Structure, Self-Assembly, and Phase Behavior of Neuroactive N-Acyl GABAs: Doxorubicin Encapsulation in NPGABA/DPPC Liposomes and Release Studies.

N-Acylated amino acids and neurotransmitters in mammals exert significant biological effects on the nervous system, immune responses, and vasculature. N-Acyl derivatives of γ-aminobutyric acid (N-acyl GABA), which belong to both classes mentioned above, are prominent among them. In this work, a homologous series of N-acyl GABAs bearing saturated N-acyl chains (C8-C18) have been synthesized and characterized with respect to self-assembly, thermotropic phase behavior, and supramolecular organization. Differential scanning calorimetric studies revealed that the transition enthalpies and entropies of N-acyl GABAs are linearly dependent on the acyl chain length. The crystal structure of N-tridecanoyl GABA showed that the molecules are packed in bilayers with the acyl chains aligned parallel to the bilayer normal and that the carboxyl groups from opposite layers associate to form dimeric structures involving strong O-H···O hydrogen bonds. In addition, N-H···O and C-H···O hydrogen bonds between amide moieties of adjacent molecules within each layer stabilize the molecular packing. Powder X-ray diffraction studies showed odd-even alternation in the d spacings, suggesting that the odd chain and even chain compounds pack differently. Equimolar mixtures of N-palmitoyl GABA and dipalmitoylphosphatidylcholine (DPPC) were found to form stable unilamellar vesicles with diameters of ∼300-340 nm, which could encapsulate doxorubicin, an anticancer drug, with higher efficiency and better release characteristics than DPPC liposomes at physiologically relevant pH. These liposomes exhibit faster release of doxorubicin at acidic pH (<7.0), indicating their potential utility as drug carriers in cancer chemotherapy.

Protein-Mediated Changes in Membrane Fluidity and Ordering: Insights into the Molecular Mechanism and Implications for Cellular Function.

Probing protein-membrane interactions is vital for understanding biological functionality for various applications such as drug development, targeted drug delivery, and creation of functional biomaterials for medical and industrial purposes. In this study, we have investigated interaction of Human Serum Albumin (HSA) with two different lipids, dipalmitoylphosphatidylglycerol (dDPPG) and dipalmitoylphosphatidylcholine (dDPPC), using Vibrational Sum Frequency Generation spectroscopy at different membrane fluidity values. In the liquid-expanded (LE) state of the lipid, HSA (at pH 3.5) deeply intercalated lipid chains through a combination of electrostatic and hydrophobic interactions, which resulted in more ordering of the lipid chains. However, in the liquid-condensed (LC) state, protein intercalation is decreased due to tighter lipid packing. Moreover, our findings revealed distinct differences in HSA's interaction with dDPPG and dDPPC lipids. The interaction with dDPPC remained relatively weak compared to dDPPG. These results shed light on the significance of protein mediated changes in lipid characteristics, which hold considerable implications for understanding membrane protein behavior, lipid-mediated cellular processes, and lipid-based biomaterial design.

Nebulization and In Vitro Upper Airway Deposition of Liposomal Carrier Systems.

Liposomal carrier systems have emerged as a promising technology for pulmonary drug delivery. This study focuses on two selected liposomal systems, namely, dipalmitoylphosphatidylcholine stabilized by phosphatidic acid and cholesterol (DPPC-PA-Chol) and dipalmitoylphosphatidylcholine stabilized by polyethylene glycol and cholesterol (DPPC-PEG-Chol). First, the research investigates the stability of these liposomal systems during the atomization process using different kinds of nebulizers (air-jet, vibrating mesh, and ultrasonic). The study further explores the aerodynamic particle size distribution of the aerosol generated by the nebulizers. The nebulizer that demonstrated optimal stability and particle size was selected for more detailed investigation, including Andersen cascade impactor measurements, an assessment of the influence of flow rate and breathing profiles on aerosol particle size, and an in vitro deposition study on a realistic replica of the upper airways. The most suitable combination of a nebulizer and liposomal system was DPPC-PA-Chol nebulized by a Pari LC Sprint Star in terms of stability and particle size. The influence of the inspiration flow rate on the particle size was not very strong but was not negligible either (decrease of Dv50 by 1.34 µm with the flow rate increase from 8 to 60 L/min). A similar effect was observed for realistic transient inhalation. According to the in vitro deposition measurement, approximately 90% and 70% of the aerosol penetrated downstream of the trachea using the stationary flow rate and the realistic breathing profile, respectively. These data provide an image of the potential applicability of liposomal carrier systems for nebulizer therapy. Regional lung drug deposition is patient-specific; therefore, deposition results might vary for different airway geometries. However, deposition measurement with realistic boundary conditions (airway geometry, breathing profile) brings a more realistic image of the drug delivery by the selected technology. Our results show how much data from cascade impactor testing or estimates from the fine fraction concept differ from those of a more realistic case.

A Sigma1 Receptor Agonist Alters Fluidity and Stability of Lipid Monolayers.

Interactions between the sigma1 receptor agonist PRE-084 and various lipid monolayers, including dipalmitoylphosphatidylcholine (DPPC), DPP-ethanolamine (DPPE), DPP-glycerol (DPPG), DPP-serine (DPPS), palmitoylsphingomyelin (PSM), and cholesterol (Ch), were investigated to elucidate the effects of PRE-084 on membrane fluidity and stability. Their interactions with sigma1 receptor agonists have potential implications for neuroprotection, antidepressant, analgesic, and cognitive enhancement effects. In this study, we observed that the presence of PRE-084 in the subphase led to increased fluidity in DPPC and DPPE monolayers, whereas decreasing fluidity was observed in DPPG, DPPS, and PSM monolayers. The interaction of PRE-084 with Ch monolayers was found to be distinct from its interaction with other lipids. Fluorescence microscopy images revealed changes in the size and shape of liquid-condensed domains in the presence of PRE-084, supporting the notion of altered membrane fluidity. Our findings provide new insights into the interaction of PRE-084 with lipid monolayers and its potential implications for biological and membrane science.

Study transport of hesperidin based on the DPPC lipid model and the BSA transport model.

Hesperidin (HE), a significant flavonoid polyphenolic compound present in citrus plants, exhibits diverse pharmacological effects. Considering the crucial involvement of biological membranes and transporter proteins in the transportation and biological processes of HE, it becomes essential to comprehend the potential mechanisms through which HE interacts with membranes and transporter proteins. In order to simulate the process of active molecule transport, a cell membrane model consisting of 1,2-dipalmitoyl-n-glycero-3-phosphatidylcholine (DPPC) and a transporter protein model of bovine serum albumin (BSA) were employed for investigation. The present study aimed to investigate the mechanism of action of hesperidin (HE) in DPPC and BSA using fluorescence quenching, Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC). The localization and interaction of HE within liposomes were also elucidated. Furthermore, the binding of BSA and HE was analyzed through UV/Vis absorption spectroscopy, fluorescence spectroscopy, infrared spectroscopy, and computational biology techniques. Computational biology analysis revealed that the binding between HE and BSA primarily occurred via hydrogen bonding and hydrophobic interactions. This study aimed to investigate the role and mechanism of HE in the DPPC cell membrane model and the BSA transporter protein model, thereby offering novel insights into the action of HE in DPPC and BSA.

Eutectic Resolves Lysolipid Paradox in Thermoresponsive Liposomes.

A better molecular understanding of the temperature-triggered drug release from lysolipid-based thermosensitive liposomes (LTSLs) is needed to overcome the recent setbacks in developing this important drug delivery system. Enhanced drug release was previously rationalized in terms of detergent-like effects of the lysolipid monostearyl lysophosphatidylcholine (MSPC), stabilizing local membrane defects upon LTSL lipid melting. This is highly surprising and here referred to as the 'lysolipid paradox,' because detergents usually induce the opposite effect─they cause leakage upon freezing, not melting. Here, we aim at better answers to (i) why lysolipid does not compromise drug retention upon storage of LTSLs in the gel phase, (ii) how lysolipids can enhance drug release from LTSLs upon lipid melting, and (iii) why LTSLs typically anneal after some time so that not all drug gets released. To this end, we studied the phase transitions of mixtures of dipalmitoylphosphatidylcholine (DPPC) and MSPC by a combination of differential scanning and pressure perturbation calorimetry and identified the phase structures with small- and wide-angle X-ray scattering (SAXS and WAXS). The key result is that LTSLs, which contain the standard amount of 10 mol % MSPC, are at a eutectic point when they release their cargo upon melting at about 41 °C. The eutectic present below 41 °C consists of a MSPC-depleted gel phase as well as small domains of a hydrocarbon chain interdigitated gel phase containing some 30 mol % MSPC. In these interdigitated domains, the lysolipid is stored safely without compromising membrane integrity. At the eutectic temperature, both the MSPC-depleted bilayer and interdigitated MSPC-rich domains melt at once to fluid bilayers, respectively. Intact, fluid membranes tolerate much less MSPC than interdigitated domains─where the latter have melted, the high local MSPC content causes transient pores. These pores allow for fast drug release. However, these pores disappear, and the membrane seals again as the MSPC distributes more evenly over the membrane so that its local concentration decreases below the pore-stabilizing threshold. We provide a pseudobinary phase diagram of the DPPC-MSPC system and structural and volumetric data for the interdigitated phase.

Targeted DPPC/DMPG surface-modified voriconazole lipid nanoparticles control invasive pulmonary aspergillosis in immunocompromised population: in-vitro and in-vivo assessment.

Invasive pulmonary aspergillosis (IPA) is the most devastating Aspergillus-related lung disease. Voriconazole (VRZ) is the first-line treatment against IPA. Despite availability in oral and parenteral dosage forms, risks of systemic toxicity dictate alternative pulmonary administration. Inspired by natural lung surfactants, dipalmitoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DPPC/DMPG) surface-modified lipid nanoparticles (LNPs) were scrutinized for pulmonary administration. DPPC/DMPG-VRZ-LNPs prepared using ultrasonication/thin film hydration were investigated for colloidal properties over 3-month shelf storage. They were stable with a slight change in entrapment efficiency. They provided a sustained VRZ release over 24 h, with a rapid initial release. In vitro aerosolization indicated higher percentages of VRZ deposited on stages corresponding to secondary bronchi and alveolar ducts. Moreover, intrapulmonary administration maintained high lung VRZ concentration (27 ± 1.14 µg/g) after 6 h. A preclinical study using a cyclophosphamide-induced neutropenic rat model demonstrated a 3-fold reduction in BALF-Galactomannan down to 0.515 ± 0.22 µg/L confirming DPPC/DMPG-VRZ-LNPs potential in hyphal growth inhibition. Histopathological examination of infected/nontreated lung sections exhibited dense fungal load inside alveoli and blood vessels indicating massive tissue and angio-invasiveness. Nevertheless, DPPC/DMPG-VRZ-LNPs-treated animals displayed minimal hyphae with no signs of invasiveness. The developed bioinspired nanoparticles serve as prospective bioactive nanocarrier candidates for pulmonary administration of VRZ in the management of IPA.

Effect of cationic dendrimer on membrane mimetic systems in the form of monolayer and bilayer.

Interactions between a zwitterionic phospholipid, 1, 2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and four anionic phospholipids dihexadecyl phosphate (DHP), 1, 2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), 1, 2-dipalmitoyl-sn-glycero-3-phosphate (DPP) and 1, 2-dipalmitoyl-sn-glycero-3-phospho ethanol (DPPEth) in combination with an additional amount of 30 mol% cholesterol were separately investigated at air-buffer interface through surface pressure (π) - area (A) measurements. π-A isotherm derived parameters revealed maximum negative deviation from ideality for the mixtures comprising 30 mol% anionic lipids. Besides the film functionality, structural changes of the monomolecular films at different surface pressures in the absence and presence of polyamidoamine (PAMAM, generation 4), a cationic dendrimer, were visualised through Brewster angle microscopy and fluorescence microscopic studies. Fluidity/rigidity of monolayers were assessed by surface dilatational rheology studies. Effect of PAMAM on the formation of adsorbed monolayer, due to bilayer disintegration of liposomes (DPPC:anionic lipids= 7:3 M/M, and 30 mol% cholesterol) were monitored by surface pressure (π) - time (t) isotherms. Bilayer disintegration kinetics were dependent on lipid head group and chain length, besides dendrimer concentration. Such studies are considered to be an in vitro cell membrane model where the alteration of molecular orientation play important roles in understanding the nature of interaction between the dendrimer and cell membrane. Liposome-dendrimer aggregates were nontoxic to breast cancer cell line as well as in doxorubicin treated MDA-MB-468 cell line suggesting their potential as drug delivery systems.

Structural changes in layers of lipid mixtures at low surface tensions.

Layers of pulmonary lipids on an aqueous substrate at non-equilibrium conditions can decrease the surface tension of water to quite low values. This is connected with different relaxation processes occurring at the interface and the associated changes in the surface layer structure. Results of measurements by the combination of methods like surface rheology, ellipsometry, Brewster angle microscopy, and IRRAS for spread layers of lipid mixtures open a possibility to specify the dynamics of structural changes at conditions close to the physiological state. At sufficiently low surface tension values (below 5 mN/m) significant changes in the ellipsometric signal were observed for pure DPPC layers, which can be related to a transition from 2D to 3D structures caused by the layer folding. The addition of other lipids can accelerate the relaxation processes connected with squeezing-out of molecules or multilayer stacks formation hampering thereby a decrease of surface tension down to low values corresponding to the folding of the monolayer.

Closer look at the calorimetric lower transition in lipid bilayers.

The thermal behavior of unilamellar vesicles has been revisited with differential scanning calorimetry to address the issue of whether it is essential to include interactions between neighboring bilayers in theories and simulations of the ripple phase. The issue focuses on the lower, aka pretransition, and the ripple phase that clearly exists between the lower and main transitions in multilamellar vesicles (MLV). We find anomalous thermal behavior in unilamellar vesicles (ULV) beginning at the same temperature as the lower transition in MLVs, but this feature is considerably broadened and somewhat weaker compared to the lower transition in MLVs. We ascribe this to the difficulty of packing a regular ripple pattern on small spheres. In agreement with a few reports of a ripple phase in direct images of single bilayers, we conclude that interactions between neighboring bilayers are not essential for the ripple phase in lipid bilayers.

Interfacial Aß fibril formation is modulated by the disorder-order state of the lipids: The concept of the physical environment as amyloid inductor in biomembranes.

The behavior of amphiphilic molecules such as lipids, peptides and their mixtures at the air/water interface allow us to evaluate and visualize the arrangement formed in a confined and controlled surface area. We have studied the surface properties of the zwitterionic DPPC lipid and Aß(1-40) amyloid peptide in mixed films at different temperatures (from 15 to 40 °C). In this range of temperature the surface properties of pure Aß(1-40) peptide remained unchanged, whereas DPPC undergoes its characteristic liquid-expanded â†’ liquid-condensed bidimensional phase transition that depends on the temperature and lateral pressure. This particular property of DPPC makes it possible to dynamically study the influence of the lipid phase state on amyloid structure formation at the interface in a continuous, isothermal and abrupt change on the environmental condition. As the mixed film is compressed the fibril-like structure of Aß(1-40) is triggered specifically in the liquid-expanded region, independently of temperature, and it is selectively excluded from the well-visible liquid condensed domains of DPPC. The Aß amyloid fibers were visualized by using BAM and AFM and they were Thio T positive. In mixed DPPC/Aß(1-40) films the condensed domains (in between 11 mN/m to 20 mN/m) become irregular probably due to the fibril-like structures is imposing additional lateral stress sequestering lipid molecules in the surrounding liquid-expanded phase to self-organize into amyloids.

Preparation of Thin Films Containing Modified Hydroxyapatite Particles and Phospholipids (DPPC) for Improved Properties of Biomaterials.

The main aims of thin biofilm synthesis are to either achieve a new form to promote the transport of drugs in oral delivery systems or as a coating to improve the biocompatibility of the implant's surface. In this study, the Langmuir monolayer technique was employed to obtain films containing Mg-doped hydroxyapatite with 0.5%, 1.0%, and 1.5% Mg(II). The obtained modified HA particles were analysed via the FT-IR, XRD, DLS, and SEM methods. It was shown that the modified hydroxyapatite particles were able to form thin films at the air/water interface. BAM microscopy was employed to characterized the morphology of these films. In the next step, the mixed films were prepared using phospholipid (DPPC) molecules and modified hydroxyapatite particles (HA-Mg(II)). We expected that the presence of phospholipids (DPPC) in thin films improved the biocompatibility of the preparing films, while adding HA-Mg(II) particles will promote antibacterial properties and enhance osteogenesis processes. The films were prepared in two ways: (1) by mixing DPPC and HA-Mg (II) and spreading this solution onto the subphase, or (2) by forming DPPC films, dropping the HA-Mg (II) dispersion onto the phospholipid monolayer. Based on the obtained π-A isotherms, the surface parameters of the achieved thin films were estimated. It was observed that the HA-Mg(II) films can be stabilized with phospholipid molecules, and a more stable structure was obtained from films synthesied via method (2).

Anomalous lateral diffusion of lipids during the fluid/gel phase transition of a lipid membrane.

A lipid membrane undergoes a phase transition from fluid to gel phase upon changing external thermodynamic conditions, such as decreasing temperature and increasing pressure. Extremophilic organisms face the challenge of preventing this deleterious phase transition. The main focus of their adaptive strategy is to facilitate effective temperature sensing through sensor proteins, relying on the drastic changes in packing density and membrane fluidity during the phase transition. Although the changes in packing density parameters due to the fluid/gel phase transition are studied in detail, the impact on membrane fluidity is less explored in the literature. Understanding the lateral diffusive dynamics of lipids in response to temperature, particularly during the fluid/gel phase transition, is albeit crucial. Here we have simulated the phase transition of a single component lipid membrane composed of dipalmitoylphosphatidylcholine (DPPC) lipids using a coarse-grained (CG) model and studied the changes of the structural and dynamical properties. It is observed that near the phase transition point, both fluid and gel phase domains coexist together. The dynamics remains highly non-Gaussian for a long time even when the mean square displacement reaches the Fickian regime at a much earlier time. This Fickian yet non-Gaussian diffusion (FnGD) is a characteristic of a highly heterogeneous system, previously observed for the lateral diffusion of lipids in raft mimetic membranes having liquid-ordered and liquid-disordered phases co-existing together. We have analyzed the molecular trajectories and calculated the jump-diffusion of the lipids, stemming from sudden jump translations, using a translational jump-diffusion (TJD) approach. An overwhelming contribution of the jump-diffusion of the lipids is observed suggesting anomalous diffusion of lipids during fluid/gel phase transition of the membrane. These results are important in unravelling the intricate nature of lipid diffusion during the phase transition of the membrane and open up a new possibility of investigating the most significant change of membrane properties during phase transition, which can be effectively sensed by proteins.

The Equilibria of Triterpene Sapogenins-Phosphatidylcholine in Monolayers at the Air/Water Interface.

Sapogenins are the non-sugar parts of saponins (aglycones), high-molecular-weight glycosides linked to one or more sugar side chains. This group of compounds presents many properties, e.g., the potent properties of reducing surface tension and foaming properties, as evidenced by the amphipathic nature of these substances. They are used in the cosmetics industry, the washing and detergent industry, and the food industry. In addition, they have many healing properties. They lower blood cholesterol but are also used to synthesize steroid drugs or hormones. As reported in the literature, saponins also show antitumor activity, leading to cell cycle inhibition and apoptosis of various neoplastic cells. In this study, the influence of two sapogenins: asiatic acid (AA) and oleanolic acid (OA), on the properties of monolayers made of phosphatidylcholine (DPPC) was investigated. The method used in these studies was the Langmuir method with Brewster angle microscopy. The interactions between the tested compounds in mixed monolayers were described. Using mathematical equations, we established that oleanolic acid and asiatic acid formed complexes with DPPC at 1:1 ratios, characterized by high stability constants. We derived the parameters characterizing the formed complexes and described the phase transitions that occur during the formation of pure and mixed monolayers.

Unraveling Complex Hysteresis Phenomenon in 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine Monolayer: Insight into Factors Influencing Surface Dynamics.

This study explores the hysteresis phenomenon in DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) monolayers, considering several variables, including temperature, compression and expansion rates, residence time, and subphase content. The investigation focuses on analyzing the influence of these variables on key indicators such as the π-A isotherm curve, loop area, and compression modulus. By employing the Langmuir-Blodgett technique, the findings reveal that all the examined factors significantly affect the aforementioned parameters. Notably, the hysteresis loop, representing dissipated energy, provides valuable insights into the monolayer's viscoelasticity, molecular packing, phase transition changes, and resistance during the isocycle process. These findings contribute to a comprehensive understanding of the structural and dynamic properties of DPPC monolayers, offering insights into their behavior under varying conditions. Moreover, the knowledge gained from this study can aid in the development of precise models and strategies for controlling and manipulating monolayer properties, with potential applications in drug delivery systems, surface coatings, as well as further investigation into air penetration into alveoli and the blinking mechanism.

Freeze-Induced Phase Transition and Local Pressure in a Phospholipid/Water System: Novel Insights Were Obtained from a Time/Temperature Resolved Synchrotron X-ray Diffraction Study.

Water-to-ice transformation results in a 10% increase in volume, which can have a significant impact on biopharmaceuticals during freeze-thaw cycles due to the mechanical stresses imparted by the growing ice crystals. Whether these stresses would contribute to the destabilization of biopharmaceuticals depends on both the magnitude of the stress and sensitivity of a particular system to pressure and sheer stresses. To address the gap of the "magnitude" question, a phospholipid, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), is evaluated as a probe to detect and quantify the freeze-induced pressure. DPPC can form several phases under elevated pressure, and therefore, the detection of a high-pressure DPPC phase during freezing would be indicative of a freeze-induced pressure increase. In this study, the phase behavior of DPPC/water suspensions, which also contain the ice nucleation agent silver iodide, is monitored by synchrotron small/wide-angle X-ray scattering during the freeze-thaw transition. Cooling the suspensions leads to heterogeneous ice nucleation at approximately -7 °C, followed by a phase transition of DPPC between -11 and -40 °C. In this temperature range, the initial gel phase of DPPC, Lß', gradually converts to a second phase, tentatively identified as a high-pressure Gel III phase. The Lß'-to-Gel III phase transition continues during an isothermal hold at -40 °C; a second (homogeneous) ice nucleation event of water confined in the interlamellar space is detected by differential scanning calorimetry (DSC) at the same temperature. The extent of the phase transition depends on the DPPC concentration, with a lower DPPC concentration (and therefore a higher ice fraction), resulting in a higher degree of Lß'-to-Gel III conversion. By comparing the data from this study with the literature data on the pressure/temperature Lß'/Gel III phase boundary and the lamellar lattice constant of the Lß' phase, the freeze-induced pressure is estimated to be approximately 0.2-2.6 kbar. The study introduces DPPC as a probe to detect a pressure increase during freezing, therefore addressing the gap between a theoretical possibility of protein destabilization by freeze-induced pressure and the current lack of methods to detect freeze-induced pressure. In addition, the observation of a freeze-induced phase transition in a phospholipid can improve the mechanistic understanding of factors that could disrupt the structure of lipid-based biopharmaceuticals, such as liposomes and mRNA vaccines, during freezing and thawing.

Investigation of the Membrane Localization and Interaction of Selected Flavonoids by NMR and FTIR Spectroscopy.

In this report, we discuss the effects of undescribed flavone derivatives, HZ4 and SP9, newly isolated from the aerial parts of Hottonia palustris L. and Scleranthus perennis L. on membranes. Interaction of flavonoids with lipid bilayers is important for medicinal applications. The experiments were performed with FTIR and NMR techniques on liposomes prepared from DPPC (dipalmitoylphosphatidylcholine) and EYPC (egg yolk phosphatidylcholine). The data showed that the examined polyphenols incorporate into the polar head group region of DPPC phospholipids at both 25 °C and 45 °C. At the lower temperature, a slight effect in the spectral region of the ester carbonyl group is observed. In contrast, at 45 °C, both compounds bring about the changes in the spectral regions attributed to antisymmetric and symmetric stretching vibrations of CH2 and CH3 moieties. Similarly, as in DPPC lipids, the tested compounds interact with the fingerprint region of the polar head groups of the EYPC lipids and cause its reorganization. The outcomes obtained by NMR analyses confirmed the localization of both flavonoids in the polar heads zone. Unraveled effects of HZ4 and SP9 in respect to lipid bilayers can partly determine their biological activities and are crucial for their usability in medicine as disease-preventing phytochemicals.

Effect of cholesterol on the mechanical stability of gel-phase phospholipid bilayers studied by AFM force spectroscopy.

The remarkably low sliding friction of articular cartilage in the major joints such as hips and knees, which is crucial for its homeostasis and joint health, has been attributed to lipid bilayers forming lubricious boundary layers at its surface. The robustness of such layers, and thus their lubrication efficiency at joint pressures, depends on the lipids forming them, including cholesterol which is a ubiquitous component, and which may act to strengthen of weaken the bilayer. In this work, a systematic study using an atomic force microscope (AFM) was carried out to understand the effect of cholesterol on the nanomechanical stability of two saturated phospholipids, DSPC (1,2-distearoyl-sn-glycero-3-phosphatidlycholine) and DPPC (1,2-dipalmitoyl-sn-glycero- phosphatidylcholine), that differ in acyl chain lengths. Measurements were carried out both in water and in phosphate buffer solution (PBS). The nanomechanical stability of the lipid bilayers was quantitatively evaluated by measuring the breakthrough force needed to puncture the bilayer by the AFM tip. The molar fractions of cholesterol incorporated in the bilayers were 10% and 40%. We found that for both DSPC and DPPC, cholesterol significantly decreases the mechanical stability of the bilayers in solid-ordered (SO) phase. In accordance with the literature, the strengthening effect of salt on the lipid bilayers was also observed. For DPPC with 10 mol % cholesterol, the effect of tip properties and the experimental procedure parameters on the breakthrough forces were also studied. Tip radius (2-42 nm), material (Si, Si3N4, Au) and loading rate (40-1000 nm/s) were varied systematically. The values of the breakthrough forces measured were not significantly affected by any of these parameters, showing that the weakening effect of cholesterol does not result from such changes in experimental conditions. As we have previously demonstrated that mechanical robustness improves the tribological performance of lipid layers, this study helps to shed light on the mechanism of physiological lubrication. Nanoindentation of SDPC bilayers.

Influence of docosahexaenoic acid on the interfacial behavior of cholesterol-containing lipid membranes: Interactions with small amphiphiles and hydration properties.

Cholesterol is known to significantly modify both the structural and the dynamical properties of lipid membranes. On one side, the presence of free cholesterol molecules has been determined to stiffen the membrane bilayer by stretching the hydrophobic tails. Additionally, recent experimental and computational findings have made evident the fact that cholesterol also alters the dynamics and the hydration properties of the polar head groups of DPPC model lipid membranes. In turn, we have recently shown that the Omega-3 fatty acid docosahexaenoic acid, DHA, counteracts the effect of cholesterol on DPPC membrane's mechanical properties by fluidizing the bilayer. However, such behavior represents in fact a global outcome dominated by the larger lipid hydrophobic tails that neither discriminates between the different parts of the membrane nor elucidates the effect on membrane hydration and binding properties. Thus, we now perform molecular dynamics simulations to scrutinize the influence of DHA on the interfacial behavior of cholesterol-containing lipid membranes by characterizing their hydration properties and their binding to amphiphiles. We find that while cholesterol destabilizes interactions with amphiphiles and slightly weakens the lipid's hydration layer, the incorporation of DHA practically restores the interfacial behavior of pure DPPC.

Determination of the Main Phase Transition Temperature of Phospholipids by Oscillatory Rheology.

Knowledge of the physical and chemical properties of phospholipids, such as phase transition temperatures (Tc), is of great importance in order to reveal the functionalities of biological and artificial membranes. Our research group developed an oscillatory rheological method for the simple and rapid determination of phase transition temperatures (Tc). The phospholipids constructing the membranes undergo conformational changes at their Tc, which cause alterations of viscoelastic properties of the molecules. The oscillatory technique recommended by us proved to be appropriate to reveal the altered molecular properties of phospholipids as tracking the slightest changes in the viscoelasticity. Our study demonstrates the abrupt changes in rheological properties at Tc for the following phospholipids: 1,2-Dimyristoyl-sn-glycero-3-Phosphocholine (DMPC), 1,2-Dipalmitoyl-sn-glycero-3-Phosphatidylcholine (DPPC), and 1,2-Distearoyl-sn-glycero-3-Phosphocholine (DSPC), proving that the applied methodology is adequate for determining the Tc of phospholipids.