Enhanced Translocation of Nanoparticles with Low Surface Coverage of Phage Display Peptide into Rat Intestinal Mucosa

Little 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 Methods The P19 sequence was optimized (P19 opt ) 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 (P19 NC ) was synthesized by replacing an aspartic acid with a neutral asparagine residue. P19 opt and P19 NC 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 P19 opt or P19 NC 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% P19 opt 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. Results NPs displaying P19 opt 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 P19 opt and either low or high density of P19 NC , NP translocation was not significantly different than control NPs. Rat intestinal ligation studies showed significantly greater mucosal concentrations of NPs with 0.5% P19 opt in the upper and mid small intestine, as well as the colon when compared to control NPs. Microscopy confirmed the presence of 0.5% P19 opt NPs in the villi after 2 hr of treatment. Conclusions Increased Caco‐2 translocation at low surface coverage of P19 opt supports the significance of controlled ligand display on the NP surface. The inactivity of the P19 NC peptide suggests the importance of a negatively‐charged residue to the functional conformation of the peptide. Due to the enhanced uptake of 0.5% P19 opt NPs into rat intestinal mucosa, we conclude that optimally displayed P19 opt NPs show considerable promise and warrant further investigation for oral drug delivery. Support or Funding Information Insight provided by Dr. Robert K. Prud'homme's lab at Princeton University; Funding by NIH Grants T32GM008339, R01AI084137, and R37AI051214