Analysis of in vitro toxicity of five cell-penetrating peptides by metabolic profiling
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Updated: Jun 19, 2026
Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures
Published on: March 28, 2017
Kalle Kilk1, Riina Mahlapuu, Ursel Soomets
1Department of Biochemistry, Medical Faculty, University of Tartu, The Centre of Excellence for Translational Medicine, 50411 Tartu, Estonia. kalle.kilk@ut.ee
Abstract:
Cell-penetrating peptides (CPPs) are promising candidates for safe and efficient delivery vectors for a wide range of cargoes. However, any compound that is internalized into cells may affect the cell homeostasis. The plethora of possible biological responses makes large scale "omics" studies appealing approaches for hunting any unsuspected side-effects and evaluate the toxicity of drug candidates. Here we have compared the alterations in cytosolic metabolome of CHO cells caused by five representatives of the most common CPPs: transportan (TP), penetratin (pAntp), HIV Tat derived peptide (pTat), nonaarginine (R(9)) and model amphipathic peptide (MAP). Analysis was done by liquid chromatography-mass spectrometry techniques, principal component analysis and heatmap displays. Results showed that the intracellular metabolome was the most affected by TP followed by pTat and MAP. Only minor changes could be associated with pAntp or R(9) treatment. The cells could recover from a treatment with 5 microM TP, but no recovery was observed at higher concentration. Both metabolomic and control experiments showed that TP affected cellular redox potential, depleted energy and the pools of purines and pyrimidines. In conclusion, we have performed a metabolomic analysis comparing the safety of cell-penetrating peptides and demonstrate the toxicity of one of them.
Insights
Cell-penetrating peptides (CPPs) show potential for drug delivery, but can impact cell health. Metabolomic analysis revealed transportan (TP) significantly altered cell metabolism and homeostasis, indicating toxicity.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Cell-penetrating peptides (CPPs) are explored as safe and efficient delivery vectors for various cargoes.
- Cellular internalization of compounds can disrupt cell homeostasis, necessitating toxicity assessments.
- Omics studies are valuable for identifying unexpected side-effects and evaluating drug candidate toxicity.
Purpose of the Study:
- To compare the effects of five common CPPs on the cytosolic metabolome of Chinese Hamster Ovary (CHO) cells.
- To identify potential toxicity and side-effects associated with CPP internalization.
- To evaluate the safety profile of different CPPs as delivery vectors.
Main Methods:
- Liquid chromatography-mass spectrometry (LC-MS) was employed to analyze the cytosolic metabolome of CHO cells.
- Principal component analysis (PCA) and heatmap displays were used to interpret the metabolomic data.
- Comparative analysis of five CPPs: transportan (TP), penetratin (pAntp), HIV Tat peptide (pTat), nonaarginine (R(9)), and model amphipathic peptide (MAP).
Main Results:
- Transportan (TP) caused the most significant alterations in the intracellular metabolome, followed by pTat and MAP.
- Penetratin (pAntp) and nonaarginine (R(9)) treatments resulted in only minor metabolic changes.
- TP treatment at 5 microM allowed cell recovery, but higher concentrations did not.
- TP was found to affect cellular redox potential, deplete energy, and alter purine and pyrimidine pools.
Conclusions:
- Metabolomic profiling is an effective approach for comparing the safety of CPPs.
- Transportan (TP) exhibits toxicity by disrupting cellular energy, redox balance, and nucleotide pools.
- The findings highlight the importance of assessing CPP-induced metabolic changes to ensure safe drug delivery applications.
