%) surface coverage of either P19opt or P19NC for 18 hours. Controls for the experiment included NPs with no displayed peptide but labeled with fluorescein. Translocation was studied by measuring fluorescence on the basolateral side. In vivo: Male Sprague‐Dawley rats were anesthetized and intestines were exposed through an abdominal incision. Sacs were formed by tying a 6 cm section at both ends with suture material in specified areas (duodenum, jejunum, and colon). Each sac was injected with NPs displaying 0.5% P19opt or no peptide and treated for 2 hr. The sacs were removed and a small section was sliced to 10 μm thickness using cryosectioning to be evaluated by fluorescent microscopy. Mucosal tissue was scraped from the remainder of each sac and assessed by fluorometric analysis.ResultsNPs displaying P19opt at a low surface density (0.5%) demonstrated ~7‐fold higher translocation (7%) across Caco‐2 cell monolayers than control NPs with no peptide (1%). At a high density (10%) of P19opt and either low or high density of P19NC, NP translocation was not significantly different than control NPs. Rat intestinal ligation studies showed significantly greater mucosal concentrations of NPs with 0.5% P19opt in the upper and mid small intestine, as well as the colon when compared to control NPs. Microscopy confirmed the presence of 0.5% P19opt NPs in the villi after 2 hr of treatment.ConclusionsIncreased Caco‐2 translocation at low surface coverage of P19opt supports the significance of controlled ligand display on the NP surface. The inactivity of the P19NC peptide suggests the importance of a negatively‐charged residue to the functional conformation of the peptide. Due to the enhanced uptake of 0.5% P19opt NPs into rat intestinal mucosa, we conclude that optimally displayed P19opt NPs show considerable promise and warrant further investigation for oral drug delivery.Support or Funding InformationInsight provided by Dr. Robert K. Prud'homme's lab at Princeton University; Funding by NIH Grants T32GM008339, R01AI084137, and R37AI051214