Development of an aprotinin-based novel nano-bioconjugate utilizing microfluidics via 3D cancer spheroid models

Abstract

Proteins are promising substances for introducing new drug carriers with efficient blood circulation due to low possibilities of clearance by macrophages. However, such natural biopolymers have highly sophisticated molecular structures, preventing them from being assembled into nano-platforms with manipulable payload release profiles. Here, we announce a novel anti-cancer nano-drug carrier moonlighting protein, Aprotinin, to be used as a newly identified carrier for cytotoxic drugs. The Aprotinin-Dox orubicin (Apr-Dox) nano-bioconjugate was prepared via a single-step microfluidics co-flow mixing technique; a feasible and simple way to synthesize a carrier-based drug design with a double-barreled approach that can release and actuate two therapeutic agents simultaneously i.e., Apr-Dox in 1:11 ratio (aprotinin an anti-metastatic carrier drug and chemotherapeutic drug DOX). With a significant stimuli-sensitive (i.e. pH) drug release ability, this nanobioconjugate achieves superior bio-performances including high cellular uptake, efficient tumor penetration and accumulation into acidic tumor microenvironment, as well as inhibiting further tumor growth by halting the urokinase plasminogen activator (uPA) involved in metastasis and tumor progression. Distinctly, in healthy human umbilical vein endothelial (HUVEC) cells, drastically lower cellular uptake of nano-bioconjugate has been observed and validated compared to anticancer agent Dox. Our findings demonstrate an enhanced cellular internalization of nano-bioconjugates towards breast cancer, prostate cancer, and lung cancer both in vitro and in physiologically relevant biological 3D-spheroid models. Consequently, the designed nano-bioconjugate shows a high potential for targeted drug delivery via natural and biocompatible moonlighting protein, thus opening a new avenue for proving aprotinin in cancer therapy both as an anti-metastatic and drug-carrying agent.