# Cell Penetration Profiling for Biotherapeutics

> **NIH NIH R01** · TUFTS UNIVERSITY MEDFORD · 2021 · $285,398

## Abstract

Cell Penetration Profiling for Biotherapeutics
Biotechnology promises the ability to control biology and disease with laser-like precision. Hundreds of
peptides, proteins and nucleic acids are being developed as diagnostics and therapies, but nearly all of these
have poor cell penetration and unpredictable subcellular localization. Currently, there are no quantitative, high-
throughput tools to measure how much of a biomolecule enters a cell and where it distributes within the cell.
The imprecision and low throughput of current methods for measuring intracellular delivery is a major
barrier for the development of biomolecule therapies.
The Kritzer lab has devised a new method for quantitating cell penetration, called the ChloroAlkane
Penetration Assay (CAPA). CAPA measures the degree to which a molecule penetrates the cytosol,
independent of the molecule's target or biological function. Our published and unpublished data show that
CAPA is quantitative, inexpensive and high-throughput, and that it exclusively measures penetration to single
cellular compartment. Based on these results, we envision that CAPA could be used for comprehensive cell
penetration profiling for a large variety of biomolecules and drug delivery systems. This will provide detailed
data on dose dependence, time course, penetration to different subcellular compartments, and cell type
specificity. It will also allow independent profiling of endocytic uptake, endosomal escape and nuclear import.
We propose applying cell penetration profiling to uncover structure-activity relationships for cell
penetration for several classes of bioactive peptides, cell-penetrant proteins, and antisense oligonucleotides.
Finally, we use cell penetration profiling to understand and improve the delivery of Cas9-sgRNA complexes by
lipid nanoparticles.
Our understanding of how all these classes of biotherapeutics reach the cell interior is severely limited by
current methods. Cell penetration profiling provides a rapid means of measuring endocytic uptake, endosomal
escape, and nuclear import, allowing critical drug delivery problems to be directly assessed and
avoided. Since intra-cellular delivery is the major hurdle for most biotherapeutics, cell penetration profiling has
the potential to become a routine and valuable part of the development process for peptide, protein and nucleic
acid therapies.

## Key facts

- **NIH application ID:** 10105335
- **Project number:** 5R01GM127585-04
- **Recipient organization:** TUFTS UNIVERSITY MEDFORD
- **Principal Investigator:** Joshua A Kritzer
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $285,398
- **Award type:** 5
- **Project period:** 2018-04-10 → 2022-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10105335

## Citation

> US National Institutes of Health, RePORTER application 10105335, Cell Penetration Profiling for Biotherapeutics (5R01GM127585-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10105335. Licensed CC0.

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