Phosphatidylethanolamine-induced cholesterol domains chemically identified with mass spectrometric imaging

Coexisting liquid phases of model membrane systems are chemically identified using imaging time-of-flight secondary ion mass spectrometry (TOF-SIMS). The systems studied were Langmuir-Blodgett (LB) model membranes of cholesterol (CH) with two different phospholipids, one a major component in the outer plasma membrane bilayer leaflet (dipalmitoylphosphatidylcholine (PC)) and the other a major component in the inner leaflet (dipalmitoylphosphatidylethanolamine (PE)). Binary mixtures of CH with each of the phospholipids were investigated, as well as a ternary system. A single homogeneous phase is evident for PC/CH, whereas both systems containing PE show lateral heterogeneity with phospholipid-rich and CH-rich regions. The interaction between CH and the two phospholipids differs due to the disparity between the phospholipid headgroups. Imaging TOF-SIMS offers a novel opportunity to chemically identify and differentiate the specific membrane locations of CH and phospholipid in membrane regions without the use of fluorescent dyes. This unique imaging method has been used to demonstrate the formation of micrometer-size CH domains in phosphatidylethanolamine-rich systems and is further evidence suggesting that CH may facilitate transport and signaling across the two leaflets of the plasma membrane.     

Mass spectral imaging of glycophospholipids, cholesterol, and glycophorin a in model cell membranes

Time of flight secondary ion mass spectrometry (ToF-SIMS) and the Langmuir-Blodgett (LB) technique have been used to create and analyze reproducible membrane mimics of the inner and outer leaflets of a cellular membrane to investigate lipid-protein and lipid-lipid interactions. Films composed of phospholipids, cholesterol and an integral membrane protein were utilized. The results show the outer membrane leaflet mimic (DPPC/cholesterol/glycophorin A LB film) consisting of a single homogeneous phase whereas the inner membrane leaflet mimic (DPPE/cholesterol/glycophorin A LB film) displays heterogeneity in the form of two separate phases. A DPPE/cholesterol phase and a glycophorin A phase. This points to differences in membrane domain formation based upon the different chemical composition of the leaflets of a cell membrane. The reliability of the measurements was enhanced by establishing the influence of the matrix effect upon the measurement and by utlilizing PCA to enhance the contrast of the images.     

Langmuir-Blodgett films formed by continuously varying surface pressure. Characterization by IR spectroscopy and epifluorescence microscopy

Monolayer films of phospholipids at the air-water interface have been transferred to solid substrates under conditions of continuously varying surface pressure, an approach termed COVASP. The molecular and supramolecular properties of the film constituents have been characterized with two complementary techniques. IR spectroscopy was used to monitor chain conformation as a function of transfer surface pressure. Results were compared to those from Langmuir films determined directly at the A/W interface by IR reflection-absorption spectroscopy (IRRAS). The methylene stretching frequencies for both proteated and acyl chain perdeuterated 1,2-dipalmitoylphosphatidylcholine (DPPC and DPPC-d62) in the transferred molecules indicate that the phospholipids retain at least, in part, their surface pressure-dependent chain-conformational order characteristics. The line widths of these modes are somewhat reduced, suggestive of slower rates of reorientational motion in the Langmuir-Blodgett (LB) films. Epifluorescence microscopy reveals a progressive condensation gradient, including nucleation and growth of probe-excluding condensed domains along the transfer line. DPPC condensation, observed along a single LB film, was qualitatively comparable to compression-driven condensation as observed in situ or in conventional LB films transferred at constant pressures. However, condensation along the compression isotherm in COVASP-LB films was reduced by 15-20% as compared to films equilibrated at different constant pressures, probably the result of kinetic differences in equilibration processes. As a preliminary demonstration of the utility of this new approach, the monolayer --> multilayer transition known to occur (Eur. Biophys. J. 2005, 34, 243) in a four-component model for pulmonary surfactant has been examined. IR parameters from both the lipid and the protein constituents of the film all indicate that the transition persists during the transfer process. This new approach for the study of transferred films will permit the efficient characterization of lipid-protein interactions and structural transitions occurring in pulmonary surfactant films subjected to dynamic compression.     

Structural effects in the analysis of supported lipid bilayers by time-of-flight secondary ion mass spectrometry

We contribute to the rapidly emerging interest in the application of time-of-flight secondary ion mass spectrometry (TOF-SIMS) for chemical analysis of biological materials by presenting a careful TOF-SIMS investigation of structurally different SiO2-supported phospholipid assemblies. Freeze-dried supported 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (POPC) bilayers, Langmuir-Blodgett POPC monolayers, and disordered thick POPC films were investigated. Compared with the two latter structures, the supported bilayer showed a strong (5-10 times) enhancement in the yield of both the molecular and the dimer ion peaks of POPC, suggesting that the molecular peak may be used as a sensitive indicator for changes in the membrane structure and, in particular, an indicator for the presence of bilayer structures in, e.g., cell and tissue samples. The detection efficiency and the useful lateral resolution indicate that a lateral resolution of around 100 nm can be obtained on all structures by imaging the phosphocholine ion at 184 u using Bi3+ primary ions. For the chemically specific molecular peak at 760 u, the measured detection efficiencies correspond to a useful lateral resolution of around 2 microm for the bilayer structure. The results are discussed in relation to recent dynamic SIMS (nano-SIMS) analysis of freeze-dried supported lipid bilayers, displaying similar or higher lateral resolution, but which in contrast to TOF-SIMS requires isotopic labeling of the analyzed lipids.     

Lateral heterogeneity of dipalmitoylphosphatidylethanolamine-cholesterol Langmuir-Blodgett films investigated with imaging time-of-flight secondary ion mass spectrometry and atomic force microscopy

To better understand the influence of cholesterol (CH) on dipalmitoylphosphatidylethanolamine (DPPE), Langmuir-Blodgett (LB) model membranes of DPPE with varying amounts of cholesterol were imaged by time-of-flight secondary ion mass spectrometry (ToF-SIMS) and atomic force microscopy (AFM). Cholesterol has a condensing effect on DPPE that at low cholesterol concentrations results in lateral heterogeneity of the LB monolayer. At 4:1 DPPE/CH, islands of DPPE/CH phase exist with a connected DPPE phase. As the concentration of cholesterol is increased, the percolation threshold is crossed and the DPPE/CH phase islands connect to separate the DPPE phase (2:1 DPPE/CH). Finally, at 50 mol % cholesterol a single homogeneous DPPE/CH phase LB monolayer exists. ToF-SIMS of the DPPE/CH phase provides a lower ion signal for the characteristic lipid fragments and substrate apparently owing to the higher molecular density induced by cholesterol. AFM data indicate that the DPPE/CH phase is lower in height than the DPPE phase. As phosphatidylethanolamine is predominant in the inner lipid leaflet of cellular membranes, this work has implications for the understanding of cholesterol domains in the inner leaflet of cells.     

Structural analysis of secondary ions by post-source decay in time-of-flight secondary ion mass spectrometry

Tandem mass spectrometry measurements have been achieved using time-of-flight secondary ion mass spectrometry (TOF-SIMS) and a post source decay (PSD)-like method. The performance of the method has been demonstrated on model molecules with well-known fragmentation pathways. Several lipids have been fragmented including the phosphocholine ion, phosphatidylcholines, cholesterol and vitamin E. Pure samples were analyzed, and the results compared with those obtained with the same compounds on a quadrupole-TOF hybrid mass spectrometer. Then, the structures of some lipids which are currently observed in the TOF-SIMS imaging of mammalian tissue sections were verified.     

Effects of lipid chain unsaturation and headgroup type on molecular interactions between paclitaxel and phospholipid within model biomembrane

Molecular interactions between paclitaxel, an anticancer drug, and phospholipids of various chain unsaturations and headgroup types were investigated in the present study by Langmuir film balance and differential scanning calorimetry. Both the lipid monolayer at the air-water interface and the lipid bilayer vesicles (liposomes) were employed as model cell membranes. It was found that, regardless of the difference in molecular structure of the lipid chains and headgroup, the drug can form nonideal, miscible systems with the lipids at the air-water interface over a wide range of paclitaxel mole fractions. The interaction between paclitaxel and phospholipid within the monolayer was dependent on the molecular area of the lipids at the interface and can be explained by intermolecular forces or geometric accommodation. Paclitaxel is more likely to form thermodynamically stable systems with 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) and 1,2-dielaidoyl-sn-glycero-3-phosphocholine (DEPC) than with 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). Investigation of the drug penetration into the lipid monolayer showed that DPPC and DEPC have higher incorporation abilities for the drug than DPPE and DSPC. A similar trend was also evidenced by DSC investigation with liposomes. While little change of DSC profiles was observed for the DPPE/paclitaxel and DSPC/paclitaxel liposomes, paclitaxel caused noticeable changes in the thermographs of DPPC and DEPC liposomes. Paclitaxel was found to cause broadening of the main phase transition without significant change in the peak melting temperature of the DPPC bilayers, which demonstrates that paclitaxel was localized in the outer hydrophobic cooperative zone of the bilayer, i.e., in the region of the C1-C8 carbon atoms of the acyl chain or binding at the polar headgroup site of the lipids. However, it may penetrate into the deeper hydrophobic zone of the DEPC bilayers. These findings provide useful information for liposomal formulation of anticancer drugs as well as for understanding drug-cell membrane interactions.     

Identification of lipid aggregate structures on TiO2 surface using headgroup IR bands

Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy was used to study the nature of the dipalmitoylphosphatidylcholine (DPPC) aggregated structures adsorbed on TiO(2). DPPC molecules were assembled on TiO(2) using Langmuir-Blodgett (LB) deposition methods or by directly flowing the DPPC liposome solution across the TiO(2)-coated ATR crystal. We found that there is a direct correlation between the intensity and frequency position of the zwitterionic headgroup IR bands and the nature of LB films. Specifically, we have shown that the bands due to PO(2)(-) modes are sensitive to changes in the degree of hydration of the LB films and that the symmetric deformation vibrational mode (delta(s) (+)N-CH(3)) is sensitive to interaction with oppositely charged surface sites. Using this information, we found that the liposomes adsorbed on TiO(2) remain intact as vesicles and that the vesicles are stable and not removed in flowing water. We have also shown that the antisymmetric deformation vibrational (delta(as) (+)N-CH(3)) modes are sensitive to changes in lateral-lateral DPPC interactions. This information was used to show that there is a lateral interaction between each positively charged (+)N(CH(3))(3) headgroup and negatively charged PO(2)(-) headgroup of the adjacent DPPC molecule in the adsorbed vesicles and LB films. This study provides a framework for the use of this IR technique in studies of adsorption and transport of molecules across membrane interfaces.     

Interfacial interaction between dextran sulfate and lipid monolayers: an electrochemical study

The interaction between dextran sulfate (DS) with zwitterionic dipalmitoylphosphatidylcholine (DPPC) and negatively charged dipalmitoylphosphatidic acid monolayers at different surface pressures at air-liquid and liquid-liquid interfaces was studied using Langmuir-Blodgett (LB) and electrochemical techniques. The negatively charged DS can bind to phospholipids via calcium ions. To investigate the mechanism of the adsorption of DS on lipid monolayers, compression isotherms (pi-A) and capacitance-potential curves were measured, and a theoretical model was developed to interpret the capacitance data. The compression of lipid monolayers in the presence of DS led to a more condensed hybrid layer, removing the LE-LC phase transition of DPPC. Lower surface pressures improved the binding of DS on the lipid monolayers via calcium bridges due to the electrostatic attraction. Alternating current voltammetry and cyclic voltammetry were used to monitor the transfer of a cationic beta-blocker (metoprolol) across lipid monolayers in the absence and presence of the polyelectrolyte and to compare with the transfer of the standard probe, tetraethylammonium cation. Results showed a strong dependence on (i) the surface pressure, (ii) the applied potential, and, (iii) in the case of the hybrid layer, the charge of the phospholipid headgroup. Finally, results were also confirmed by attenuated total reflection Fourier transform infrared spectroscopy, performed after transferring lipid multilayers onto a solid substrate by the LB method.     

The cis-bis(decanoate)tin phthalocyanine/DPPC film at the air/water interface

Films made of cis-bis-decanoate-tin(IV) phthalocyanine (PcSn10) and racemic dipalmitoylphosphatidylcholine (DPPC) are studied with compression isotherms and Brewster angle microscopy (BAM) at the air/water interface. Films enriched in PcSn10 present phase separation elliptical-shaped domains. These domains present optical anisotropy and molecular order. They are enriched in PcSn10, and the film outside these domains is enriched in DPPC, as shown in by high-angle annular dark-field transmission electron microscopy on Langmuir-Blodgett (LB) transferred films. Film collapse area and atomic force microscopy images of LB transferred films on mica indicate that the films are actually multilayers. A computational survey was performed to determine how the PcSn10 molecules prefer to self-assemble, in films basically made of PcSn10. The relative energetic stability for several dimeric assemblies was obtained, and a crystal model of the film was developed through packing and repeating the PcSn10 molecules, along the crystallographic directions of the unit cell. Our results contribute to understanding the strong interaction between PcSn10 and DPPC at the air/water interface, where even small quantities of DPPC (~1-2%) can modify the film in an important way.     

Studies on molecular structure of Schiff base derivatives in LB films

The present paper investigated the molecular configurations of the two novel amphiphiles with Schiff base moiety as headgroup in LB films. FTIR-ATR spectra and UV-vis electronic absorption spectra revealed the different isomers between the two amphiphiles LB film. Since z.sbnd;OH groups situate in different position of the aromic rings in Schiff base amphiphiles, the proton transfer by different ways leads to different isomers, which were reflected by monolayer and LB films behavior.     

Reorganization and caging of DPPC, DPPE, DPPG, and DPPS monolayers caused by dimethylsulfoxide observed using Brewster angle microscopy

The interaction between dimethylsulfoxide (DMSO) and phospholipid monolayers with different polar headgroups was studied using "in situ" Brewster angle microscopy (BAM) coupled to a Langmuir trough. For a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayer, DMSO was shown to significantly impact the structure of the liquid expanded (LE) and gaseous phases. The domains reorganized to much larger domain structures. Domains in the liquid condensed (LC) phase were formed on the DMSO-containing subphase at the mean molecular area where only gaseous and LE phases were previously observed on the pure water subphase. These results clearly demonstrate the condensing and caging effect of DMSO molecules on the DPPC monolayer. Similar effects were found on dipalmitoyl phosphatidyl ethanolamine, glycerol, and serine phospholipids, indicating that the condensing and caging effect is not dependent upon the phospholipid headgroup structure. The DMSO-induced condensing and caging effect is the molecular mechanism that may account for the enhanced permeability of membranes upon exposure to DMSO.     

Spectroscopic studies on molecular recognition capabilities of a nucleolipid bearing thymine headgroup to adenosine

The Langmuir-Blodgett (LB) films of octadecanoyl ester of 1-(2-carboxyethyl) thymine deposited from pure water and aqueous adenosine subphases were investigated by ultraviolet-visible (UV-vis), Fourier transform infrared-attenuated total reflection (FTIR-ATR), and Fourier transform surface-enhanced Raman scattering (FT-SERS) spectroscopy. The obtained spectral results indicate that the adenosine molecules in the subphase can be transferred onto solid substrates by LB techniques as a result of the formation of base pairs at the air/water interface. UV-vis spectra alternations indicated that, with increasing adenosine concentration in subphase, more adenosine molecules were recognized by nucleolipid monolayer and were transferred onto the quartz substrates. The closed-packing of the constituent molecules facilitates the photodimerization of the thymine moieties in the headgroup under ultraviolet irradiation. FTIR-ATR results suggest that the hydrocarbon chains of nucleolipid in the LB films deposited from pure water and aqueous adenosine take on a close-packed all trans conformation. By analyzing the FT-SERS spectra results, it can be deduced that the orientation of nucleobase in the headgroup is different before and after the recognition effect occurred. For LB film deposited from pure water, the nucleobases are lying flat on the silver substrates; whereas for LB film deposited from aqueous adenosine, the base pairs take an end-on adsorption on silver substrate.     

Surface forces in foam films from DPPC and lung surfactant phospholipid fraction

Surface properties of foam films formed from aqueous dispersions of dipalmitoyl-phosphatidylcholine (DPPC) and from solutions of a phospholipid fraction of lung surfactant (TPL) are studied employing the foam film method. Experiments are carried out within a wide range of NaCl concentrations (C_el) and the ranges of C_el determining formation of common films (CF), common black films (CBF) and bilayer Newton black films (NBF) are found. The thickness (h) of the CF and CBF decreases with the increase of C_el until the critical electrolyte concentrations (C_{el, cr}) is reached. The determined C_{el, cr} that characterize the transition to NBF show that C_{el, cr} of the TPL films is an order of magnitude higher than that of the DPPC films. The measured h of the TPL films is higher than that of the DPPC films in the whole C_el range. Besides, only the h(C_el) curve of the DPPC films outlines a metastable C_el range where both CF and NBF are obtained. Both the h(C_el) curves and the direct measurements of the disjoining pressure isotherms of the DPPC films (Π(h) isotherms) demonstrate the role of electrostatic repulsive forces for the stability of the phospholipid films The obtained results are compared with the DLVO theory equations and the evaluated potentials of the diffuse electric layer φ₀  20 mV for the DPPC films and φ₀  100 mV for TPL films show the strong effect of the charged phospholipids in the TPL mixture on the electric properties at the film interfaces.     

LB膜中的缺陷畴区及其诱导形成环形草酸钙图形

利用存在缺陷的LB膜来模拟受损伤的肾上皮细胞膜诱导肾结石矿物草酸钙(CaOxa)晶体生长. 经2.5 mmol·L-1的草酸钾溶液对二棕榈酰磷脂酰胆碱(DPPC)的LB膜进行处理后, 可强化LB膜中液态扩张相(LE)和液态凝聚相(LC)之间的结构差异, 原子力显微镜(AFM)证实了圈状缺陷的存在. 以LB膜的圈状缺陷为模板, 诱导了一水草酸钙(COM)晶体环状图案. 相比之下, 未经草酸钾处理的LB膜只诱导生成零散的六边形COM晶体.     

Direct investigation of the vectorization properties of amphiphilic cyclodextrins in phospholipid films

Recently, new cyclodextrin derivatives were synthesized and shown to exhibit strong amphiphilic properties. In this paper, we study the action of these new amphiphilic cyclodextrins on phospholipids. Mixed phospholipid/cyclodextrin derivative films were prepared and studied using X-ray reflectivity for various phospholipid/cyclodextrin ratios. A molar ratio of 3 provides a highly stable film the molecular structure of which has been investigated in detail. The cholesterol tail of the cyclodextrin molecule was found to be anchored into the phospholipid film. The cyclodextrin moieties exposed to the aqueous medium are prone to the addition of the guest molecule Dosulepin, making them of high interest for drug delivery. For this purpose and as an example of a potential application, this cyclodextrin molecular carrier property is also addressed to this complex film architecture.     

The interactions between cholesterol and phospholipids located in the inner leaflet of humane erythrocytes membrane (DPPE and DPPS) in binary and ternary films—The effect of sodium and calcium ions

The studies on the influence of cholesterol on phospholipids accumulated in inner leaflet of membrane are performed rather rarely, especially in the presence of electrolytes, which are present in membrane environment. Therefore, in this work the interactions between cholesterol and saturated phosphatidylethanolamine (PE) and phosphatidylserine (PS) were studied in binary (phospholipid/cholesterol) and ternary (PS/PE/cholesterol) monolayers in the presence and absence of sodium and calcium ions. The composition of ternary films was estimated to reflect the proportion of PSs to PEs in inner layer of human erythrocyte membrane. The influence of electrolytes on pure PS and PE films was also analyzed. It was found that both sodium and calcium ions affect the condensation of DPPS films, and influence the interactions in DPPS/cholesterol monolayers. On the other hand, no effect of these ions on DPPE films as well as on DPPE/cholesterol interactions in the mixed systems was observed. The results obtained for ternary mixtures prove that in the presence of Na⁺ the interactions between the lipids are more favorable than in the absence of these ions. This is in contrast to the effect of Ca²⁺. All the results were thoroughly analyzed in the context of the structure of polar heads of the investigated phospholipids.     

Mixed monolayers of Bauhinia monandra and concanavalin A lectins with phospholipids, part II

Isotherms of surface pressure and surface potential versus mean molecular area for dibehenoylphosphatidylcholine (DBPC), dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), and dioleoylphosphatidylcholine (DOPC) monolayers were shown to be greatly modified when these lipids were cospread with either Bauhinia monandra (BmoLL) or Concanavalin A (Con A) lectins. For the binary films of DBPC, DPPC, and DPPE cospread with each of these two lectins, there was both a displacement of the Pi-A and DeltaV-A isotherms toward higher molecular areas relative to pure lipids and an increase in the maximum surface potential values relative to the DeltaV-A relationships observed for the corresponding single-lectin systems. Both effects can be understood in terms of the occurrence of an explicit interaction between the lipids and the lectins. The plots of the corresponding compressibilities versus molecular areas reveal that, for all lipids but DOPC, the extent of this interaction was always larger for BmoLL than for Con A. The DPPC and DPPE mixed films with BmoLL differed in compressibility. Owing to the small DPPE polar headgroup, the DPPE-BmoLL film was much more incompressible than the DPPC-BmoLL mixed monolayer. However, for the DOPC-BmoLL and DOPC-Con A mixed films there was no evidence that an interaction between the lectins and the lipid took place, a fact attributed to the unsaturated character in the DOPC aliphatic chains, which leads to an expanded Pi-A isotherm.     

Influence of dipalmitoylphosphatidylcholine (or dioleoylphosphatidylcholine) and phospholipase A2 enzyme on the properties of emulsions

The properties of n-tetradecane emulsions with dipalmitoylphosphatidylcholine (DPPC) or dioleoylphosphatidylcholine (DOPC) in 1M ethanol were investigated at 20 and 37°C. The zwitterionic phospholipids having the same headgroup bound to the apolar tail composed of two saturated or unsaturated chains were used as stabilizing agents. Both phospholipids may self-organize into aggregates, which possess different sizes and surface affinities. Electrokinetic properties of the systems at natural pH or pH 8 were investigated taking into account the effective diameter of the droplets as well as the zeta potentials using the dynamic light scattering technique. The effect of both phospholipids decreases the initially negative zeta potential of the n-tetradecane emulsion and is more evident in the case of DPPC especially at a physiological temperature near its main temperature transition. The change of zeta potential by DOPC is visible at both temperatures probably as an effect of a loose packing of this phospholipid on n-tetradecane droplets, because of the presence of double bonds in its molecule. Also, the role of ethanol dipoles on the stability of oil/phospholipid emulsions is obvious. The other aim of paper was the characterization of the phospholipase A(2) influence on DOPC hydrolysis in the emulsion environment in order to emphasize the importance of such methodology. The present work is the first study that explores the effects of both electrolyte ions and ethanol molecules on DOPC hydrolysis by phospholipase. The effect of enzyme on the n-tetradecane/DOPC emulsions was investigated at pH 8 with Na(+) or Ca(2+) ions, which occur in the physiological fluids. The effective diameters do not always correlate with the zeta potentials. A possible reason of such behavior might a mechanism different from the electrostatic stabilization. The particular role of Ca(2+) ions in the emulsions with phospholipids was confirmed. Those investigations provide insight into the properties of the PLA(2) hydrolysis process enhanced by added ethanol. It is believed that the enzyme effect on the phospholipid aggregation behavior at the oil-water interface will be helpful for understanding other biological phenomena.     

Circular patterns of calcium oxalate crystals induced by defective Langmuir-Blodgett film

The injury of the renal epithelial cell membrane can promote the nucleation of nascent crystals, as well as adhesion of crystals on it. It thus accelerates the formation of renal calculi. In this paper, the defective Langmuir-Blodgett (LB) films were used as a model system to simulate the injured renal epithelial cell membrane. The microcosmic structure of the defective LB film and the molecular mechanism of the effect of this film on nucleation, growth, deposited patterns and adhesion of calcium oxalate monohydrate (COM) were investigated. The circular defective domains were formed in dipalmitoylphosphatidylcholine (DPPC) LB film after the film was treated by potassium oxalate. These domains could induce ring-shaped patterns of COM crystals. In comparison, the LB film without pretreatment by potassium oxalate only induced random growth of hexagonal COM crystals. As the crystallization time increased, the size of COM crystals in the patterns increased, the crystal patterns changed from empty circles to solid circles, and the number of the circular patterns with small size (5-20 μm) increased. The results would shed light on the molecular mechanism of urolithiasis induced by injury of the renal epithelial membrane at the molecular and supramolecular level.