Cell-penetrating peptides (CPPs) are able to traverse the obstacle presented by the cell membrane to deliver membrane impermeable drugs to intracellular targets. Given that cellular entry mechanisms including both energy-independent direct translocation and energy-dependent endocytic processes involve permeation through cellular membranes, the general biophysical properties of membranes can substantially influence the efficiency of cellular entry and thus the biological effects and therapeutic potential of CPP-cargo complexes. In this review, we summarized the entry mechanisms of CPPs and the general principles how the biophysical properties of membranes can alter these processes. Furthermore, we provide a brief overview of disorders such as diabetes mellitus, Alzheimer’s disease and various tumors, in which CPP-mediated drug delivery shows great therapeutic promise. However, we argue that alterations in the membrane lipid composition intrinsically associated with these disorders and consequent changes in bilayer properties could extensively reduce therapeutic efficacy of CPP-cargo complexes, and therefore, a beneficial combination with membrane lipid therapies can be expected to enhance the drug delivery mediated by CPPs.
The most common method of measuring FRET involves measuring the donor
and acceptor intensities separately. To calculate the FRET efficiency, the two
intensity values obtained are calibrated using a complex system of equations.
Our aim was to determine the exact ratio of the intensities of the excited
donor and excited acceptor, denoted by alpha, required for the calibration.
Traditionally, in FRET measurements, alpha is determined by determining the intensity of samples labelled with the same number of donors and acceptors. However, if it is determined on a small number of cells, e.g., in a microscope, this shortcoming introduces a large statistical error (A). The statistical error can be reduced by measuring the cells by flow cytometry, as much more cells can be examined in a short period of time.
One of the innovations introduced in our experiments was to use microbeads with a calibrated number of antibody binding sites, labelled with fluorescently stained antibodies, instead of cells. Much more accurate measurement results are obtained using the microbeads (B).
Further improvement in reproducibility was achieved by labeling cell or bead samples with a mixture of antibodies containing both donor and acceptor. This modification gave even better results as it eliminated the need for comparing the intensities of two different samples (C). Since the proposed method does not require specialized instrumentation, it can enjoy widespread application in the calibration of intensity-based FRET measurements.
This chapter in Current Protocols in Cytometry describes the principles of Förster resonance energy transfer (FRET) in a way suitable for helping researchers understand the pros and cons, the pitfalls and recent advancements in the technique.
experiments reveal a protective role of protein phosphatase Z1 against
oxidative damage of the cell membrane in Candida albicans
Hajdu T, Szabó K, Jakab Á, Pócsi I, Dombrádi V, Nagy P
Free Rad Biol Med, 176: 222-227. (2021)
In a collaborative study, the fungus-specific protein phosphatase Z1 (Ppz1) enzyme has been investigated and it was found to be crucial in the defence against oxidative stress. Researchers in the Department of Biophysics and Cell Biology, Department of Medical Chemistry and Department of Molecular Biotechnology and Microbiology proved that the deletion of the gene of Ppz1 leads to low membrane compactness and an increase in the sensitivity to oxidative challenge in Candida albicans cells. Examining the membrane fluidity-related generalized polarization reveals that while most native cells (WT) expressing Ppz1 were able to neutralize the effect of the applied oxidative agent, tert-butyl-hydroperoxide (tBOOH), mutant cells lacking Ppz1 expression (KO) suffered from latent oxidative damage and were characterized by incompact membrane. The lack of Ppz1 also manifests in increased membrane lipid peroxidation. Investigation of the lateral mobility of lipids revealed that the lack of Ppz1 combined with oxidative stress reduces the lateral diffusion of lipids resulting in reduced competitive fitness. According to these data, applying mild oxidative treatments together with prospective Ppz1 inhibitor molecules may tackle fungal infections caused by drug-resistant Candida albicans species. The study also revealed that changes in membrane characteristics may be used by fungal cells as a cue for responding to changes in the environment.
Statin-boosted cellular uptake and endosomal escape of penetratin due to reduced membrane dipole potential
Batta G, Kárpáti L, Henrique GF, Tóth G, Tarapcsák S, Kovács T, Zákány F, Mándity IM, Nagy P
Br J Pharmacol, 178: 3667-3681. (2021)
Research carried out in collaboration between the Department of Biophysics and Cell Biology, and the Department of Genetics of Applied Microbiology at the University of Debrecen, and the Department of Organic Chemistry, Semmelweis University revealed that cellular uptake of cell penetrating peptides (CPP) depends on the membrane dipole potential and that it can be enhanced by reducing the dipole potential. Cell penetrating peptides are molecules capable of traversing the intact cell membrane even when loaded with cargo, e.g. membrane impermeable drugs. The research group of Peter Nagy found that this uptake takes place by endocytosis followed by endosomal escape. Since most cell penetrating peptides are positively charged, the intramembrane, positive dipole potential generated by the ordered arrangement of dipoles in the membrane, inhibits their membrane crossing. Atorvastatin, widely applied in the treatment of high blood cholesterol levels, decreases the membrane dipole potential and consequently enhances the cellular uptake of cell penetrating peptides. The findings lay the foundation for applying the revealed principle for boosting the cellular uptake of drugs in the treatment of human diseases.
ω-3, but Not an ω-6 Polyunsaturated Fatty Acid Decreases Membrane
Dipole Potential and Stimulates Endo-Lysosomal Escape of Penetratin
Zákány F, Szabó M, Batta G, Kárpáti L, Mándity M. István, Fülöp P, Varga Z, Panyi G, Nagy P, Kovács T
Frontiers in Cell and Developmental Biology 9: 647300. (2021)
3-O-gallate induces 67 kDa laminin receptor-mediated cell death
accompanied by downregulation of ErbB proteins and altered lipid raft
clustering in mammary and epidermoid carcinoma cells
Mocanu MM, Ganea C, Georgescu L, Váradi T, Shrestha D, Baran I, Katona E, Nagy P, Szöllősi J.
J Nat Prod. 77(2):250-7 (2014)