RESUMO
The solubilization of poorly water-soluble progesterone derivatives into micelles of a gemini surfactant was investigated in an aqueous medium. The aqueous solubility at different temperatures was determined spectroscopically using an ultraviolet visible light spectrophotometer. Thermodynamic parameters for the solubilization were calculated under the basis of the solubility change against temperature. The solubility of progesterone was quite low and remained constant below the critical micelle concentration (cmc) of the surfactant. On the other hand, the solubility increased considerably with increasing surfactant concentration above the cmc. It was suggested that the solubilizates were located in the vicinity of polar regions of micelles.
Assuntos
Micelas , Progesterona/química , Tensoativos , Solubilidade , Soluções , Espectrofotometria Ultravioleta , Temperatura , Termodinâmica , ÁguaRESUMO
The high costs of artificial pulmonary surfactants, ranging in hundreds per kilogram of body weight, used for treating the respiratory distress syndrome (RDS) premature babies have limited their applications. We have extensively studied soy lecithins and higher alcohols as lipid alternatives to expensive phospholipids such as DPPC and PG. As a substitute for the proteins, we have synthesized the peptide Hel 13-5D3 by introducing D-amino acids into a highly lipid-soluble, basic amphiphilic peptide, Hel 13-5, composed of 18 amino acid residues. Analysis of the surfactant activities of lipid-amphiphilic artificial peptide mixtures using lung-irrigated rat models revealed that a mixture (Murosurf SLPD3) of dehydrogenated soy lecithin, fractionated soy lecithin, palmitic acid (PA), and peptide Hel 13-5D3 (40:40:17.5:2.5, by weight) superior pulmonary surfactant activity than a commercially available pulmonary surfactant (beractant, Surfacten®). Experiments using ovalbumin-sensitized model animals revealed that the lipid-amphiphilic artificial peptide mixtures provided significant control over an increase in the pulmonary resistance induced by premature allergy reaction and reduced the number of acidocytes and neutrophils in lung-irrigated solution. The newly developed low-cost pulmonary surfactant system may be used for treatment of a wide variety of respiratory diseases.
Assuntos
Aminoácidos/metabolismo , Asma/tratamento farmacológico , Hemólise/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Peptídeos/farmacologia , Surfactantes Pulmonares/farmacologia , Animais , Dicroísmo Circular , Interações Hidrofóbicas e Hidrofílicas , Pulmão/patologia , Peptídeos/química , Ratos , Ratos Endogâmicos BN , Ratos WistarRESUMO
Pulmonary surfactant model peptide, Hel 13-5, in binary and ternary lipid mixtures has been characterized employing the polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS) in situ at the air-water interface for a monolayer state and the polarized ATR-FTIR for a bilayer film. In the bilayer form, Hel 13-5 predominantly adopts an α-helical secondary structure in the lipid mixtures. It had been made clear from CD measurements that the Hel 13-5 structure is mainly in the α-helical form in aqueous solutions. In the monolayer state, however, the secondary structure of Hel 13-5 exhibits an interconversion of the α-helix into ß-sheet with increasing surface pressures. The difference in the secondary structure is attributed to formation of a surface-associated reservoir just below the surface monolayer. The reservoir formation is a key function of pulmonary surfactants and is induced by a squeeze-out of the fluid components in their monolayers. Compression and expansion cycles of the monolayers generate a hysteresis in molecular orientation of the lipid monolayer as well as in peptide structure. The formation and deformation of reservoirs are, in common, deeply related to the hysteresis behavior. Thus, the transition of peptide structures across the interface is a quite important matter to clarify the role and its mechanism of the reservoirs in pulmonary functions. The present study primarily reveals roles of the anionic lipids in control of the peptide secondary structure. Accordingly, it is demonstrated that they prevent the protein structure transition from α-helix into ß-sheet by incorporating the peptide during the squeeze-out event.
Assuntos
Peptídeos/química , Surfactantes Pulmonares/química , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Sequência de Aminoácidos , Dados de Sequência Molecular , Estrutura Secundária de ProteínaRESUMO
Effective additives to pulmonary surfactant (PS) preparations for therapy of respiratory distress syndrome (RDS) are being intensively sought. We report here the investigation of the effects of partially fluorinated amphiphiles (PFA) on the surface behavior of a model PS formulation. When small amounts of a partially fluorinated alcohol C(8)F(17)C(m)H(2m)OH (F8HmOH, m = 5 and 11) are added to the PS model preparation (a dipalmitoylphosphatidylcholine (DPPC)/Hel 13-5 peptide mixture) considered here, the effectiveness of the latter in in vitro pulmonary functions is enhanced. The mechanism for the improved efficacy depends on the hydrophobic chain length of the added PFA molecules. The shorter PFA, F8H5OH, when incorporated in the monolayer of the PS model preparation, promotes a disordered liquid-expanded (LE) phase upon lateral compression (fluidization). In contrast, the addition of the longer PFA, F8H11OH, reduces the disordered LE/ordered liquid-condensed (LC) phase transition pressure and promotes the growth of ordered domains (solidification). Furthermore, compression-expansion cycles suggest that F8H5OH, when incorporated in the PS model preparation, undergoes an irreversible elimination into the subphase, whereas F8H11OH enhances the squeeze-out phenomenon of the SP-B mimicking peptide, which is important in pulmonary functions and is related to the formation of a solid-like monolayer at the surface and of a surface reservoir just below the surface. F8H11OH particularly reinforces the effectiveness of DPPC in terms of minimum reachable surface tension, and of preservation of the integrated hysteresis area between compression and expansion isotherms, the two latter parameters being generally accepted indices for assessing PS efficacy. We suggest that PFA amphiphiles may be useful potential additives for synthetic PS preparations destined for treatment of RDS in premature infants and in adults.
Assuntos
Fluorocarbonos/uso terapêutico , Surfactantes Pulmonares/química , 1,2-Dipalmitoilfosfatidilcolina/química , Adulto , Materiais Biocompatíveis/química , Biofísica , Desenho de Fármacos , Humanos , Recém-Nascido , Teste de Materiais , Microscopia de Força Atômica/métodos , Microscopia de Fluorescência/métodos , Peptídeos/química , Síndrome do Desconforto Respiratório/tratamento farmacológico , Propriedades de SuperfícieRESUMO
In situ polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) at the air-water interface has been used to determine secondary structure of the pulmonary surfactant model peptide, Hel 13-5, in the absence and the presence of phospholipid monolayers. Herein, fully saturated phospholipids of DPPC and DPPG are utilized to understand the effect of specific interaction between anionic DPPG and cationic Hel 13-5 on the peptide secondary structure. The spectrum frequency in the amide region (1500-1700cm(-1)) obtained from PM-IRRAS has been confirmed by comparing with that from ATR-FTIR for the corresponding bulk films. The PM-IRRAS spectra of single Hel 13-5 monolayers indicate the alpha-helical contour in the amide region, which coincides with the result from CD measurements in aqueous solutions. In the presence of phospholipid monolayers, however, Hel 13-5 changes its conformation from the alpha-helix to the extended beta-sheet as surface pressure increases upon compression at the interface, and this interconversion is found to be irreversible even during expansion process of monolayers. Furthermore, it is notable that the electrostatic interaction between DPPG and Hel 13-5 inhibits to some extent the interconversion to the beta-sheet during compression. These features are completely different from the bulk behavior, which demonstrates different roles of native proteins in the bulk phase and at the interface for pulmonary functions. In addition, the conformational variation of Hel 13-5 does not indicate close correlation with surface activity, which is common characteristic even for reversible hysteresis curves in pulmonary surfactant systems. This suggests that the secondary structure of native proteins is not strongly related to the surface activity during respiration. This work contributes to secondary structure determination of Hel 13-5 in the phospholipid domains in situ at the air-water interface and will provide insight into the molecular and physiological mechanism for SP-B and SP-C actions across the interface.
Assuntos
Membranas Artificiais , Peptídeos/química , Fosfolipídeos/química , Surfactantes Pulmonares/química , Estrutura Secundária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier/métodosRESUMO
Interfacial behavior was studied in pulmonary surfactant model systems containing an amphiphilic alpha-helical peptide (Hel 13-5), which consists of 13 hydrophobic and five hydrophilic amino acid residues. Fully saturated phospholipids of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) were utilized to understand specific interactions between anionic DPPG and cationic Hel 13-5 for pulmonary functions. Surface pressure (pi)-molecular area (A) and surface potential (DeltaV)-A isotherms of DPPG/Hel 13-5 and DPPC/DPPG (4:1, mol/mol)/Hel 13-5 preparations were measured to obtain basic information on the phase behavior under compression and expansion processes. The interaction leads to a variation in squeeze-out surface pressures against a mole fraction of Hel 13-5, where Hel 13-5 is eliminated from the surface on compression. The phase behavior was visualized by means of Brewster angle microscopy, fluorescence microscopy, and atomic force microscopy. At low surface pressures, the formation of differently ordered domains in size and shape is induced by electrostatic interactions. The domains independently grow upon compression to high surface pressures, especially in the DPPG/Hel 13-5 system. Under the further compression process, protrusion masses are formed in AFM images in the vicinity of squeeze-out pressures. The protrusion masses, which are attributed to the squeezed-out Hel 13-5, grow larger in lateral size with increasing DPPG content in phospholipid compositions. During subsequent expansion up to 35 mN m(-1), the protrusions retain their height and lateral diameter for the DPPG/Hel 13-5 system, whereas the protrusions become smaller for the DPPC/Hel 13-5 and DPPC/DPPG/Hel 13-5 systems due to a reentrance of the ejected Hel 13-5 into the surface. In this work we detected for the first time, to our knowledge, a remarkably large hysteresis loop for cyclic DeltaV-A isotherms of the binary DPPG/Hel 13-5 preparation. This exciting phenomenon suggests that the specific interaction triggers two completely independent processes for Hel 13-5 during repeated compression and expansion: 1), squeezing-out into the subsolution; and 2), and close packing as a monolayer with DPPG at the interface. These characteristic processes are also strongly supported by atomic force microscopy observations. The data presented here provide complementary information on the mechanism and importance of the specific interaction between the phosphatidylglycerol headgroup and the polarized moiety of native surfactant protein B for biophysical functions of pulmonary surfactants.
Assuntos
1,2-Dipalmitoilfosfatidilcolina/química , Peptídeos/química , Fosfatidilgliceróis/química , Surfactantes Pulmonares/química , Microscopia de Força Atômica , Microscopia de Fluorescência , Modelos Biológicos , Pressão , Conformação Proteica , Eletricidade EstáticaRESUMO
The artificial pulmonary surfactant composition in the present study is characterized by a lipid mixture system composed of higher aliphatic alcohol, egg yolk phosphatidylcholine (egg PC), soy lecithin and higher aliphatic acid as the major components or a peptide-lipid mixture system composed of a combination of the lipid mixture system to which a peptide is added. Three peptides with amphiphilic surface-staying, membrane spanning, and both properties were designed and synthesized. The evaluation of pulmonary surfactant assay was performed by a hysteresis curve drawn upon the measurement for the surface tension-area curve with the Wilhelmy surface tensometer in vitro and the recovery of lung compliance for the pulmonary surfactant-deficient rat models in vivo. Lipid-mixture systems composed of octadecanol or soy lecithins containing no peptide were favorable hysteresis curves as compared with commercially available Surfacten, but were not prominent. The peptide-lipid mixture systems composed of a combination of the lipid mixture of alkyl alcohol or soy lecithin to which peptides designed were added were desirable hysteresis curves similar to Surfacten and amphiphilic Hel 13-5 peptide-lipids mixture systems were much more effective than the lipid mixture system. Particularly, the recovery of lung compliance treated with hydrogenated soy lecithin-fractionated soy lecithin PC70-palmitic acid-peptide Hel 13-5 (40:40:17.5:2.5, w/w) was comparable to that with Surfacten. Because the artificial pulmonary surfactant compositions of this study can be prepared at lower costs, they are useful for the treatment of respiratory distress syndrome and acute respiratory distress syndrome as well as for inflammatory pulmonary diseases, dyspnea caused by asthma, etc.
Assuntos
Álcoois/química , Lecitinas/química , Lipídeos/química , Peptídeos/química , Surfactantes Pulmonares/administração & dosagem , Surfactantes Pulmonares/síntese química , Síndrome do Desconforto Respiratório/tratamento farmacológico , Animais , Modelos Animais de Doenças , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Injeções Intraperitoneais , Instilação de Medicamentos , Surfactantes Pulmonares/economia , Ratos , Ratos Wistar , Testes de Função Respiratória , Glycine max/química , Tensão Superficial , Fatores de TempoRESUMO
Interfacial behavior was studied on the pulmonary lipid mixture containing a newly designed amphiphilic alpha-helical peptide (Hel 13-5) that consists of 13 hydrophobic and 5 hydrophilic amino acid residues. Moreover, the data obtained were compared with those of commercially available Surfacten (Surfactant TA) which has been clinically used for neonatal respiratory distress syndrome (NRDS) in Japan. Surface pressure (pi)-A and surface potential (DeltaV)-area (A) isotherms were measured for our synthetic preparations and Surfacten. Herein, a mixture of dipalmitoylphosphatidylcholine (DPPC)/egg-phosphatidylglycerol (PG)/palmitic acid (PA) (68:22:9 by weight) was used as the constituent of basic preparations. Monolayers were spread on 0.02 M Tris buffer (pH 7.4) with 0.13 M NaCl at the air/liquid interface, and the surface behavior was investigated by employing the Wilhelmy method, an ionizing electrode method, and fluorescence microscopy (FM). Cyclic compression and expansion isotherms of the prepared materials (or products) (DPPC/PG/PA/Hel 13-5) were examined to confirm the spreading and respreading ability. For the prepared products, a plateau region exists on pi-A and DeltaV-A isotherms at approximately 42 mN m(-1), indicating that Hel 13-5 is squeezed out of surface monolayers together with fluid components (PG) upon lateral compression. That is, the squeeze-out phenomenon induces a 2D-3D phase transformation. In particular, the inclination of the pi-A isotherms at X(Hel 13-5) = 0.1 in the plateau region was almost zero irrespective of the molecular area. As proposed in the earlier report (Nakahara, H.; Lee, S.; Sugihara, G.; Shibata, O. Langmuir 2006, 22, 5792-5803), an observed refluorescence phenomenon was discussed for FM measurements. This phenomenon provides evidence of the squeeze-out motion with fluid molecules. Furthermore, the cyclic pi-A and DeltaV-A isotherms show larger hysteresis areas and better respreading abilities in comparison with the previous ternary systems (DPPC/PG/Hel 13-5 and DPPC/PA/Hel 13-5) that are very important properties in pulmonary functions. FM photographs and the temperature dependence of pi-A and DeltaV-A isotherms suggest that the phase behavior of the present preparation product is very similar to that of Surfacten in terms of the domain size and in parameters such as collapse pressures, maximum DeltaV values, and so on. These results demonstrate that PG and PA even in the present preparations work well for compression-expansion cycling as is the case in the previous ternary systems, and the present preparations show comparable properties to Surfacten in vitro.
Assuntos
Produtos Biológicos/química , Lipídeos/química , Peptídeos/química , Surfactantes Pulmonares/química , 1,2-Dipalmitoilfosfatidilcolina , Ar , Modelos Biológicos , Ácido Palmítico , Transição de Fase , Fosfatidilgliceróis , Propriedades de Superfície , ÁguaRESUMO
Solubilization of benzene, toluene, ethylbenzene, n-propylbenzene, n-butylbenzene, and n-pentylbenzene into the micelles of octaethylene glycol monotetradecyl ether (C(14)E(8)) was studied, where equilibrium concentrations of all the solubilizates were determined spectrophotometrically at 298.2, 303.2, and 308.2 K. The concentration of the above solubilizates except benzene remained constant below the critical micelle concentration (cmc) and increased linearly with an increase in C(14)E(8) concentration above the cmc, whereas benzene concentration was found to remain constant over the whole concentration range of C(14)E(8). The Gibbs energy change (DeltaG(0)) for their solubilization was evaluated by the partitioning of the solubilizates between the aqueous phase and the micellar phase because of the large aggregation number of the C(14)E(8) micelle. Furthermore, enthalpy and entropy changes for their solubilization were evaluated from the temperature dependence of the DeltaG(0) values. From these thermodynamical parameters and the change in absorption spectra of the solubilizates due to their incorporation into the micelles, the solubilization site was found to move into the inner core of the micelle with increasing alkyl chain length of the solubilizates.
Assuntos
Derivados de Benzeno/química , Detergentes/química , Polietilenoglicóis/química , Alquilação , Carbono/química , Micelas , Solubilidade , Espectrofotometria Ultravioleta , TermodinâmicaRESUMO
Surface pressure (pi)-, surface potential (DeltaV)-, dipole moment (mu( perpendicular))-area (A) isotherms and morphological behavior at the air-water interface were obtained for multicomponent monolayers of two different systems for dipalmitoylphosphatidylcholine (DPPC)/egg-phosphatidylglycerol (PG) (= 68:22, by weight)/Hel 13-5 and DPPC/palmitic acid (PA) (= 90:9, by weight)/Hel 13-5 (Hel 13-5 is a newly designed 18-mer amphiphilic alpha-helical peptide with 13 hydrophobic and 5 hydrophilic amino acid residues). The phase behavior of these model systems was investigated on a subsolution of 0.02 M tris(hydroxymethyl)aminomethane (Tris) buffer (pH 8.4) with 0.13 M NaCl at 298.2 K by employing the Wilhelmy method, the ionizing electrode method, and fluorescence microscopy. Especially, the present study focuses on the interfacial effect of the addition of Hel 13-5 on two binary systems, DPPC/egg-PG and DPPC/PA monolayers, as the substitute for pulmonary surfactant proteins, and on the respective roles of PG and PA for the monolayers in the three-component systems. Constant kink points ( approximately 42 mN m(-1)) clearly appear on the pi-A isotherms, independent of the compositions in the ternary systems, which corresponds to the Hel 13-5 collapse pressure similar to that of SP-B and SP-C as functions in multicomponent monolayers. This implies that Hel 13-5 is squeezed out of ternary monolayers above approximately 42 mN m(-1), resulting in two- to three-dimensional phase transformation. Furthermore, Langmuir isotherms clearly show that Hel 13-5 with egg-PG is squeezed out of the DPPC/egg-PG/Hel 13-5 system, whereas only Hel 13-5 is squeezed out of the DPPC/PA/Hel 13-5 system. Cyclic compression and expansion isotherms of these systems were carried out to confirm the spreading and respreading capacities. In addition, the interfacial behavior of the ternary mixtures has been analyzed by the additivity rule. Morphological examinations and comparisons have verified the interactions of Hel 13-5 with the representative miscible mixture (DPPC/PA system) by fluorescence microscopy. Consequently, distinct morphological variations corresponding to the squeeze-out behavior are observed as a fluorescent contrast recovery. Herein, a new mechanism of the refluorescent phenomenon is proposed by varying the surface composition of Hel 13-5.
Assuntos
1,2-Dipalmitoilfosfatidilcolina/química , Ácido Palmítico/química , Peptídeos/química , Fosfatidilgliceróis/química , Ar , Sequência de Aminoácidos , Interações Hidrofóbicas e Hidrofílicas , Substâncias Macromoleculares/química , Microscopia de Fluorescência , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Tensoativos/química , Termodinâmica , ÁguaRESUMO
Surface pressure (pi)-, surface potential (deltaV)-, and dipole moment (mu(perpendicular))-area (A) isotherms and morphological behavior were examined for monolayers of a newly designed 18-mer amphiphilic alpha-helical peptide (Hel 13-5), DPPC, and DPPC/egg-PC (1:1) and their combinations by the Wilhelmy method, ionizing electrode method, fluorescence microscopy (FM), and atomic force microscopy (AFM). The newly designed Hel 13-5 showed rapid adsorption into the air-liquid interface to form interfacial films such as a SP-B function. Regardless of the composition and constituents in their multicomponent system of DPPC/egg-PC, the collapse pressure (pi(c); approximately 42 mN m(-1)) was constant, implying that Hel 13-5 with the fluid composition of egg-PC is squeezed out of Hel 13-5/DPPC/egg-PC monolayers accompanying a two- to three-dimensional phase transformation. FM showed that adding a small amount of Hel 13-5 to DPPC induced a dispersed pattern of ordered domains with a "moth-eaten" appearance, whereas shrinkage of ordered domains in size occurred for the DPPC/egg-PC mixture with Hel 13-5. Furthermore, AFM indicated that (i) the intermediate phase was formed in pure Hel 13-5 systems between monolayer states and excluded nanoparticles, (ii) protrusions necessarily located on DPPC monolayers, and (iii) beyond the collapse pressure of Hel 13-5, Hel 13-5 was squeezed out of the system into the aqueous subphase. Furthermore, hysteresis curves of these systems nicely resemble those of the DPPC/SP-B and DPPC/SP-C mixtures reported before.
Assuntos
Ar , Peptídeos/química , Fosfatidilcolinas/química , Água/química , Adsorção , Sequência de Aminoácidos , Microscopia de Força Atômica , Microscopia de Fluorescência , Dados de Sequência MolecularRESUMO
Evaporation rates were determined for water-ethylene glycol liquid mixtures with different mole fractions, where the evaporation rate expressed as mg min(-1)/area was used because of the presence of two kinds of molecular species. The rate increased with increasing temperature and decreased with increasing mole fraction of ethylene glycol, almost obeying ideal mixing of the two components, although a small positive deviation was observed over the mole fraction from 0 to 0.5 of ethylene glycol at higher temperatures. The activation energy of evaporation was determined from the temperature dependence of the evaporation rate, where the energy was an apparent one because the composition of evaporated species was not determined. The activation energy increased with decreasing temperature and with increasing mole fraction of ethylene glycol, where the energy obeyed the ideal mixing at lower temperatures while it positively deviated at higher temperatures. The evaporation rates were examined by surface tension of the liquid mixture, but any definite relation between them was not found. Both the evaporation rate and the activation energy were found to be determined mainly by the mole fraction in the surface layer from which the evaporation takes place. Finally, the new concept of surface excess was presented, where the surfactant molecules were concentrated and formed a bimolecular layer at a certain distance beneath the air/solution interface.