Self-assembled cationic lipopeptides are membrane-active amphiphilic systems whose biological responses depend on molecular composition and supramolecular organization. Here, we investigated the structure-dependent cytotoxicity, hemocompatibility, cellular association/internalization, and cell-death mechanisms of four cationic lipopeptides with distinct peptide sequences and lipidation patterns: arginine-containing PRWG derivatives and guanidinocarbonylpyrrole (GCP)-containing PK(GCP)WG derivatives. Cytotoxicity assays showed concentration-dependent reductions in cell viability, with cell-line-dependent responses between HeLa and HEK293 cells. The double-lipidated GCP-containing lipopeptide showed the most favorable differential viability trend between these cell lines under the tested conditions, whereas PRWG-based lipopeptides induced more pronounced apoptosis-associated responses in HeLa cells. Hemolysis assays showed low hemolytic activity after 3 h, supporting preliminary short-term hemocompatibility; however, 24 h exposure revealed concentration- and structure-dependent hemolysis, particularly for PRWG-C18H37. Cellular association/internalization studies using coumarin-6 as a physically incorporated hydrophobic fluorescent probe revealed higher cell-associated fluorescence in HeLa cells than in HEK293 cells, suggesting that membrane interactions contribute to the biological response. Because coumarin-6 was physically incorporated rather than covalently attached, these data should be interpreted as cell-associated fluorescence from coumarin-6-loaded assemblies rather than direct quantitative evidence of intact lipopeptide uptake. Annexin V/7-AAD staining and JC-1 analysis indicated that PRWG-based lipopeptides predominantly induced apoptosis-associated cell death and mitochondrial membrane depolarization in HeLa cells, particularly the double-lipidated PRWG derivative. These findings show that peptide sequence, lipidation pattern, apparent surface charge, supramolecular morphology, and membrane-associated fluorescence collectively modulate the biological activity of cationic lipopeptides, highlighting their relevance as supramolecular platforms for investigating tunable lipopeptide-membrane interactions and cell-death responses.
Integral membrane proteins (IMPs) are challenging, yet highly relevant targets for structural biology. Selection of the appropriate membrane mimic environment necessitates extensive optimization using a combination of biochemical and biophysical techniques. We report here on the expanding role of mass photometry (MP) as an in-solution technique to enable single-particle analysis of IMPs. To showcase MP versatility, we present here a selection of examples of MP applications for IMP analysis, using nicotinic acetylcholine receptor (nAChR) extracted from Torpedo marmorata as case study, together with additional literature examples. We further illustrate how MP supports the screening of purification conditions, and the quality control of samples prior to cryo-electron microscopy grid preparation or native mass spectrometry. The benefits of MP for IMP characterization are illustrated through its ability to quantify single-molecule populations, study membrane biomolecular assemblies, and characterize membrane-associated phenomena from very low amounts of starting material. MP therefore stands out as a unique biophysical tool that combines rapid analysis with broad adaptability to most solubilizing environments, enabling simultaneous relative quantification of all detected species in a highly user-friendly manner.
The chemokine CCL20/MIP3α plays an important role in the migration of dendritic cells and several lymphocytes. This 70-residue protein is also implicated in various diseases, such as rheumatoid arthritis, psoriasis, sepsis and numerous forms of cancer. In addition, as the most basic human chemokine, CCL20 also displays direct antimicrobial and antibiofilm activities. The binding and oligomerization of chemokines to cell surface glycosaminoglycans (GAG) can play an important role during receptor activation; therefore we have studied the interactions of CCL20 with fondaparinux. This anticoagulant drug is a uniform sulfated linear pentasaccharide that structurally closely resembles the heterogenous low molecular weight heparins that have often been used to study GAG-protein binding in solution. Using multidimensional multinuclear NMR spectroscopy, we could map out the distinct GAG/fondaparinux binding sites on the monomeric (pH 5.5) and dimeric (pH 7.5) forms of the CCL20 protein surface. In the second part of this study, we showed that CCL20-(51-70), the recombinant 20-residue carboxyterminal helical peptide of CCL20, interacted weakly with fondaparinux. We also surveyed several host-defence properties of this peptide. In comparison to several other well-characterized peptides, CCL20-(51-70) displayed potent antibiofilm and antimicrobial activities against a pathogenic Gram-positive Staphylococcus aureus MRSA strain. It also displayed somewhat lower activities against the Gram-negative Pseudomonas aeruginosa PAO1 strain. These results highlight the potential of CCL20-(51-70) as a potent host-defence peptide, that could play a role in eradicating bacterial biofilms, particularly for Gram-positive pathogenic bacteria.
Regulation of Na+,K+-ATPase activity plays a critical role in maintaining ionic homeostasis and Ca2+-dependent contractility in smooth muscle, yet the contribution of selective sodium pump modulators to myometrial function remains insufficiently understood. In this study, we investigated the effects of calix[4]arene C-1220 (25,27-dipropoxycalix[4]arene-bis-N-tolylsulfonylaminomethylphosphonic acid) and its structural analogue C-99 on ATP-hydrolyzing activities, intracellular Ca2+ dynamics, mitochondrial function, and contractile behavior of uterine smooth muscle. Enzymatic assays showed that C-1220 is a highly potent and selective inhibitor of Na+,K+-ATPase (IC₅₀ = 48 ± 2 nM) without influencing other ATPases in the plasma membrane. Confocal imaging demonstrated that C-1220 elevates cytosolic Ca2+ in myocytes, whereas flow cytometry and Ca2+-accumulation assays indicated that mitochondrial membrane polarization and matrix Ca2+ levels remain unaffected. Tensometric measurements revealed that both C-1220 and C-99 enhance spontaneous contractions and force-, velocity-, and impulse-related mechanokinetic parameters; however, normalization of velocity parameters suggests that these changes do not reflect direct modulation of Ca2+-transporting systems. Comparative analysis supports the conclusion that inhibition of Na+,K+-ATPase and consequent disturbance of Na+/Ca2+ exchange underlie the contractile activation induced by both compounds. These findings provide new insights into membrane mechanisms governing electro- and pharmacomechanical coupling in uterine smooth muscle and highlight C-1220 as a useful molecular tool for probing Na+,K+-ATPase-dependent Ca2+ regulation.
Apicomplexan parasites, including Toxoplasma gondii and Plasmodium falciparum, reside within a specialized compartment known as the parasitophorous vacuole (PV) during their intracellular life cycle. The PV membrane (PVM), which derives from the host plasma membrane upon invasion, serves as a selective barrier that permits nutrient acquisition while shielding the parasite from host defense mechanisms. Although the protein composition of the PVM has been studied extensively, its lipid organization remains poorly understood. Using the quick-freeze, freeze-fracture replica labeling (QF-FRL) method, we quantitatively analyzed the transbilayer distribution of phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEtn), and GM3 ganglioside in the PVM of T. gondii and P. falciparum. Unlike host cell plasma membranes, where these lipids exhibit strict asymmetry-PtdSer and PtdEtn confined to the cytoplasmic leaflet and GM3 to the exoplasmic leaflet-we found that all three lipids were symmetrically distributed across both leaflets of the PVM. This striking loss of lipid asymmetry suggests that the PVM undergoes profound remodeling during infection. The presence of PtdSer and PtdEtn in the luminal leaflet may facilitate the binding of perforin-like proteins (PLP1s) during egress. These findings reveal a unique feature of the PVM that redefines our understanding of host-parasite membrane biology.
Enterobacter cloacae ATCC13047 encodes the EmmdR efflux protein, which belongs to the Multidrug And Toxic compound Extrusion (MATE) transporter family (Transporter Classification ID 2.A.66.1). The protein has a predicted existence in the Uniprot database owing to the lack of structure elucidation and experimental evidence of membrane localization. This study experimentally validated the predicted topology of EmmdR and ascertained the role of charged amino acids in membrane insertion. The experimental strategy included qualitative and quantitative assays of dual reporter PhoA-LacZ enzymes coupled with microscopic analysis to ascertain the localization of EmmdR. The topology of periplasmic loop 1 was confirmed by immunofluorescence analysis. The results of this study corroborated the predicted 12 transmembrane segment architecture of EmmdR. Colocalization of green fluorescent protein (GFP) with membrane staining dye FM4-64 confirmed cytoplasmic membrane localization and cytoplasmic orientation of C and N-terminus of EmmdR. Immunofluorescence microscopy of spheroplasts confirmed the periplasmic localization of loop 1. The membrane insertion of EmmdR concurred with the 'positive inside' rule, and deletion of critical positive amino acids in the cytoplasmic loop resulted in altered topology. Thus, this study is the first to report the experimental topology determination and membrane localization of EmmdR, which validates the predicted existence and supports the predicted structure of EmmdR.
Biomolecular condensates are vital organizers of cellular space. These membraneless organelles wet and remodel membranes to orchestrate many critical physiological processes. While the molecular interactions driving condensate formation and their material properties are increasingly well understood, their emergent electrochemical environment has only recently come into focus. In contrast to membrane electrostatics, which has been extensively characterized for decades, the surface charge, interphase potentials and dielectric properties of condensates are only now beginning to be investigated. In this mini-review we examine how electrostatics drive and shape condensate-membrane interactions and critically assess the advantages and limitations of the currently used methods to characterize condensate electrical properties. Finally, we identify the challenges and open questions in the field.
Many antimicrobial peptides act by disrupting cellular membranes, with therapeutic selectivity arising from their ability to discriminate between bacterial and mammalian cells, a distinction largely governed by membrane surface charge. Here, we investigate the interfacial organisation and membrane insertion of the cationic antimicrobial peptide magainin 2 using specular neutron reflectometry on lipid monolayers that mimic bacterial and mammalian membranes. Negatively charged dipalmitoylphosphatidylglycerol (DPPG) and zwitterionic dipalmitoylphosphatidylcholine (DPPC) were used as respective representative model systems. Experimental conditions were adjusted to compensate for the markedly different membrane association constants of magainin 2, enabling direct comparison of peptide behavior at the two interfaces.Despite similar peptide coverages, magainin 2 exhibits strikingly different interfacial behavior. At the DPPG interface, the magainin 2 α-helix inserts deeply and adopts an orientation parallel to the membrane surface, with hydrophobic residues facing the lipid chains and cationic residues interacting with the polar headgroups. In contrast, at the DPPC interface, the peptide displays limited interaction with the monolayer and a diffuse intensity distribution across the headgroup and acyl-chain regions, consistent with a largely disordered conformation.These results demonstrate that electrostatic interactions not only promote membrane binding but also dictate peptide insertion depth and interfacial organisation. By directly linking membrane charge to peptide penetration and structural arrangement, this work provides mechanistic insight into the selective antibacterial activity of magainin 2 and establishes a general framework for understanding the membrane-disruptive action of cationic antimicrobial peptides.
Laurdan (6-dodecanoyl-2-(dimethylamino)-naphthalene) is a solvatochromic fluorescent probe widely used for investigating membrane biophysical properties. Since its first application in phospholipid bilayers, laurdan has become a valuable tool in this field, owing to its sensitivity to the mobility and dynamics of surrounding lipid carbonyl groups and its ability to report on membrane phase behavior through the generalized polarization (GP) parameter. GP, a ratiometric empirical parameter, is derived from the ratio of emission intensities at approximately 440 and 490 nm, providing a measure of membrane fluidity ranging from rigid gel phases to more hydrated liquid-crystalline states. This review outlines the spectroscopic evolution of laurdan applications, beginning with steady-state fluorescence measurements in model membranes and progressing through more modern methodologies including anisotropy measurements, two-photon excitation microscopy, fluorescence correlation spectroscopy, and spectral phasor analysis. Key developments in laurdan's application to biological systems are discussed, including investigations of lipid raft-like domains, heavy metal-membrane interactions, and cellular membrane organization. Practical considerations for the exogenous incorporation of laurdan into membrane systems are also addressed, including the influence of solvent vehicle on probe aggregation and incorporation kinetics. Laurdan derivatives such as C-laurdan, CAPRYDAA, and organelle-targeted variants have further extended the versatility of this probe family. Together, these advances illustrate how laurdan has evolved from a simple polarity sensor into a multifaceted platform for characterizing membrane structure, lateral heterogeneity, and dynamics across model and biological systems.
This mini-review examines how molecular dynamics simulations reshape the view of annexin-mediated plasma membrane repair from simple recruitment to coupled protein-membrane states. Molecular dynamics simulations show that annexin-induced curvature is shaped by oligomerization, cholesterol, and anionic lipid chemistry, while engineering normally non-trimerizing annexin A3 into a trimer-forming state demonstrates that curvature generation alone does not ensure repair competence. Membrane-active perturbants such as trifluoperazine further suppress repair by altering bilayer thickness, lipid packing, phosphatidylserine mobility, and annexin binding. These findings shift the question from whether annexins arrive to how protein-membrane states generate repair-relevant remodeling.
The thermodynamic phase behavior of lipid bilayers formed by R-dipalmitoylphosphatidylcholine (R-DPPC), its enantiomer S-DPPC, and racemic DPPC (rac-DPPC) was investigated by differential scanning calorimetry and high-pressure light transmittance measurements. After annealing, the R-DPPC and S-DPPC bilayers exhibited subtransition, pretransition, and main transition, whereas the rac-DPPC bilayers showed no subtransition and failed to form the lamellar crystalline phase. The thermodynamic parameters of the main transition in the rac-DPPC bilayers were nearly identical to the ideal mixing values, while those of the pretransition exhibited clear negative deviations, indicating that the nonideality of racemic mixing becomes more pronounced in highly ordered bilayer phases. In addition, the rac-DPPC bilayers exhibited broadened transition peaks, indicating reduced cooperativity and less collective bilayer ordering. Furthermore, the R-DPPC bilayers were thermodynamically more stable than the S-DPPC bilayers in all phase states despite their enantiomeric relationship, demonstrating that enantiomeric lipids can exhibit nonequivalent stability in the aggregated bilayer state. In contrast, pressure-induced formation of the interdigitated gel phase occurred at nearly identical pressures for all bilayers, indicating minimal chirality dependence of interdigitation. These results demonstrate that lipid chirality modulates bilayer phase stability through collective bilayer ordering, molecular packing, and cooperativity, and that its effects are amplified in condensed membrane phases.
The hydrophobic tail structure of lipids or amphiphiles plays a critical role in governing the self-assembly, structure, dynamics, and function. In the present work, β-alaninol-based amphiphiles bearing cholesterol, saturated, unsaturated, and branched fatty acid-based tails were synthesized in N-acyl and N-cholesteroyl forms and systematically investigated to elucidate how tail architecture controls physicochemical properties. Results revealed that variations in amphiphile tail structure influenced pronounced differences in phase transition behavior and supramolecular packing. Amphiphiles bearing saturated, unsaturated, or branched tails favored more tilted or less-ordered bilayer arrangements than the cholesterol-based amphiphile, which lead to lower phase transition temperatures of fatty acid-based amphiphiles than the cholesterol-based amphiphile. Thermal analysis studies revealed that N-acylation substantially enhanced thermal stability, with decomposition temperatures increased by 50-70 °C compared to the corresponding parent fatty acids; however, the cholesteroyl-based amphiphile exhibited lower enhancement than cholesterol. Laurdan and ANS (8-anilinonapthalene-1-sulphonic acid) fluorescence studies revealed a strong correlation between tail structure, membrane packing and interfacial hydration, in which unsaturation in tail lowers the gel-to-liquid-crystalline transition temperature and enhances ANS accessibility. Dynamic light scattering and field-emission scanning electron microscopy confirmed the formation of unilamellar vesicles, where tail architecture significantly influences vesicle size, polydispersity, and zeta potential. Comparison of bilayer-related properties between matched-chain saturated ethanolamine- and β-alaninol-based amphiphiles revealed only moderate effects arising from the headgroup substitution. Overall, these findings establish amphiphile tail design as a key structural parameter for controlling amphiphile packing, phase behavior, and hydration, offering molecular-level insights for tuning properties of lipid-based assemblies for various applications.
Large-conductance Ca2+- and voltage-activated potassium (BK) channels display a near-linear current-voltage relationship in excised membrane patches but exhibit inward rectification in intact cells due to block by intracellular cations. This rectification arises from electrostatic interactions between endogenous cationic blockers and negatively charged residues lining the inner pore. Here, we examined how the introduction of an additional negative charge within the inner vestibule affects BK channel permeation. Phenylalanine 315 in the S6 segment of the BK α-subunit was substituted with aspartate (F315D), and channel properties were examined using single-channel patch-clamp recordings in HEK293 cells.F315D channels exhibited reduced single-channel conductance, with a greater reduction in outward than inward current, resulting in strong inward rectification in the cell-attached configuration. Patch excision into Mg2+-free solutions substantially increased both inward and outward current amplitudes, indicating strong modulation by intracellular cations. Reintroduction of Mg2+ restored current suppression in a concentration-dependent manner, with mutant channels showing greatly enhanced Mg2+ sensitivity compared with wild-type BK channels. In addition, F315D channels exhibited reduced voltage dependence of activation and high open probability even at strongly negative potentials under low Ca2+ conditions.These data are consistent with the introduction of an additional electrostatic determinant within the inner vestibule that strengthens intracellular cation block and converts BK channels into strong inward rectifiers. Because F315 is also part of the hydrophobic deep-pore region that controls BK gating, we interpret the F315D phenotype as the combined result of altered pore electrostatics, local hydration/permeation, and modified energetic coupling between the pore and activation sensors.
The LRLLR cell-penetrating motif can be transferred to confer membrane translocation activity, but only to compatible recipient peptides. Using umbrella sampling molecular dynamics simulations, we show that C-terminal LRLLR addition to the pro-apoptotic smacN peptide eliminates its translocation barrier, transforming a + 65 kJ/mol barrier into a - 50 kJ/mol energy well. In contrast, N-terminal LRLLR addition to the neuroprotective NR2B9c peptide increases the barrier from +85 to +100 kJ/mol. The LRLLR pentapeptide, identified through systematic screening as essential for spontaneous translocation, represents a minimal penetrating element whose transferability was unknown. We appended this motif to both peptides and calculated potential of mean force profiles across a POPC/POPG bilayer. Analysis of hydrogen bonding patterns, secondary structure, and conformational dynamics reveals the structural basis for these divergent outcomes. Successful transfer to smacN reflects favorable charge complementarity: the hydrophobic, neutral AVPI tetrapeptide provides an ideal platform for the charged, amphipathic LRLLR motif, enabling simultaneous interaction with both membrane leaflets. Transfer failure with NR2B9c stems from its positive charge and conformational rigidity imposed by intramolecular hydrogen bonding, preventing optimal membrane insertion. These findings establish that CPP motif transfer requires compatibility in charge distribution, hydrophobicity, and conformational flexibility, and demonstrate the value of computational screening to identify compatible motif-cargo pairings prior to experimental investment.
Mg2+ and Ca2+ are involved in nearly every aspect of cellular function. These divalent cations can bind to the same protein binding sites, often with significantly binding affinities, resulting in distinct conformational changes in proteins. Recent studies demonstrated that both Mg2+ and Ca2+ can influence lipid-dependent folding and insertion of various membrane-active peptides and non-constitutive membrane proteins and peptides. Specifically, cardiolipin, can recruit various apoptotic regulators of the Bcl-2 family (e.g., pro-apoptotic BAX and anti-apoptotic Bcl-xL) to the model membranes in the absence of canonical protein activators, like the BH3-only proteins. This interaction can only occur in the presence of divalent cations, such as Mg2+ which is constantly present outside of the targeted mitochondrial membrane in the cell, or Ca2+, which is released from the mitochondria at the early stages of apoptosis. Here, we use all-atom molecular dynamics simulations to provide the first atomistic-level characterization of the dynamic protein-lipid-cation interaction of the Bcl-xL, anchored to the cardiolipin-containing lipid bilayer, in the presence of either Mg2+ or Ca2+. Our results show that both ions interact with Bcl-xL and membrane lipids in the membrane-anchored state, but through distinct modes. Whereas Mg2+ primarily forms water-mediated interactions with both the protein and the lipids, Ca2+ establishes direct contacts with both. These differences led to more constrained conformational dynamics of Bcl-xL in the presence of Ca2+, particularly in the α1-α2 loop and the BH3-binding groove involved in inhibitory interactions with pro-apoptotic BAX.
The cell envelope of Synechocystis sp. PCC 6803 has a Gram-negative-like organization, with inner and outer membranes and an outermost surface layer. A comprehensive characterization of its layered structure, considering the presence of pigments and distinctive proteins, has not yet been established. Here, we isolated cell envelope fragments and characterized them by electrophoretic and proteomic analyses identifying a recurring set of inner membrane, outer membrane, and S-layer proteins, including six conserved porins and additional associated proteins with repeated beta-strand motifs, among which several contain a SLH domain. Further characterization by absorption spectroscopy, to investigate the typical orange color of this cell envelope, revealed extended retention of carotenoids. These results provide an integrated and preliminary view of the Synechocystis sp. PCC 6803 cell envelope and its characteristic proteome.
Antimicrobial peptides (AMPs) are promising alternatives to conventional antibiotics due to their broad-spectrum activity and low tendency to induce resistance. Based on their modes of action, AMPs are generally classified as membrane-active or intracellular-targeting peptides. Rational design of next-generation AMPs with enhanced efficacy and reduced cytotoxicity requires a detailed understanding of their antimicrobial mechanisms. Solid-state nuclear magnetic resonance (ssNMR) has emerged as a powerful tool for probing AMP-target interactions, providing high-resolution structural and dynamic information under native-like membrane conditions. This review summarizes recent advances in ssNMR methodologies applicable in model membranes and intact cells and highlights their contributions to elucidating AMP antimicrobial mechanisms. Representative investigations reveal that the membrane-active peptide protegrin-1 disrupts lipid bilayers via a toroidal pore mechanism, whereas lipid II-targeting AMPs inhibit cell wall biosynthesis by immobilizing lipid II through supramolecular assembly.
Staphylococcus aureus (S. aureus) is an opportunistic pathogen that is a global health concern for its ability to cause a wide spectrum of clinical infections ranging from minor skin abscesses to systemic conditions such as sepsis and pneumonia. Due to the emergence of resistance to commonly used antibiotics, there has been interest in exploring the use of antimicrobial peptides to treat S. aureus infections. However, changes in the lipid composition of the lipid bilayer membrane can alter the activity of peptides, and S. aureus is able to induce variations in lipid composition in response to environmental stress. Here, we explore how the main lipid components in S. aureus are altered when exposed to LL-37, a human cathelicidin involved in primary immune response, and ATRA-1, a short antimicrobial peptide derived from the snake Naja atra venom. A lipidomic study is conducted through HPLC-MS-MS (LC-ESI-MS/MS) to quantify phosphatidylglycerol, cardiolipin, lysylphosphatidylglycerol, monogalacto-and digalacto-diacylglycerol, and carotenoids. In addition, menaquinones, responsible for electron transport during oxidative phosphorylation, were also quantified. Biophysical properties such as membrane electric surface potential and lipid packing were assessed. We find that lipid adaptation is specific to the type of antimicrobial peptide, where ATRA-1 mainly induces changes in the electric surface potential through variations in Lysyl-PG, while exposure to LL-37 changes carotenoid levels, inducing an increase in membrane rigidity as measured by FTIR. In addition, both peptides induce a reduction in menaquinone and DGDG levels. These findings highlight the role of membrane lipid remodeling as a peptide-specific response mechanism in S. aureus, with implications for the development of AMP-based therapies.
We compare biophysical and microbiological results to link structure and function in determining the mechanism of bacterial killing by two antimicrobial peptides (AMPs). Two 14-mer peptides were compared, each containing 6 tryptophans, in addition to 8 lysines (W6K8) or 8 arginines (W6R8). MIC values show W6K8 is more effective at killing bacteria, and less toxic to red blood cells than is W6R8. Small angle X-ray scattering (SAXS) shows W6K8 is more efficient at fusing unilamellar vesicles (ULVs) that mimic bacterial lipid model membranes (LMMs), and W6R8 is more efficient at fusing ULVs that mimic eukaryotic cells, suggesting that membrane destabilization is a requirement for both activities. Circular dichroism (CD) exhibits excitonic coupling suggesting that AMP aggregation is involved in the killing mechanism, while secondary structure is mostly random coil and beta-sheet. Differential interference contrast (DIC) microscopy confirms that W6K8 aggregates GUVs to a greater extent than does W6R8 in bacterial LMMs. X-ray diffuse scattering (XDS) indicates that W6K8 penetrates more deeply than W6R8 into bacterial LMMs compared to W6R8. XDS also reports that W6K8 condenses the lipid area more than W6R8 in bacterial LMMS, but that the opposite is true in eukaryotic LMMs. Fourier Transform Infrared Spectoscopy (FTIR) indicates that W6K8 destabilizes the gel phase of DPPG, while W6R8 does not. These five biophysical methods indicate differences that could be responsible for the different microbiological results.
The design of effective non-viral nucleic acid delivery systems based on cationic amphiphilic compounds remains a major challenge in biomedical research. In this study, cationic benzimidazolium surfactants (BI-n) and cationic liposomes composed of BI-n in combination with auxiliary lipids (DOPE, DOTAP, and cholesterol) were investigated and compared as potential nucleic acid carriers. Physicochemical and biological experiments were performed using a model double-stranded oligonucleotide, with additional transfection studies conducted using plasmid DNA. Both BI-n and liposomal systems effectively bound nucleic acids to form compact complexes (60-250 nm). Complex formation was driven primarily by electrostatic interactions, with additional contributions from hydrophobic and intercalation effects. The lipoplexes exhibited a predominantly spherical morphology, and showed high internalization efficiency in M-HeLa, A549, and HuTu 80 cancer cell lines. The N/ P charge ratio was identified as a key factor regulating both cellular uptake and hemagglutination activity. Liposomal formulations containing auxiliary lipids demonstrated enhanced cellular internalization compared to surfactant-based systems. Importantly, transfection experiments using plasmid DNA (pEGFP-N2) revealed that BI-12-based systems enable functional nucleic acid delivery, resulting in efficient gene expression. BI-12 exhibited transfection efficiency comparable to a commercial reagent (Lipomaster 2000) and higher than that of DOTAP, while maintaining activity in the presence of serum. Notably, increased cellular uptake of liposomal systems did not result in improved transfection efficiency, highlighting the critical role of intracellular release. These findings demonstrate that benzimidazolium surfactants represent a promising platform for the development of non-viral delivery systems and provide insight into the structure-activity relationships governing nucleic acid transport.
