Effects of block copolymer's architecture on its association with lipid membranes: experiments and simulations

Triblock copolymers of the form poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) have been shown to effectively interact with and restore activity of damaged cell membranes. To better understand the interaction between these polymers and cell membranes, we have modeled the outer leaflet of a cell membrane with a lipid monolayer spread at the air-water interface and injected poloxamers of varying architectures into the subphase beneath the monolayer. Subsequent interactions of the polymer with the monolayer upon compression were monitored with concurrent Langmuir isotherm and fluorescence microscopy measurements. Monte Carlo simulations were run in parallel using a coarse-grained model to capture interactions between lipids and poloxamers. Changing the ratio of the PEO to PPO block lengths (NPEO:NPPO) affects the equilibrium spreading pressure of the polymer. Poloxamers with a relatively longer central hydrophobic block are less soluble, resulting in more polymer adsorbed to the interface and therefore a higher equilibrium spreading pressure. Simulation results show that changing the poloxamer structure effectively affects its solubility. This is also reflected in the degree of lipid corralling as poloxamers with a higher chemical potential (and resulting higher equilibrium spreading pressure) cause the neighboring lipid domains to be more ordered. Upon lateral compression of the monolayers, the polymer is expelled from the film beyond a certain squeeze-out pressure. A poloxamer with a higher NPEO:NPPO ratio (with either NPEO or NPPO held constant in each series) has a lower squeeze-out pressure. Likewise when the total size of the polymer is varied with a constant hydrophilic:hydrophobic ratio, smaller poloxamers are squeezed out at a lower pressure. Our simulation results capture the trends of our experimental observations, both indicating how the interactions between lipids and poloxamers can be tuned by the polymer architecture.     

Interactions of Pluronics with phospholipid monolayers at the air-water interface

Pluronics are triblock copolymers which are extensively applied excipients shown to interact with cell membranes. The aim of our study was to apply monolayer techniques and epifluorescence microscopy to investigate the interaction behavior between selected Pluronics and phospholipid monolayers which serve as a model of cell membranes. The results showed that Pluronic L61 with hydrophobic proportions much larger than those of F68 demonstrated condensed film-like surface behavior while F68 exhibited more expanded behavior. The increments of surface pressure and the changes of image were more obvious in adding Pluronic L61 than F68 to the subphase of dipalmitoylphosphatidylcholine (DPPC) monolayers, which indicated that the interaction may be related to van der Waals forces and hydrophobic interaction. Pluronics selected with higher hydrophobicities demonstrated larger surface activities and penetration abilities while being added to the subphase of DPPC and dimyristoylphosphatidylcholine (DMPC) monolayers. Pluronic P85 and F68 were found to be squeezed to subphase at higher surface pressures, which may be attributed to their relatively higher hydrophilicities.     

Insertion mechanism of a poly(ethylene oxide)-poly(butylene oxide) block copolymer into a DPPC monolayer

Interactions between amphiphilic block copolymers and lipids are of medical interest for applications such as drug delivery and the restoration of damaged cell membranes. A series of monodisperse poly(ethylene oxide)-poly(butylene oxide) (EOBO) block copolymers were obtained with two ratios of hydrophilic/hydrophobic block lengths. We have explored the surface activity of EOBO at a clean interface and under 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayers as a simple cell membrane model. At the same subphase concentration, EOBO achieved higher equilibrium surface pressures under DPPC compared to a bare interface, and the surface activity was improved with longer poly(butylene oxide) blocks. Further investigation of the DPPC/EOBO monolayers showed that combined films exhibited similar surface rheology compared to pure DPPC at the same surface pressures. DPPC/EOBO phase separation was observed in fluorescently doped monolayers, and within the liquid-expanded liquid-condensed coexistence region for DPPC, EOBO did not drastically alter the liquid-condensed domain shapes. Grazing incidence X-ray diffraction (GIXD) and X-ray reflectivity (XRR) quantitatively confirmed that the lattice spacings and tilt of DPPC in lipid-rich regions of the monolayer were nearly equivalent to those of a pure DPPC monolayer at the same surface pressures.     

亚相pH对气水界面上VE/DPPC单分子膜的影响

通过表面压－分子面积等温线的测定，考察了亚相ｐＨ对气水界面上的维生素Ｅ（ＶＥ）／二棕榈酰基磷脂酰胆碱单分子膜的影响。亚相ｐＨ降低不改变ＤＰＰＣ单分子膜的崩裂压，但使ＶＥ单分子膜的崩裂压明显增大，不改变ＶＥ单分子膜的平均分子面积，但使ＤＰＰＣ单分子膜凝缩，低表面压下，ＶＥ对ＤＰＰＣ单分子膜的膨胀作用在纯水上很小，在ｐＨ为１的亚相上则很明显，这提示在低ｐＨ的亚相上，ＶＥ／ＤＰＰＣ单分子膜中的极性头基间     

Interfacial properties of Pluronics and the interactions between Pluronics and cholesterol/DPPC mixed monolayers

Pluronics are triblock copolymers of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) with wide range of hydrophilic-lipophilic balance. In order to investigate the relationship between the chemical structures of Pluronics and the interfacial properties at the air-water interface by monolayer techniques, Pluronics L61, P65, F68, P84, P123, L35, and P105 were selected. Since cholesterol influenced substantially the molecular packing stage and the characteristics of cell membranes, the interactions between Pluronics and model cell membranes in the absence and presence of cholesterol were compared. The results of pi-A isotherms and surface elasticities of Pluronic monolayers indicated that the first and second transition like stage were mainly affected by the numbers of EO and PO monomers, respectively. Pluronics with higher hydrophobicities demonstrated larger surface activities and penetration abilities to dipalmitoylphosphatidylcholine (DPPC) monolayers, which might be due to hydrophobic interactions and van der Waals forces. In the presence of cholesterol, hydrogen bonding effects was supposed to exist between the 3beta-hydroxy group of cholesterol and ether oxygen of PEO chains, which led Pluronic F68, with the longest PEO chain herein, to exhibit significantly higher penetration ability. Our findings proposed a theoretical basis for selection of optimized drug carriers and the starting point for further investigations.     

Dynamic elasticity of triblock copolymer of poly(ethylene oxide) and poly(propylene oxide) on a water surface

Dilatational viscoelasticity of adsorbed and spread films of the poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer at the air-water interface is studied by the capillary waves and oscillating barrier techniques. At the surface pressure below 10 mN/m, dynamic surface properties of these films coincide with those of poly(ethylene oxide). At higher surface pressures, the results obtained indicate the desorption of poly(propylene oxide) segments from the monolayer and their interaction with poly(ethylene oxide) segments in an aqueous phase. At a surface pressure close to 19 mN/m, the behavior of adsorbed and spread poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) films becomes different. The real part of dynamic surface elasticity of spread films tends toward its maximum value (20 mN/m) and, upon further compression, films begin to dissolve. At the same time, the surface elasticity of adsorbed films decreases nearly twofold upon the achievement of the maximum value that testifies the formation of looser structure of the surface layer.     

Pressure- and temperature-induced association of arborescent polystyrene-graft-poly(ethylene oxide) copolymers at the air-water interface

The influence of surface pressure and subphase temperature on the association of arborescent polystyrene- graft-poly(ethylene oxide) (PS- g-PEO) copolymers at the air-water interface was investigated using the Langmuir balance and atomic force microscopy (AFM) techniques. These dendritic molecules form stable condensed monolayers with surface compressional moduli >250 mN/m. The variation in film thickness observed as a function of surface pressure suggests that at low surface pressures (gaslike phase) the PEO chains remain adsorbed at the air-water interface. At higher surface pressures (condensed phase), the PEO chains partially desorb into the subphase and adopt a more brushlike conformation. Large islandlike clusters with a broad size distribution were observed for samples with PEO contents of up to 15% by weight. In contrast, copolymers with PEO contents of 22-43% displayed enhanced side-by-side association into ribbonlike superstructures upon compression. The same effect was observed even in the absence of compression when the subphase temperature was increased from 12 to 27 degrees C. The temperature-induced association was attributed to increased van der Waals attractive forces between the PS cores relative to the steric repulsive forces between PEO chains in the coronas because the solvent quality for the PEO segments decreased at higher temperatures. The restricted number of superstructures observed for arborescent copolymers as compared with linear- and star-branched PS-PEO block copolymers is attributed to the enhanced structural rigidity of the molecules due to branching.     

Halofantrine-phospholipid interactions: monolayer studies.

The antimalarial agent halofantrine penetrates dipalmitolylphosphatidylcholine (DPPC) monolayers resulting in an increase in surface pressure and an expansion in area occupied by the lipid components of the monolayer. This phenomenon is observed at concentrations (0.05-0.2 microm) of halofantrine that have no surface activity. Penetration increases with drug concentration and is greatest at low initial surface pressures of the monolayer. A critical surface pressure of the DPPC monolayer has been determined from constant area and constant pressure conditions. The magnitude of these values support the hypothesis that halofantrine readily penetrates the DPPC monolayers. The presence of cholesterol in the DPPC monolayer hampers penetration and a lower critical surface pressure is obtained under such conditions. Even then, a slower rate of penetration is observed only in monolayers maintained at high initial surface pressures (10, 15 mN/m), corresponding to the liquid condensed phase of the monolayer, and not at low surface pressures (2.5, 5.0 mN/m). These results help to give a better understanding of the dynamics of the halofantrine-phospholipid interaction as well as the pharmacodynamic character of the drug.     

Miscibility behavior of dipalmitoylphosphatidylcholine with a single-chain partially fluorinated amphiphile in Langmuir monolayers

Surface pressure-area, surface potential-area, and dipole moment-area isotherms were obtained for monolayers made from a partially fluorinated surfactant, (perfluorooctyl)undecyldimorpholinophosphate (F8H11DMP), dipalmitoylphosphatidylcholine (DPPC), and their combinations. Monolayers, spread on a 0.15 M NaCl subphase, were investigated at the air/water interface by the Wilhelmy method, ionizing electrode method, and fluorescence microscopy. Surface potentials were analyzed using the three-layer model proposed by Demchak and Fort. The contribution of the dimorpholinophosphate polar head group of F8H11DMP to the vertical component of the dipole moment was estimated to be 4.99 D. The linear variation of the phase transition pressure as a function of F8H11DMP molar fraction (X(F8H11DMP)) demonstrated that DPPC and F8H11DMP are miscible in the monolayer. This result was confirmed by deviations from the additivity rule observed when plotting the molecular areas and the surface potentials as a function of X(F8H11DMP) over the whole range of surface pressures investigated. Assuming a regular surface mixture, the Joos equation, which was used for the analysis of the collapse pressure of mixed monolayers, allowed calculation of the interaction parameter (xi=-1.3) and the energy of interaction (Delta epsilon =537 Jmol(-1)) between DPPC and F8H11DMP. The miscibility of DPPC and F8H11DMP within the monolayer was also supported by fluorescence microscopy. Examination of the observed flower-like patterns showed that F8H11DMP favors dissolution of the ordered LC phase domains of DPPC, a feature that may be key to the use of phospholipid preparations as lung surfactants.     

Nanostructured latex films from poly(butyl methacrylate) latex cross-linked with poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene oxide) triblock macro-cross-linker

Biphasic polymer latexes were synthesized by a seeded swelling and polymerization method. The latexes were composed of a poly(butyl methacrylate) core and a poly(ethylene oxide) rich shell cross-linked with poly(ethylene oxide)-poly (propylene oxide)-poly(ethylene oxide) triblock diol diacrylate macro-cross-linker. Nanostructured films were obtained by annealing the biphasic polymer latexes at a temperature between the glass-transition temperatures of the core latex and the cross-linked poly(ethylene oxide) based shell. Atomic force microscope images of the latex film revealed that the poly(butyl methacrylate) core phase is confined in the poly(ethylene oxide)-rich continuous phase with the form of separate nanosized spheres.     

Effect of temperature on the interfacial behavior of a polystyrene-b-poly(methyl methacrylate) diblock copolymer at the air/water interface

Monolayers of a polystyrene-poly(methyl methacrylate) (PS-PMMA) diblock copolymer at the air-water interface were studied by measuring the surface pressure-area isotherms at several temperatures. Langmuir film balance experiments and atomic force microscopy showed that the diblock copolymer molecules formed surface micelles. In the plot of the surface pressure versus surface area per repeating unit, the monolayer changed from the gas phase to the liquid expanded phase at lower surface pressure for systems at low temperature compared to those at high temperature. In addition, a plateau, corresponding to the transition from the liquid expanded to liquid condensed phase, appeared in that plot at lower surface pressure for systems with a higher subphase (water) temperature. Hysteresis was observed in the compression-expansion cycle process. Increasing the subphase temperature alleviated this hyteresis gap, especially at low surface pressures. The minimum in the plot of the surface pressure versus surface area per repeating unit in the expansion process (which arises from the transition) and the transition plateau appeared more vividly at higher water temperature. These dynamic experimental results show that PS-PMMA diblock copolymers, in which both blocks are insoluble in water, do not form complicated entanglements in two-dimensional space. Although higher water temperature provided more entropy to the chains, and thus more conformational freedom, it did not change the surface morphology of the condensed film because both blocks of PS-PMMA are insoluble in water.     

Continuous synthesis of CdSe(x)Te(1-x) nanocrystals: chemical composition gradient and single-step capping

Thermosensitive poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymer, Pluronic F68, containing a hydrophobic unit, oligo-(lactic acid)(oligo-LA) or oligo-caprolactone (oligo-CL), 2-META and RGD as side groups was successfully synthesized and characterized by (1)H NMR, FTIR, and elemental analysis. Their aqueous solution displayed special gel-sol-gel phase transition behavior with increasing temperature from 10 to 70°C, when the polymer concentration was above critical micelle concentration (CMC). The gel-sol phase diagram was investigated using tube inversion method, rheological measurement, and dynamic light scattering. Based on these results, the gelation properties of modified F68 were affected by several factors such as the composition of the substituents, chain length of oligo L-LA or oligo ε-CL, and the concentration of the polymer solutions. The unique phase transition behavior with temperature was observed by modified F68 triblock copolymer, composed of the PPO blocks core and the PEO blocks shell in aqueous solution. This phenomenon was elucidated using (1)H NMR data; the alteration of hydrophobic interaction and chain mobility led to the formation of transparent gel, coexistence of gel-sol, and opaque gel. These hydrogels may be useful in drug delivery and tissue engineering.     

髓鞘碱性蛋白对细胞膜脂质分子DPPC、DPPE单层膜影响机理研究

本文通过Langmuir单层膜的表面压力-平均分子面积(π-A)曲线的测定与分析,分别对髓鞘碱性蛋白(MBP)与细胞膜中不同头部基团脂质分子二棕榈酰基磷脂胆碱(DPPC)和二棕榈酰基磷脂酰乙醇胺(DPPE)在空气/液体界面上的相互作用过程进行了系统研究.实验结果表明:(1)当界面上脂质含量一定时,亚相中随着MBP浓度的增大,DPPC、DPPE单层膜的等温线向平均分子面积较大的方向移动;(2)在单层膜表面压力为10 mN/m时,一个MBP分子分别结合140±3个DPPC分子和100±3个DPPE分子,随着表面压力增大,当MBP分子分别与两种磷脂分子相互作用时,MBP插入到磷脂单层界面的个数逐渐减少;(3)随着蛋白质浓度的增加,脂分子形成的单层膜变得较为疏松,且MBP分子易于插入到分子头部较小的DPPE单层膜中;(4)蛋白质的存在使DPPC单层膜的表面压力逐渐减小,且蛋白质浓度越大表面压力降低越多,DPPC被MBP带入到亚相中越多;(5)对于DPPE单层膜,蛋白质通过与DPPE相互作用插入到界面膜中,引起表面压力增大,且蛋白质浓度越高,压力变化量越大.     

Mixed DPPC/DPTAP monolayers at the air/water interface: influence of indolilo-3-acetic acid and selenate ions on the monolayer morphology

The interactions of mixed monolayers of two lipids, zwitterionic 1,2-dipalmitoyl-phosphatidylcholine (DPPC) and positively charged 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), with phytohormone indolilo-3-acetic acid (IAA) and selenate anions in the aqueous subphase were studied. For this purpose, isotherms of the surface pressure versus the mean molecular area were recorded. Domain formation was investigated by using Brewster angle microscopy (BAM). The method of grazing incidence X-ray diffraction (GIXD) was also applied for the characterization of the organization of lipid molecules in condensed monolayers. It was found that selenate ions contribute to monolayer condensation by neutralizing the positive net charge of mixed monolayers whereas IAA molecules penetrated the lipid monolayer, causing its expansion/fluidization. When both solutes were introduced into the subphase, a competition between them for interaction with the positively charged lipids in the monolayer was observed.     

Temperature,concentration and salt effect on F68 tri-block copolymer in aqueous solution: Rheological study

The rheology of the aqueous solution of poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) (PEO?PPO?PEO) triblock copolymer, Pluronic F68 in the presence of KF was studied in the temperature range from 15 to 60°C. The variation of the shear stress according to the shear rate shows that independently from the temperature and concentration, the F68 solutions exhibit a Newtonian behavior. The results show that the Critical Micelle Temperature of Pluronic F68 in a KF aqueous solution decreases with the increase in the salt concentration.     

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.     

Salt induced micellization of very hydrophilic PEO-PPO-PEO block copolymers in aqueous solutions

Symmetrical poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide), PEO-PPO-PEO, triblock copolymers with 80% polyethylene oxide (PEO, the hydrophilic end blocks) and polypropylene oxide (PPO, the hydrophobic middle block) usually remain as molecularly dissolved at ambient temperature even at fairly high-concentrations (2 wt.% or more). However, the micellization is induced at lower concentration/temperature in the presence of salts. The results on salt induced micellization from four such hydrophilic copolymers Pluronic^® F38, F68, F88 and F108 obtained from several independent techniques are described. FTIR and fluorescence results provide essentially identical critical micelle temperatures (CMTs) showing marked decrease with increase in PPO molecular weight and in the presence of salt. These copolymers were weakly surface active and did not show a clear break point in surface tension concentration plot typical of surfactants. While addition of salt decreases the cloud point, no significant micelle growth was observed even at temperature close to cloud point (CP). Marked increased in solubilization of an oil dye was observed in presence of KCl. Different methods showed good agreement in temperature/salt-induced micellization of these hydrophilic copolymers.     

Surface rheology of PEO-PPO-PEO triblock copolymers at the air-water interface: comparison of spread and adsorbed layers

The dilatational rheological properties of monolayers of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide)-type block copolymers at the air-water interface have been investigated by employing an oscillating ring trough method. The properties of adsorbed monolayers were compared to spread layers over a range of surface concentrations. The studied polymers were PEO26-PPO39-PEO26 (P85), PEO103-PPO40-PEO103 (F88), and PEO99-PPO65-PEO99 (F127). Thus, two of the polymers have similar PPO block size and two of them have similar PEO block size, which allows us to draw conclusions about the relationship between molecular structure and surface dilatational rheology. The dilatational properties of adsorbed monolayers were investigated as a function of time and bulk solution concentration. The time dependence was found to be rather complex, reflecting structural changes in the layer. When the dilatational modulus measured at different concentrations was replotted as a function of surface pressure, one unique master curve was obtained for each polymer. It was found that the dilatational behavior of spread (Langmuir) and adsorbed (Gibbs) monolayers of the same polymer is close to identical up to surface concentrations of approximately 0.7 mg/m2. At higher coverage, the properties are qualitatively alike with respect to dilatational modulus, although some differences are noticeable. Relaxation processes take place mainly within the interfacial layers by a redistribution of polymer segments. Several conformational transitions were shown to occur as the area per molecule decreased. PEO desorbs significantly from the interface at segmental areas below 20 A(2), while at higher surface coverage, we propose that segments of PPO are forced to leave the interface to form a mixed sublayer in the aqueous region.     

Nonequilibrium 2-hydroxyoctadecanoic acid monolayers: effect of electrolytes

2-Hydroxyacids display complex monolayer phase behavior due to the additional hydrogen bonding afforded by the presence of the second hydroxy group. The placement of this group at the position α to the carboxylic acid functionality also introduces the possibility of chelation, a utility important in crystallization including biomineralization. Biomineralization, like many biological processes, is inherently a nonequilibrium process. The nonequilibrium monolayer phase behavior of 2-hydroxyoctadecanoic acid was investigated on each of pure water, calcium chloride, sodium bicarbonate and calcium carbonate crystallizing subphases as a precursor study to a model calcium carbonate biomineralizing system, each at a pH of ～6. The role of the bicarbonate co-ion in manipulating the monolayer structure was determined by comparison with monolayer phase behavior on a sodium chloride subphase. Monolayer phase behavior was probed using surface pressure/area isotherms, surface potential, Brewster angle microscopy, and synchrotron-based grazing incidence X-ray diffraction and X-ray reflectivity. Complex phase behavior was observed for all but the sodium chloride subphase with hydrogen bonding, electrostatic and steric effects defining the symmetry of the monolayer. On a pure water subphase hydrogen bonding dominates with three phases coexisting at low pressures. Introduction of calcium ions into the aqueous subphase ensures strong cation binding to the surfactant head groups through chelation. The monolayer becomes very unstable in the presence of bicarbonate ions within the subphase due to short-range hydrogen bonding interactions between the monolayer and bicarbonate ions facilitated by the sodium cation enhancing surfactant solubility. The combined effects of electrostatics and hydrogen bonding are observed on the calcium carbonate crystallizing subphase.     

Surface phase behavior in Gibbs monolayers of bis(ethylene glycol) mono-n-tetradecyl ether at the air-water interface

We present the adsorption kinetics and surface morphology of the adsorbed monolayers of bis(ethylene glycol) mono-n-tetradecyl ether (C14E2) by film balance and Brewster angle microscopy. A cusp point followed by a plateau region in the pressure (pi)-time (t) adsorption isotherm indicates a first-order phase transition in the coexistence region between a lower density liquid expanded (LE) phase and a higher density liquid condensed (LC) phase. A variety of condensed phase domains surrounded by the homogeneous LE phase are observed just after the appearance of the phase transition. The domains are of a spiral or striplike structure at lower temperatures. This characteristic shape of the domains is because of strong dipole-dipole repulsion between the molecules. At 18 degrees C, the domains are found to be quadrant structures. A slight increase in subphase temperature (around 1 degrees C) brings about a quadrant-to-circular shape transition in the domains. The circular domains return to quadrant structures as the subphase temperature is lowered. The domains completely disappear when the temperature is increased beyond 19 degrees C, suggesting that the critical temperature for the condensed domain formation is 19 degrees C. Above this temperature, the hypothetical surface pressure necessary for the phase transition exceeds the actual surface pressure attainable from a solution of concentration greater than or equal to the critical micelle concentration. An increase in molecular motion with increasing temperature results in a higher degree of chain flexibility. As a result, the molecules cannot accumulate in the condensed phase form when the subphase temperature is above 19 degrees C.