Membrane-impermeable Molecules Research Articles

Photoswitchable Detergents for Light-Controlled Liposome Lysis and Channel Gating.

Modulation of membrane properties via photoswitchable lipids has attracted attention due to the unparalleled spatiotemporal resolution of their functional control. Beside lipids, detergents are another prominent class for selective membrane perturbations owing to their ease of handling and spontaneous insertion in lipid bilayers. Herein, we describe the synthesis and characterization of three classes of visible light-sensitive surfactants with various azobenzene tail chain lengths. The photoswitchable detergents show water-solubility and micellization as well as undergo reversible isomerization under blue-/green light illumination. We demonstrate that the light-induced structural change of azobenzene can lead to vesicle rupture, making them a tool for controlled cargo release from vehicles. Via spontaneous insertion into the plasma membrane of mammalian cells transiently transfected with MscL, we used the azobenzene-derived detergents to optically activate the transmembrane mechanosensitive channel. This led to the rapid controlled uptake of membrane-impermeable molecules. Since detergents are extensively used in biochemistry and biotechnology, we propose that the photoswitchable detergents will be useful tools for the spatiotemporal modulation of membrane properties. Additionally, our work provides a design strategy for new detergents in membrane (protein) research.

Bidirectional transfer of a small membrane-impermeable molecule between the C. elegans intestine and germline

The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) is a positive regulator of cell proliferation often upregulated in cancer. Its C. elegans ortholog MPK-1 stimulates germline stem cell (GSC) proliferation non-autonomously, from the intestine or somatic gonad. How MPK-1 can perform this task from either of these two tissues however remains unclear. We reasoned that somatic MPK-1 activity could lead to the generation of pro-proliferative small molecules that could transfer from the intestine and/or somatic gonad to the germline. Here, in support of this hypothesis, we demonstrate that a significant fraction of the small membrane-impermeable fluorescent molecule, 5-carboxyfluorescein (5-CF), transfers to the germline after its microinjection in the animal’s intestine. The larger part of this transfer targets oocytes and requires the germline RME-2 yolk receptor. A minor quantity of the dye is however distributed independently from RME-2 and more widely in the animal, including the distal germline, gonadal sheath, coelomocytes and hypodermis. We further show that the intestine-to-germline transfer efficiency of this RME-2 independent fraction does not vary together with GSC proliferation rates or MPK-1 activity. Therefore, if germline proliferation was influenced by small membrane-impermeable molecules generated in the intestine, it is unlikely that proliferation would be regulated at the level of molecule transfer rate. Finally, we show that conversely, a similar fraction of germline injected 5-CF transfers to the intestine, demonstrating transfer bidirectionality. Altogether, our results establish the possibility of an intestine-to-germline signaling axis mediated by small membrane-impermeable molecules that could promote GSC proliferation cell non-autonomously downstream of MPK-1 activity.

Nanoparticle-mediated photoporation - an emerging versatile physical drug delivery method.

Facilitating the delivery of impermeable molecules into cells stands as a pivotal step for both basic research and therapeutic delivery. While current methods predominantly use nanoparticles or viral vectors, the exploration of physical phenomena, particularly light-based techniques, remains relatively under-explored. Photoporation, a physical method, employs either pulsed or continuous wave lasers to create transient pores in cell membranes. These openings enable the entry of exogenous, membrane-impermeable molecules into the cytosol while preserving cell viability. Poration can either be achieved directly through focusing a laser beam onto a cell membrane, or indirectly through the addition of sensitizing nanoparticles that interact with the laser pulses. Nanoparticle-mediated photoporation specifically has recently been receiving increasing attention for the high-throughput ability to transfect cells, which also has exciting potential for clinical translation. Here, we begin with a snapshot of the current state of direct and indirect photoporation and the mechanisms that contribute to cell pore formation and molecule delivery. Following this, we present an outline of the evolution of photoporation methodologies for mammalian and non-mammalian cells, accompanied by a description of variations in experimental setups among photoporation systems. Finally, we discuss the potential clinical translation of photoporation and offer our perspective on recent key findings in the field, addressing unmet needs, gaps, and inconsistencies.

Na+/K+ carrier ionophore antibiotics valinomycin and monensin enhance the antibacterial activity of fluoride.

Fluoride is routinely used as a highly effective antibacterial agent that interferes with bacterial metabolism through fundamentally different mechanisms. One of the major bacterial evasion mechanisms against fluoride is the impermeability of cell envelope to the anion that limits its cellular uptake. Therefore, translating such compounds to clinical settings requires novel mechanisms to facilitate the uptake of membrane-impermeant molecules. Published data have indicated antibiotic synergy between fluoride and membrane destabilizing agents that induce strong fluoride toxicity in bacteria via enhancing the permeability of bacterial membranes to fluoride. Here, we report a similar mechanism of antibiotic synergy between fluoride and potassium ion carriers, valinomycin and monensin against Gram-positive bacteria, B. subtilis and S. aureus. Molecular dynamics simulations were performed to understand the effect of potassium on the binding affinity of fluoride to monensin and valinomycin. The trajectory results strongly indicated that the monensin molecules transport fluoride ions across the cell membrane via formation of ion-pair between the monensin-K+ complex and a fluoride. This study provides new insights to design novel compounds to enhance the uptake of small toxic anions via synergistic interactions and thus exert strong antibacterial activity against a wide variety of pathogens.

Gap Junction-Mediated Delivery of Polymeric Macromolecules.

Cellular delivery of therapeutic macromolecules such as proteins, peptides, and nucleic acids remains limited due to inefficient transport across the cellular plasma membrane. Gap junction channels, composed of connexin proteins, provide a mechanism for direct transfer of small molecules across membranes, and recent evidence suggests that the transfer of larger, polymer-like molecules such as microRNAs may be possible. Here, we report direct evidence of gap junction-mediated transfer of polymeric macromolecules. Specifically, we examined the transport of dextran chains with molecular weights ranging from 10 to 70 kDa. We found that dextran chains of up to 40 kDa can diffuse through at least five cell layers in a gap junction-dependent manner within a 30 min time frame. Further, we evaluated the ability of connectosomes, cell-derived vesicles containing functional connexin proteins, to be loaded with dextran chains. By opening connexon hemichannel pores within the membranes of connectosomes, we found that 10 kDa dextran was loaded into more than 90% of vesicles, with reduced levels of loading for dextran chains of larger molecular weight. Upon delivering 10 kDa dextran-loaded connectosomes to cells, we further found that connectosomes transferred these membrane-impermeable molecules to the cellular cytosol with dramatically improved efficiency in comparison to the delivery of free, unencapsulated dextran. Collectively, these results reveal that polymeric macromolecules can be delivered to cells via gap junctions, suggesting that the gap junction route may be useful for the delivery of polymeric therapeutic molecules, such as nucleic acids and peptides.

Electroporation: An Effective Method For In Vivo Gene Delivery

Background: Gene therapy is a promising approach for the treatment of various diseases, including cancer, hereditary disorders, and some viral infections. The development of efficient and safe gene delivery systems is essential for facilitating gene trans-fer to various organs and tissues in vivo. Objective: In this review, we briefly describe the principal mechanisms of gene delivery systems, particularly electroporation, and discuss the latest advancements in the application of electro-poration for in vivo gene transfer. Methods: A narrative review of all the relevant publication known to the authors was conducted. Results: In recent years, electroporation-based strategies have emerged as an auspicious and versa-tile platform for efficient and controlled delivery of various biomolecules, including nucleic acids. Applying electric pulses of enough magnitude leads to the formation of hydrophilic pores in the cell membrane and allows the entry of otherwise membrane-impermeant molecules, such as DNA. Alt-hough electroporation has been initially developed for in vitro transfection of cells, it has recently advanced to preclinical in vivo applications and finally to clinical trials. Conclusion: Electroporation has already entered the clinical practice for antitumor therapy and may be an essential part of future personalized treatments. Given the ability of electroporation to deliver multiple genes in a single event, it will also certainly be further developed both as a stand-alone de-livery approach and when coupled with other technologies.

Intracellular Cargo Delivery Induced by Irradiating Polymer Substrates with Nanosecond-Pulsed Lasers.

There is a great need in the biomedical field to efficiently, and cost-effectively, deliver membrane-impermeable molecules into the cellular cytoplasm. However, the cell membrane is a selectively permeable barrier, and large molecules often cannot pass through the phospholipid bilayer. We show that nanosecond laser-activated polymer surfaces of commercial polyvinyl tape and black polystyrene Petri dishes can transiently permeabilize cells for high-throughput, diverse cargo delivery of sizes of up to 150 kDa. The polymer surfaces are biocompatible and support normal cell growth of adherent cells. We determine the optimal irradiation conditions for poration, influx of fluorescent molecules into the cell, and post-treatment viability of the cells. The simple and low-cost substrates we use have no thin-metal structures, do not require cleanroom fabrication, and provide spatial selectivity and scalability for biomedical applications.

Transient Destabilization of Biological Membranes Contributes to the Superior Performance of Star-Shaped PDMAEMA in Delivering pDNA.

Nonviral DNA vectors are promising alternatives to viral ones. Their use in DNA medicine is limited by an inability to transfect, for example, nondividing or suspension cells. In recent years, star-shaped synthetic polycationic vectors, so called “Nanostars”, have shown some promise in this regard, at least when compared to the “gold standard” in nonviral vectors, namely, linear poly(ethyleneimine) (l-PEI). It has been hypothesized that an ability to transiently destabilize cellular membranes is partially responsible for the phenomenon. This hypothesis is investigated here, taking human leukemia suspension cells (Jurkat cells) as an example. Contrary to l-PEI, the Nanostars promote the cellular uptake of small, normally membrane-impermeant molecules (trypan blue and propidium iodide) as well as that of fluorescent polystyrene beads (average diameter 100 nm). Since Nanostars, but not l-PEI, are apparently able to deliver DNA to nuclei of nondividing cells, nuclear uptake is, in addition, investigated with isolated cell nuclei. Our results provide evidence that Nanostars are more efficient than l-PEI in increasing the nuclear membrane association/permeability, allowing accumulation of their cargo on/in the nucleus.

Actin networks regulate the cell membrane permeability during electroporation

Transient physical disruption of cell membranes by electric pulses (or electroporation) has significance in biomedical and biological applications requiring the delivery of exogenous (bio)molecules to living cells. We demonstrate that actin networks regulate the cell membrane permeability during electroporation. Disruption of actin networks increases the uptake of membrane-impermeable molecules such as propidium iodide during electroporation. Our experiments at different temperatures ranging from 11 °C to 37 °C show that molecular uptake during electroporation increases with temperature. Furthermore, by examining the temperature-dependent kinetics of propidium iodide uptake, we infer that the activation energy barrier of electroporation is lowered when the actin networks are disrupted. Our numerical calculations of transmembrane voltage show that the reduced activation energy barrier for the cells with disrupted actin is not a consequence of the changes in transmembrane voltage associated with changes in the cell shape due to the disruption of actin, indicating that this could be due to changes in membrane mechanical properties. Our results suggest that the current theoretical models of electroporation should be advanced further by including the contributions of the cytoskeletal networks on the cell membrane permeability during the delivery of exogenous materials.

Laser-Assisted Delivery of Molecules in Fungal Cells.

Fungal infections are becoming a global health problem. A major limiting factor for the development of antifungals is the high impermeability of the rigid and thick fungal cell wall. Compared to mammalian cells, fungal cells are more resilient to perforation due to the presence of this carbohydrate armor. While a few methods have been reported to penetrate the fungal cell wall, such as electroporation, biolistics, glass beads, and the use of monovalent cations, such methods are generally time-consuming, compromise cell viability, and often lead to low permeation rates. In addition, their use remains limited to in vitro applications due to the collateral damage that these techniques could cause to healthy living tissues. Presented in this study is a delivery approach based on the generation of transient breaks, or pores, in the cell wall. Breaks are generated by cavitation and shock waves resulting from the irradiation of gold nanoparticles with a femtosecond infrared laser. Such an approach enabled the delivery of membrane impermeable molecules (i.e., calcein and plasmid DNA) into Saccharomyces cerevisiae, a fungal model organism. This method is expected to exhibit high biocompatibility and holds potential for clinical applications for the treatment of fungal infections given that neither the laser irradiation nor the nanoparticles have been found to damage cells. Mechanistical aspects of photoporation, such as the proximity needed between the nanoparticle and the cell membrane for these processes to take place, are also discussed. Hence, the laser-assisted drug delivery approach described here is suitable for further preclinical evaluation in oral, vaginal, and skin mycoses where current treatments are insufficient due to host-related adverse reactions, poor fungal cell penetration, or risk of developing antifungal resistance.

Decoding the proteome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for cell-penetrating peptides involved in pathogenesis or applicable as drug delivery vectors

Synthetic or natural derived cell-penetrating peptides (CPPs) are vastly investigated as tools for the intracellular delivery of membrane-impermeable molecules. As viruses are intracellular obligate parasites, viral originated CPPs have been considered as suitable intracellular shuttling vectors for cargo transportation. A total of 310 CPPs were identified in the proteome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Screening the proteome of the cause of COVID-19 reveals that SARS-CoV-2 CPPs (SCV2-CPPs) span the regions involved in replication, protein-nucleotide and protein-protein interaction, protein-metal ion interaction, and stabilization of homo/hetero-oligomers. However, to find the most appropriate peptides as drug delivery vectors, one might face several hurdles. Computational analyses showed that 94.3% of the identified SCV2-CPPs are non-toxins, and 38% are neither antigenic nor allergenic. Interestingly, 36.70% of SCV2-CPPs were resistant to all four groups of protease families. Nearly 1/3 of SCV2-CPPs had sufficient inherent or induced helix and sheet conformation leading to increased uptake efficiency. Heliquest lipid-binding discrimination factor revealed that 44.30% of the helical SCV2-CPPs are lipid-binding helices. Although Cys-rich derived CPPs of helicase (NSP13) can potentially fold into a cyclic conformation in endosomes with a higher rate of endosomal release, the most optimal SCV2-CPP candidates as vectors for drug delivery were SCV2-CPP118, SCV2-CPP119, SCV2-CPP122, and SCV2-CPP129 of NSP12 (RdRp). Ten experimentally validated viral-derived CPPs were also used as the positive control to check the scalability and reliability of our protocol in SCV2-CPP retrieval. Some peptides with a cell-penetration ability known as bioactive peptides are adopted as biotherapeutics themselves. Therefore, 59.60%, 29.63%, and 32.32% of SCV2-CPPs were identified as potential antibacterial, antiviral, and antifungals, respectively. While 63.64% of SCV2-CPPs had immuno-modulatory properties, 21.89% were recognized as anti-cancers. Conclusively, the workflow of this study provides a platform for profound screening of viral proteomes as a rich source of biotherapeutics or drug delivery carriers.

Potassium channels as potential drug targets for limb wound repair and regeneration.

BackgroundIon channels are a large family of transmembrane proteins, accessible by soluble membrane-impermeable molecules, and thus are targets for development of therapeutic drugs. Ion channels are the second most common target for existing drugs, after G protein-coupled receptors, and are expected to make a big impact on precision medicine in many different diseases including wound repair and regeneration. Research has shown that endogenous bioelectric signaling mediated by ion channels is critical in non-mammalian limb regeneration. However, the role of ion channels in regeneration of limbs in mammalian systems is not yet defined.MethodsTo explore the role of potassium channels in limb wound repair and regeneration, the hindlimbs of mouse embryos were amputated at E12.5 when the wound is expected to regenerate and E15.5 when the wound is not expected to regenerate, and gene expression of potassium channels was studied.ResultsMost of the potassium channels were downregulated, except for the potassium channel kcnj8 (Kir6.1) which was upregulated in E12.5 embryos after amputation.ConclusionThis study provides a new mouse limb regeneration model and demonstrates that potassium channels are potential drug targets for limb wound healing and regeneration.

Survival of membrane-damaged Escherichia coli in a cytosol-mimicking solution

Selective permeability of cell membrane is critically important for cell survival. The damage caused to cell membrane by pore-forming antimicrobial peptides may result in the loss of selective permeability and leakage of intracellular molecules, eventually leading to cell death. Here, we examined whether the membrane-damaged Escherichia coli cells survive in a cytosol-mimicking solution (CMS), which compensates for the lethal leakage of intracellular molecules. We prepared a CMS comprising 34 low molecular weight compounds from the cytosol and found that the cells were able to grow in CMS even in the presence of a pore-forming peptide, melittin. We confirmed that the melittin-treated cells lost selective membrane permeability by staining with membrane-impermeable dyes, propidium iodide and SYTOX green. Some stained cells maintained the colony formation ability in CMS. These results provide an evidence that E.coli cells can at least partially survive in the CMS even after the temporary impairment of membrane selective permeability. This study demonstrates a technique that allows temporal loss of the selective permeability of the cell membrane while maintaining the viability of cells that may be useful for the introduction of membrane-impermeable molecules into E.coli cells.

An Assay to Assess Gap Junction Communication in Cell Lines.

This protocol was developed to assess communication in tumor cells and to provide a dependable and standardized assay for the in vitro determination of gap junction function. The method is noninvasive; in this method, the cell population under study is divided such that 1 fraction is loaded with a lipophilic cell plasma membrane permeable dye, calcein acetoxymethyl ester, that is hydrolyzed upon cellular uptake by cytoplasmic esterases to yield calcein, a fluorescent and membrane-impermeable molecule. The other fraction is loaded with 1,1'-dioctadecyl-3,3,3',3' tetramethylindodicarbocyanine perchlorate (DiD)/1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate [Dil; DilC18(3)], which is a lipophilic membrane dye that diffuses laterally to stain the entire cell membrane, is impermeable, and attains an orange-red fluorescence upon incorporation into membranes. The 2 fractions are mixed and incubated under coculture conditions. Calcein with MW 890 kD is transferred to the DiD/DiI-stained cells through gap junctions. The assessment of this uptake is made with confocal imaging and quantitated using flow cytometry. Cell lines representing cancer of the breast as well as a nontransformed cell line developed from the buccal mucosa were analyzed for gap junction competency. Confocal imaging with acquisition at specific time points during the in vitro treatment and flow cytometry gave a qualitative and quantitative analysis of the passage of molecules through the gap junctions. Here, the method has been combined to obtain images as well as quantitation and is a simple and effective approach in assessing the functional competency of gap junction in epithelial cells.

The cell resealing technique for manipulating, visualizing, and elucidating molecular functions in living cells

BackgroundCell-based assays are essential for analyzing molecular functions and spatiotemporal information. The cell resealing technique, in which pore-forming toxins are used to permeabilize cell membranes, enables the delivery of various membrane-impermeable molecules inside cells. Scope of reviewWe review the basics of the resealed cell system, including optimized protocols, assessment of cellular damage, and recovery following permeabilization of the membrane. Additionally, we introduce the streptolysin O (SLO)-type and listeriolysin O (LLO)-type resealing techniques. In SLO, the formation of larger pores (~30 nm) enables the passage of a wider range of molecules. Then, we discuss the advantages and applications of the semi-intact cell system, in which ongoing permeabilization is selected to maintain and analyze a specific cellular environment. Major conclusionsAs confirmed by the effective use of quantitative image analysis, the SLO-type resealing system is successful for establishing and phenotyping diabetic model cells by introducing cytosol from diabetic mice. The LLO-type resealing technique enables the delivery of mid-sized molecules with high efficiency and low damage. As each technique has specific advantages, understanding the characteristics of LLO and SLO is necessary for choosing the appropriate technique. General significanceSLO-type resealing is optimal for creating disease model cells and drug screening, especially lifestyle-related diseases. LLO-type resealing is expected to be suitable for screening mid-sized biological drugs. Semi-intact cells can contribute to elucidating various cellular phenomena that have remained intractable due to their complexity.

Assessing foraminifera biomineralisation models through trace element data of cultures under variable seawater chemistry

The process by which foraminifera precipitate calcite from seawater has received much attention, in part because a mechanistic basis for empirical calibrations between shell chemistry and environmental parameters is desirable given their widespread application in palaeoceanography. The incorporation of fluorescent membrane-impermeable molecules into the shell demonstrates that seawater, transported by vacuolisation, is present at the site of calcification. However, recent discussion has focused on whether the calcium required for chamber formation is sourced predominantly by transmembrane Ca transport (TMT), with seawater vacuolisation playing a passive role, or vice versa. This debate has arisen in part because of the need to explain the low Mg/Ca ratio of most foraminifera compared to inorganic calcite. Here, we present trace element data of Operculina ammonoides and Globigerinoides ruber, a high-Mg shallow benthic, and low-Mg planktonic species respectively, cultured under variable seawater carbonate and elemental chemistries. We find that Mg incorporation in high and low-Mg species is characterised by an opposite response to the carbonate system, demonstrating that the negative relationship between Mg/Ca and pH or CO32- in several low-Mg foraminifera is not an intrinsic feature of calcite precipitation. Therefore, any biomineralisation model must be able to explain why the mechanism by which seawater Mg/Ca is reduced is impacted by the carbonate system. Moreover, we show that trace element incorporation in G. ruber is consistent with Rayleigh fractionation from unmodified seawater except for Mg-removal, but in very poor agreement with a biomineralisation site [Ca] substantially elevated above that of seawater as required by the TMT hypothesis. In addition, any biomineralisation model must explain the nonlinear relationship between seawater and shell Mg/Ca, and the large number of seawater vacuoles observed in some species. Although there are important inter-species differences in biomineralisation, evident from the observed range of shell Mg/Ca ratios, we argue that these differences are mechanistically related to the degree of Mg exclusion prior to chamber formation. Indeed, whilst our data for both low-Mg and high-Mg species are consistent with biomineralisation via ions sourced through seawater vacuolisation, it is difficult to reconcile many of these observations with a model based on significant transmembrane Ca transport.

LLO-mediated Cell Resealing System for Analyzing Intracellular Activity of Membrane-impermeable Biopharmaceuticals of Mid-sized Molecular Weight

Cell-based assays have become increasingly important in the preclinical studies for biopharmaceutical products such as specialty peptides, which are of interest owing to their high substrate specificity. However, many of the latter are membrane impermeable and must be physically introduced into cells to evaluate their intracellular activities. We previously developed a “cell-resealing technique” that exploited the temperature-dependent pore-forming activity of the streptococcal toxin, streptolysin O (SLO), that enabled us to introduce various molecules into cells for evaluation of their intracellular activities. In this study, we report a new cell resealing method, the listeriolysin O (LLO)-mediated resealing method, to deliver mid-sized, membrane-impermeable biopharmaceuticals into cells. We found that LLO-type resealing required no exogenous cytosol to repair the injured cell membrane and allowed the specific entry of mid-sized molecules into cells. We use this method to introduce either a membrane-impermeable, small compound (8-OH-cAMP) or specialty peptide (Akt-in), and demonstrated PKA activation or Akt inhibition, respectively. Collectively, the LLO-type resealing method is a user-friendly and reproducible intracellular delivery system for mid-sized membrane-impermeable molecules into cells and for evaluating their intracellular activities.

Simultaneous membrane interaction of amphipathic peptide monomers, self-aggregates and cargo complexes detected by fluorescence correlation spectroscopy

Peptides able to translocate cell membranes while carrying macromolecular cargo, as cell-penetrating peptides (CPPs), can contribute to the field of drug delivery by enabling the transport of otherwise membrane impermeable molecules. Formation of non-covalent complexes between amphipathic peptides and oligonucleotides is driven by electrostatic and hydrophobic interactions. Here we investigate and quantify the coexistence of distinct molecular species in multiple equilibria, namely peptide monomer, peptide self-aggregates and peptide/oligonucleotide complexes. As a model for the complexes, we used a stearylated peptide from the PepFect family, PF14 and siRNA. PF14 has a cationic part and a lipid part, resembling some characteristics of cationic lipids. Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) were used to detect distinct molecular entities in solution and at the plasma membrane of live cells. For that, we labeled the peptide with carboxyrhodamine 6G and the siRNA with Cyanine 5. We were able to detect fluorescent entities with diffusional properties characteristic of the peptide monomer as well as of peptide aggregates and peptide/oligonucleotide complexes. Strategies to avoid peptide adsorption to solid surfaces and self-aggregation were developed and allowed successful FCS measurements in solution and at the plasma membrane. The ratio between the detected molecular species was found to vary with pH, peptide concentration and the proximity to the plasma membrane. The present results suggest that the diverse cellular uptake mechanisms, often reported for amphipathic CPPs, might result from the synergistic effect of peptide monomers, self-aggregates and cargo complexes, distributed unevenly at the plasma membrane.

Soft electroporation for delivering molecules into tightly adherent mammalian cells through 3D hollow nanoelectrodes

Electroporation of in-vitro cultured cells is widely used in biological and medical areas to deliver molecules of interest inside cells. Since very high electric fields are required to electroporate the plasma membrane, depending on the geometry of the electrodes the required voltages can be very high and often critical to cell viability. Furthermore, in traditional electroporation configuration based on planar electrodes there is no a priori certain feedback about which cell has been targeted and delivered and the addition of fluorophores may be needed to gain this information. In this study we present a nanofabricated platform able to perform intracellular delivery of membrane-impermeable molecules by opening transient nanopores into the lipid membrane of adherent cells with high spatial precision and with the application of low voltages (1.5–2 V). This result is obtained by exploiting the tight seal that the cells present with 3D fluidic hollow gold-coated nanostructures that act as nanochannels and nanoelectrodes at the same time. The final soft-electroporation platform provides an accessible approach for controlled and selective drug delivery on ordered arrangements of cells.

Identification of Short Hydrophobic Cell-Penetrating Peptides for Cytosolic Peptide Delivery by Rational Design.

Cell-penetrating peptides (CPPs) enhance the cellular uptake of membrane-impermeable molecules. Most CPPs are highly cationic, potentially increasing the risk of toxic side effects and leading to accumulation in organs such as the liver. As a consequence, there is an unmet need for less cationic CPPs. However, design principles for effective CPPs are still missing. Here, we demonstrate a design principle based on a classification of peptides according to accumulated side-chain polarity and hydrophobicity. We show that in comparison to randomly selected peptides, CPPs cover a distinct parameter space. We designed peptides of only six to nine amino acids with a maximum of three positive charges covering this property space. All peptides were tested for cellular uptake and subcellular distribution. Following an initial round of screening we enriched the collection with short and hydrophobic peptides and introduced d-amino acid substitutions and lactam bridges which increased cell uptake, in particular for long-term incubation. Using a GFP complementation assay, for the most active peptides we demonstrate cytosolic delivery of a biologically active cargo peptide.