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Updated: Jul 19, 2026
Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers
Published on: October 17, 2013
A novel assay for monitoring internalization of nanocarrier coupled antibodies
Ulrik B Nielsen1, Dmitri B Kirpotin, Edward M Pickering
1Department of Anesthesia, University of California San Francisco, San Francisco, CA 94110, USA. unielsen@merrimackpharma.com
Background:
Discovery of tumor-selective antibodies or antibody fragments is a promising approach for delivering therapeutic agents to antigen over-expressing cancers. Therefore it is important to develop methods for the identification of target- and function specific antibodies for effective drug delivery. Here we describe a highly selective and sensitive method for characterizing the internalizing potential of multivalently displayed antibodies or ligands conjugated to liposomes into tumor cells. The assay requires minute amounts of histidine-tagged ligand and relies on the non-covalent coupling of these antibodies to fluorescent liposomes containing a metal ion-chelating lipid. Following incubation of cells with antibody-conjugated liposomes, surface bound liposomes are gently removed and the remaining internalized liposomes are quantitated based on fluorescence in a high throughput manner. We have termed this methodology "Chelated Ligand Internalization Assay", or CLIA.
Results:
The specificity of the assay was demonstrated with different antibodies to the ErbB-2 and EGF receptors. Antibody-uptake correlated with receptor expression levels in tumor cell lines with a range of receptor expression. Furthermore, Ni-NTA liposomes containing doxorubicin were used to screen for the ability of antibodies to confer target-specific cytotoxicity. Using an anti-ErbB2 single chain Fv (scFv) (F5) antibody, cytotoxicity could be conferred to ErbB2-overexpressing cells; however, a poly(ethylene glycol)-linked lipid (DSPE-PEG-NTA-Ni) was necessary to allow for efficient loading of the drug and to reduce nonspecific drug leakage during the course of the assay.
Conclusion:
The CLIA method we describe here represents a rapid, sensitive and robust assay for the identification and characterization of tumor-specific antibodies capable of high drug-delivery efficiency when conjugated to liposomal nanocarriers.
Insights
A new assay, Chelated Ligand Internalization Assay (CLIA), rapidly identifies tumor-specific antibodies for efficient liposomal drug delivery. This method enables high-throughput screening of antibody-drug conjugates for cancer therapy.
Area of Science:
- Biotechnology
- Nanomedicine
- Oncology
Background:
- Targeted drug delivery to cancer cells using tumor-specific antibodies is a key strategy in oncology.
- Developing methods to identify antibodies with specific targeting and functional capabilities is crucial for effective cancer therapeutics.
- Characterizing the internalization potential of antibody-drug conjugates is essential for optimizing drug delivery efficiency.
Purpose of the Study:
- To develop a highly selective and sensitive method for characterizing the internalization potential of antibodies conjugated to liposomes.
- To establish a high-throughput assay for identifying and evaluating tumor-specific antibodies for drug delivery applications.
- To assess the ability of antibody-conjugated liposomes to deliver therapeutic agents specifically to cancer cells.
Main Methods:
- Developed the Chelated Ligand Internalization Assay (CLIA), a method utilizing histidine-tagged ligands and fluorescent liposomes.
- Employed non-covalent coupling of antibodies to liposomes containing a metal ion-chelating lipid for sensitive detection.
- Quantified internalized liposomes via fluorescence after removing surface-bound liposomes, enabling high-throughput screening.
Main Results:
- Demonstrated assay specificity using antibodies against ErbB-2 and EGF receptors, correlating antibody uptake with receptor expression levels.
- Successfully screened for antibody-mediated cytotoxicity using doxorubicin-loaded liposomes, confirming target-specific drug delivery.
- Identified the necessity of DSPE-PEG-NTA-Ni for efficient drug loading and reduced leakage in antibody-drug conjugates.
Conclusions:
- The CLIA method is a rapid, sensitive, and robust assay for identifying and characterizing tumor-specific antibodies.
- CLIA facilitates the evaluation of antibody-drug conjugates for efficient drug delivery via liposomal nanocarriers.
- This assay is valuable for advancing targeted cancer therapies and optimizing antibody-based drug delivery systems.
