For decades, the question seemed straightforward but defied a clean answer: how do cell-penetrating peptides (CPPs), short sequences of amino acids that can carry therapeutic payloads into cells, actually get through the plasma membrane?
The answer matters enormously for drug delivery. CPPs are one of the most promising strategies for getting large, charged molecules (proteins, nucleic acids, nanoparticles) into the cytoplasm of diseased cells. But without knowing the mechanism, rational design of better delivery vehicles has been guesswork. Competing theories, endocytosis, inverted micelle formation, carpet-like membrane destabilization, each had their proponents, and each had their gaps.
Now, a team of French researchers has provided what appears to be the definitive answer. In a study published in the Proceedings of the National Academy of Sciences, Evgeniya Trofimenko and colleagues from five French research institutions show that CPPs and homeoproteins cross the membrane through transient, submillisecond pores, and they have the electrophysiological recordings to prove it.
Direct evidence at last
The team used a double patch-clamp technique on individual cells, essentially poking two electrodes into a single cell, one to hold the membrane voltage and one to detect currents, while applying CPPs to the external surface. What they recorded were brief, unitary current spikes, each corresponding to a single pore-opening event.
These pores formed and closed faster than any known endocytic event, on the order of hundreds of microseconds to a few milliseconds. Crucially, the same translocation was observed at 11 °C, a temperature at which endocytosis is completely blocked. The process is energy-independent, ruling out active transport entirely.
The pores are transient: they open, allow the peptide to pass through, and reseal. The membrane remains intact, the process is not cytotoxic.
GAG-dependent and voltage-sensitive
The mechanism has two critical dependencies.
First, cell-surface glycosaminoglycans (GAGs), long, negatively charged sugar chains that coat most cell types, are mandatory. The peptides must bind to heparan sulfate-like GAGs to nucleate pore formation. In cells genetically lacking GAGs (CHO-psgA-745), no translocation occurred at all. This resolves a long-standing puzzle in the field: why CPP uptake varies dramatically between cell types. The answer is simply GAG density.
Second, membrane voltage strongly modulates the process. Hyperpolarization, making the inside of the cell more negative, enhanced translocation frequency 10- to 100-fold in a supralinear fashion. Depolarization had little effect. This voltage sensitivity provides a potential handle for targeted delivery: cells with more negative resting potentials (such as neurons) may be naturally more receptive to CPP-mediated cargo delivery.
A unified mechanism
One of the study’s most striking findings is that the same transient pore mechanism applies across a broad range of peptides. The team tested four CPPs, Tat (from HIV), polyarginine R9, penetratin, and R6W3, and two homeoproteins (Otx2 and Engrailed-2). All showed the same basic mechanism with similar kinetics.
This unification is important. Homeoproteins are naturally occurring transcription factors that can travel between cells in living organisms, a process called “messenger protein” signaling that has been observed in development and neurobiology. The new study suggests that these proteins use exactly the same GAG-dependent, voltage-sensitive pore mechanism as synthetic CPPs, opening the possibility that artificial delivery vehicles can be designed around a natural biological process.
Implications for drug delivery
The findings transform the practical landscape for CPP-based therapeutics in at least four ways:
1. Rational design: Instead of screening thousands of peptide variants empirically, delivery vehicles can now be engineered to optimize GAG binding affinity and voltage sensitivity.
2. Endosomal escape solved: One of the major failures of endocytosis-dependent delivery is that internalized cargo gets trapped in endosomes and degraded. The transient pore mechanism bypasses endosomes entirely, cargo enters directly into the cytosol.
3. Cargo delivery validated: The team demonstrated that a CPP-conjugated toxic cargo (the pro-apoptotic peptide KRAKLAK) successfully entered cells and killed them via the transient pore route, proving the mechanism is capable of therapeutically relevant delivery.
4. CNS potential: The same pore mechanism was observed in brain cortical pyramidal cells from acute slices, suggesting that homeoprotein-based carriers could be developed for central nervous system drug delivery, a notoriously difficult therapeutic frontier.
What remains to be explored
The study establishes the mechanism definitively for the peptides tested, but several questions remain. How does the pore actually form at the molecular level, what lipid rearrangements occur? Can the pore size be modulated to accommodate larger cargoes, such as antibodies or gene-editing complexes? And crucially, will this mechanism translate from cultured cells and brain slices into living organisms for therapeutic use?
The answers to those questions will determine whether the transient pore mechanism becomes the basis of a new generation of delivery technologies. But the mechanism itself is no longer in doubt.
Funding: Agence Nationale de la Recherche (ANR-17-CE11-0050-CROSS, ANR-20-CE44-0018-GLYCOTARGET).
Source
Trofimenko, E., Gervasi, N., Perez, S., Rodriguez, N., Ravault, D., Cribier, S., Berry, H., Venance, L., and Sagan, S. “Transient pores account for cell-penetrating peptide and homeoprotein translocation.” Proceedings of the National Academy of Sciences 123(26), e2602649123 (2026). DOI: 10.1073/pnas.2602649123