Targeted delivery of doxorubicin toward 4 T1 cells via in situ binding between maleimide functionalized AS1411 − NH2 aptamer and endogenous albumin

Abstract

Trojan horse strategy is a targeted delivery system that directs therapeutic agents toward specific sites through in situ binding with circulating endogenous albumin. This study developed a Trojan horse system utilizing AS1411 − NH₂ aptamer (Apt), Mal-PEG_3.5-NHS ester (PEG), human/mouse serum albumin (HSA/MSA), and doxorubicin (DOX). The formation of Apt−PEG conjugate was confirmed via agarose gel electrophoresis, while its attachment to HSA was validated using SDS-PAGE. The loading capacity for transporting DOX was assessed by quenching the fluorescence intensity of DOX. The PEG's maleimide formed an in situ bond with albumin's cysteine-34, producing DOX − Apt−PEG−HSA compound in vitro/DOX − Apt−PEG−MSA complex in vivo. The DLS revealed proper size and negative charge distribution, and the drug release profile demonstrated stability and pH-sensitivity of the compound. The functionality and internalization of DOX − Apt−PEG composite were validated through in vitro assays, including MTT, live/dead, flow cytometry, apoptosis/necrosis, and fluorescence imaging. In vivo assessments of DOX within the complex on six 4 T1 tumor-bearing mice presented four cases of tumor shrinkage and two cases of tumor ablation. Histological studies and ex vivo/in vivo fluorescence imaging on tumor-bearing mice exhibited significant accumulation of complex, leading to massive tumor necrosis and less accumulation in other organs, validating a noticeable reduction in overall toxicity.