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Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers
Published on: October 17, 2013
Comparing Cell Penetration of Biotherapeutics across Human Cell Lines
Nefeli Batistatou1, Joshua A Kritzer1
1Department of Chemistry, Tufts University, Medford, Massachusetts 02155, United States.
Abstract:
A major obstacle in biotherapeutics development is maximizing cell penetration. Ideally, assays would allow for optimization of cell penetration in the cell type of interest early in the drug development process. However, few assays exist to compare cell penetration across different cell types independent of drug function. In this work, we applied the chloroalkane penetration assay (CAPA) in seven mammalian cell lines as well as primary cells. Careful controls were used to ensure that data could be compared across cell lines. We compared the nuclear penetration of several peptides and drug-like oligonucleotides and saw significant differences among the cell lines. To help explain these differences, we quantified the relative activities of endocytosis pathways in these cell lines and correlated them with the penetration data. Based on these results, we knocked down clathrin in a cell line with an efficient permeability profile and observed reduced penetration of peptides but not oligonucleotides. Finally, we used small-molecule endosomal escape enhancers and observed enhancement of cell penetration of some oligonucleotides, but only in some of the cell lines tested. CAPA data provide valuable points of comparison among different cell lines, including primary cells, for evaluating the cell penetration of various classes of peptides and oligonucleotides.
Insights
The chloroalkane penetration assay (CAPA) effectively measures cell penetration for biotherapeutics across diverse cell types. This assay helps optimize drug delivery by identifying cell-specific uptake differences for peptides and oligonucleotides.
Area of Science:
- Biotechnology
- Drug Delivery
- Cell Biology
Background:
- Maximizing cell penetration is a critical challenge in biotherapeutics development.
- Existing assays often fail to compare cell penetration across different cell types independently of drug function.
- A need exists for robust assays to optimize cell penetration early in the drug development pipeline.
Purpose of the Study:
- To apply and validate the chloroalkane penetration assay (CAPA) for comparing cell penetration across various mammalian cell lines and primary cells.
- To investigate the differential nuclear penetration of peptides and oligonucleotides in different cell types.
- To correlate cell penetration efficiency with endocytosis pathway activity and explore strategies for enhancing delivery.
Main Methods:
- Utilized the chloroalkane penetration assay (CAPA) across seven mammalian cell lines and primary cells.
- Quantified the activity of different endocytosis pathways to understand their role in cellular uptake.
- Performed clathrin knockdown and utilized endosomal escape enhancers to modulate and assess cell penetration.
Main Results:
- Significant differences in nuclear penetration of peptides and oligonucleotides were observed among the tested cell lines.
- Endocytosis pathway activity correlated with observed cell penetration variations.
- Clathrin knockdown reduced peptide penetration but not oligonucleotide penetration, while endosomal escape enhancers showed variable effects on oligonucleotide delivery.
Conclusions:
- The CAPA assay provides a valuable tool for comparing cell penetration across diverse cell types, including primary cells.
- Cell-specific differences in endocytosis pathways significantly influence the penetration of peptides and oligonucleotides.
- Targeting specific endocytosis pathways or utilizing endosomal escape enhancers may offer strategies for optimizing biotherapeutic delivery, though efficacy is cell-type dependent.
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