Folate-Conjugated Halloysite Nanotubes, an Efficient Drug Carrier, Deliver Doxorubicin for Targeted Therapy of Breast Cancer

Abstract

To carry doxorubicin (DOX) on breast cancer site effectively, halloysite nanotubes conjugated with poly(ethylene glycol) and folate (HNTs-PEG-FA) is designed as a targeted drug delivery system. Halloysite nanotubes (HNTs) are shortened to ∼200 nm by ultrasonic scission and functionalized with amide groups to conjugate with N-hydroxylsuccinimide-polyethylene glycol carboxylic acid (NHS-PEG-COOH) and folate (FA). DOX@HNTs-PEG-FA is prepared by loading DOX on HNTs-PEG-FA via physical adsorption. The sustained and controlled release of DOX from DOX@HNTs-PEG-FA is up to 35 h in an acidic environment (pH 5.3). DOX@HNTs-PEG-FA, performed as a new nanodelivery system, shows significant inhibition of proliferation and induction of death in MCF-7 cells with positive FA receptor but not in L02 cells with negative FA receptor. Results of acridine orange/ethidium bromide and flow cytometric assay indicate that DOX@HNTs-PEG-FA induces cell death through apoptosis. Compared to the same dose of DOX, DOX@HNTs-PEG-FA generates more reactive oxygen species (ROS) in MCF-7 cells, which lead to mitochondrial damage and apoptosis. Furthermore, with fluorescence images and transmission electron microscopy, uptake of DOX@HNTs-PEG-FA by tumor cells is both through endocytosis and direct penetration mechanism. The in vivo antibreast cancer activity of DOX@HNTs-PEG-FA is further confirmed in 4T1-bearing mice. In contrast to DOX, DOX@HNTs-PEG-FA effectively reduces heart toxicity and inhibits solid tumor growth with higher cleaved caspase-3 protein level in tumor tissue of 4T1-bearing mice. DOX@HNTs-PEG-FA reveals a higher DOX fluorescence intensity in tumor tissue than in other normal tissues including heart, spleen, lung, and kidney at different time points. All these results suggest that FA-conjugated HNTs may be designed to be a novel drug delivery system for targeted therapy of breast cancer via intravenous injection.