Engineering Nano‐Based Delivery Systems for a Rectal Pre‐Exposure Prophylaxis Treatment for HIV Prevention
Authors: Nelson Antoinette , Szekely Zoltan , Zhang Xiaoping , Sinko Patrick J 
Published: November 2023

IntroductionCurrent FDA approved pre‐exposure prophylaxis (PrEP) treatment for HIV prevention, Truvada, requires patients to follow a strict once‐a‐day oral dosing regimen that raises concerns about systemic toxicity. Patient adherence is also a limiting factor. Our goal is to develop a mucosal PrEP approach to address these concerns by delivering antiretrovirals directly to the colorectal tissue in a sustained manner. However, a major obstacle to drug delivery in the colorectum is penetration of the protective epithelial cell barrier and its mucus lining. The objective of our study was to engineer nano‐sized delivery systems labeled with a cell penetrating peptide to translocate through colorectal mucus into the tissue and be retained in the rectum and distal colon.Materials and MethodsTwo types of nano‐sized delivery systems were investigated. Fluorescently‐labeled 3.4 kDa poly(ethylene glycol) (PEG) was conjugated to the antiretroviral drug amprenavir or cell‐penetrating peptide, bac7, forming two conjugates, APF and BPF respectively. The second delivery system consisting of fluorescently‐labeled polycaprolactone–poly(ethylene glycol) nanoparticles (NPs) was formulated by Flash Nanoprecipitation. These NPs, about 60 nm in diameter, were conjugated with bac7 to achieve low (1%) or high (10%) surface coverage.In vitro cellular uptake and transportCaco‐2 intestinal cells were treated with APF or BPF and analyzed for cell uptake after two hours by confocal microscopy and flow cytometry. Caco‐2 monolayers were grown for 21‐days on transwell inserts and then treated with plain NPs, 1% bac7 NPs, or 10% bac7 NPs for 18 hours. Transport of NPs across the cell barrier was investigated. Mucus penetration studies were also performed using fluorescence recovery after photobleaching (FRAP).In vivo efficacyMale CD1 mice were treated with enemas containing APF or BPF. Mice were euthanized up to 5 days post dose. Tissue retention in the rectum and distal colon was investigated by fluorescence analysis. Similar testing was done for bac7 NPs and samples were collected at 2 and 24 hours.ResultsIn vitro, we found that bac7 is able to significantly increase cellular uptake and transport of both PEG conjugates and NPs. We report a 4‐fold increase in uptake for BPF compared to APF in vitro. 10%‐labeled bac7 NPs showed the greatest transport across Caco‐2 cells in vitro with an approximately 10‐fold increase in translocation across the monolayer when compared to plain NPs. In vivo, BPF was found within the colorectal tissue at a significantly higher concentration than APF at all time points. BPF was detected within colorectal tissue for up to 5 days. This suggests transport of the conjugate into the lamina propria, a prime target for PrEP treatment. For bac7‐conjugated NPs, we report a 3‐fold increase in uptake for NPs with 1% bac7 surface coverage compared to plain and 10% covered NPs. All NPs were significantly cleared from the colon after 24 hours.ConclusionsBac7 is effective in increasing the transport of PEG conjugates and NPs across a cell barrier in vitro and in vivo. PEG conjugates appear to show greater tissue absorption and retention compared to larger NPs. Charge and steric hindrance may limit the translocation of 10% labeled NPs across the mucus and epithelial layer in vivo. Future work will investigate the use of branched PEG conjugates to increase nano‐carrier drug loading.Support or Funding InformationFunding by NIH Grants T32GM008339, R01AI084137 and R37AI051214