Cancer cell-selective, clathrin-mediated endocytosis of aptamer decorated nanoparticles

Abstract

Lung cancer is the leading cause of cancer mortality worldwide, resulting in 88% deaths of all diagnosed patients. Hence, novel therapeutic modalities are urgently needed. Single-stranded oligonucleotide-based aptamers (APTs) are excellent ligands for tumor cell targeting. However, the molecular mechanisms underlying their internalization into living cells have been poorly studied. Towards the application of APTs for active drug targeting to cancer cells, we herein studied the mechanism underlying S15-APT internalization into human non-small cell lung cancer A549 cells. We thus delineated the mode of entry of a model nanomedical system based on quantum dots (QDs) decorated with S15-APTs as a selective targeting moiety for uptake by A549 cells. These APT-decorated QDs displayed selective binding to, and internalization by target A549 cells, but not by normal human bronchial epithelial BEAS2B, cervical carcinoma (HeLa) and colon adenocarcinoma CaCo-2 cells, hence demonstrating high specificity. Flow cytometric analysis revealed a remarkably low dissociation constant of S15-APTs-decorated QDs to A549 cells (Kd = 13.1 ± 1.6 nM). Through the systematic application of a series of established inhibitors of known mechanisms of endocytosis, we show that the uptake of S15-APTs proceeds via a classical clathrin-dependent receptor-mediated endocytosis. This cancer cell-selective mode of entry could possibly be used in the future to evade plasma membrane-localized multidrug resistance efflux pumps, thereby overcoming an important mechanism of cancer multidrug resistance.

Selective eradication of human non-small cell lung cancer cells using aptamer-decorated nanoparticles harboring a cytotoxic drug cargo

“…We found ( Fig. 7) that APT(30)-NPs are taken up via classical clathrin-mediated endocytosis (CME), as we have recently reported for S15-APT-decorated QDs that possess a smaller size than the APT-PEG-PCL NPs 37 . In the clinical setting, it is expected that internalization via CME will enable an efficient cellular uptake of PTX-loaded APT-NPs and achieve targeted drug delivery, resulting in efficacious therapeutic treatment.…”

“…Furthermore, the specificity of S15-APT was explored previously by our group in human cell lines 37 . Hence, these APT-NPs hold great promise towards eradication of NSCLC cells.…”

“…We next undertook experiments to verify that the uptake of APT-NPs proceeds via endocytosis, as we previously found for quantum dots (QDs)-labeled APTs 37 . We used inhibitors of several processes related to endocytosis.…”

“…A549 target cells were subjected to various established endocytosis inhibitors and analyzed by confocal laser microscopy ( Fig. 7b-d) 37 . Neither pretreatment with filipin ( Fig.…”

Aptamer-Conjugated Superparamagnetic Ferroarabinogalactan Nanoparticles for Targeted Magnetodynamic Therapy of Cancer

“…Aptamers are considered to be a synthetic chemical product, rather than biological because they are chemically synthesized in high purity by an automated procedure. Functionalization of nanoparticles with the aptamers improves their biocompatibility, colloidal stability, and increases the circulation time in vivo [19][20][21].…”