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Updated: May 16, 2026
A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer
Published on: September 13, 2022
Target-specific delivery of doxorubicin to retinoblastoma using epithelial cell adhesion molecule aptamer
Nithya Subramanian1, Vaishnavi Raghunathan, Jagat R Kanwar
1Larsen & Toubro Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, Chennai, India.
Purpose:
To study target-specific delivery of doxorubicin (Dox) using an RNA aptamer against epithelial cell adhesion molecule (EpCAM) in retinoblastoma (RB) cells.
Methods:
The binding affinity of the EpCAM aptamer to RB primary tumor cells, Y79 and WERI-Rb1 cells, and Müller glial cell lines were evaluated with flow cytometry. Formation of physical conjugates of aptamer and Dox was monitored with spectrofluorimetry. Cellular uptake of aptamer-Dox conjugates was monitored through fluorescent microscopy. Drug efficacy was monitored with cell proliferation assay.
Results:
The EpCAM aptamer (EpDT3) but not the scrambled aptamer (Scr-EpDT3) bound to RB tumor cells, the Y79 and WERI-Rb1 cells. However, the EpCAM aptamer and the scrambled aptamer did not bind to the noncancerous Müller glial cells. The chimeric EpCAM aptamer Dox conjugate (EpDT3-Dox) and the scrambled aptamer Dox conjugate (Scr-EpDT3-Dox) were synthesized and tested on the Y79, WERI-Rb1, and Müller glial cells. The targeted uptake of the EpDT3-Dox aptamer caused cytotoxicity in the Y79 and WERI-Rb1 cells but not in the Müller glial cells. There was no significant binding or consequent cytotoxicity by the Scr-EpDT3-Dox in either cell line. The EpCAM aptamer alone did not cause cytotoxicity in either cell line.
Conclusions:
The results show that the EpCAM aptamer-Dox conjugate can selectively deliver the drug to the RB cells there by inhibiting cellular proliferation and not to the noncancerous Müller glial cells. As EpCAM is a cancer stem cell marker, this aptamer-based targeted drug delivery will prevent the undesired effects of non-specific drug activity and will kill cancer stem cells precisely in RB.
Insights
This study developed an RNA aptamer to deliver doxorubicin (Dox) specifically to retinoblastoma (RB) cells by targeting EpCAM. This targeted drug delivery effectively killed RB cancer cells while sparing healthy cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Retinoblastoma (RB) is a pediatric eye cancer.
- Targeted drug delivery aims to improve treatment efficacy and reduce side effects.
- Epithelial cell adhesion molecule (EpCAM) is a potential target in cancer therapy.
Purpose of the Study:
- To develop and evaluate an RNA aptamer for targeted delivery of doxorubicin (Dox) to retinoblastoma (RB) cells.
- To assess the specificity and efficacy of EpCAM aptamer-doxorubicin conjugates in RB cells.
- To investigate the potential of this approach for targeting cancer stem cells in RB.
Main Methods:
- Synthesized an RNA aptamer targeting EpCAM and conjugated it with doxorubicin (Dox).
- Evaluated aptamer binding affinity to RB cells (Y79, WERI-Rb1) and Müller glial cells using flow cytometry.
- Assessed cellular uptake and cytotoxicity of the aptamer-Dox conjugate via fluorescent microscopy and cell proliferation assays.
Main Results:
- The EpCAM aptamer specifically bound to RB cells (Y79, WERI-Rb1) but not to Müller glial cells.
- The EpCAM aptamer-doxorubicin conjugate (EpDT3-Dox) demonstrated targeted uptake and induced cytotoxicity in RB cells.
- No significant binding or cytotoxicity was observed with a scrambled aptamer or with the aptamer alone in either cell type.
Conclusions:
- EpCAM aptamer-doxorubicin conjugates enable selective drug delivery to RB cells, inhibiting proliferation.
- This targeted approach minimizes toxicity to noncancerous Müller glial cells.
- The EpCAM aptamer shows promise for precise targeting of cancer stem cells in retinoblastoma, reducing off-target effects.
