Selective protein delivery to acidic tumor and inflammatory microenvironment by pH sensitive linker application

Abstract

Inflammatory region and tumor microenvironment (TME) have lower pH than normal tissues due to increased cell density to produce acidic waste. Therefore, a targeting strategy for acidic microenvironment to specific drug distribution in vivo could be considered with a CPP (cell-permeable peptide) mediate protein delivery in tumor or inflammatory disease model. Here, we designed pH sensitive linker (HE) which could attenuate a CPP function by charge interaction. We generated a recombinant protein containing pH-sensitive linker and AP (Astrotactin 1–derived peptide)-CPP and screened its protein delivery efficiency in various pH environment. In vitro, we optimized proper pH sensitive sequence (AP-10HE) and confirmed that the protein delivery efficiency was increased pH 6.5 or 6 than 7.4 in several cell lines like Hela, U271, U343, Hacat. Next, we would like to confirm pH linker prevent AP-dTomato protein delivery in liver, kidney, spleen tissue in normal mice and hypothesized it could be more specific in tumor or diseased tissue. We found AP-10HE shows specific delivery to tumor region in subcutaneously implanted B16F10 cells of mice and it also specifically delivered in spinal cord tissue rather than liver or kidney in EAE (Experimental autoimmune encephalomyelitis) model. These results suggest that the pH sensitive linker combined to AP-CPP could be a specific targeting tool for delivering of biologic drug like a protein into acidic microenvironment, so could be utilized for tumor therapy or immune modulatory drug development research.