Programmable Peptide-Cross-Linked Nucleic Acid Nanocapsules as a Modular Platform for Enzyme Specific Cargo Release.

Herein we describe a modular assembly strategy for photo-cross-linking peptides into nucleic acid functionalized nanocapsules. The peptides embedded within the nanocapsules form discrete nanoscale populations capable of gating the release of molecular and nanoscale cargo using enzyme-substrate recognition as a triggered release mechanism. Using photocatalyzed thiol-yne chemistry, different peptide cross-linkers were effectively incorporated into the nanocapsules and screened against different proteases to test for degradation specificity both in vitro and in cell culture. By using a combination of fluorescence assays, confocal and TEM microscopy, the particles were shown to be highly specific for their enzyme targets, even between enzymes of similar protease classes. The rapid and modular nature of the assembly strategy has the potential to be applied to both intracellular and extracellular biosensing and drug delivery applications.

Lateral Pressure Profile and Free Volume Properties in Phospholipid Membranes Containing Anesthetics.

The effect of four general anesthetics, namely chloroform, halothane, diethyl ether, and enflurane on the free volume fraction and lateral pressure profiles in a fully hydrated dipalmitoylphosphatidylcholime (DPPC) membrane is investigated by means of computer simulation. In order to find changes that can be related to the molecular mechanism of anesthesia as well as its pressure reversal, the simulations are performed both at atmospheric and high (1000 bar) pressures. The obtained results show that the additional free volume occurring in the membrane is localized around the anesthetic molecules themselves. Correspondingly, the fraction of the free volume is increased in the outer of the two membrane regions (i.e., at the outer edge of the hydrocarbon phase) where anesthetic molecules prefer to stay in every case. As a consequence, the presence of anesthetics decreases the lateral pressure in the nearby region of the lipid chain ester groups, in which the anesthetic molecules themselves do not penetrate. Both of these changes, occurring upon introducing anesthetics in the membrane, are clearly reverted by the increase of the global pressure. These findings are in accordance both with the more than 60 years old "critical volume hypothesis" of Mullins, and with the more recent "lateral pressure hypothesis" of Cantor. Our results suggest that if anesthesia is indeed caused by conformational changes of certain membrane-bound proteins, induced by changes in the lateral pressure profile, as proposed by Cantor, the relevant conformational changes are expected to occur in the membrane region where the ester groups are located.

Conformational Exploration of Enflurane in Solution and in a Biological Environment.

Enflurane is a fluorinated volatile anesthetic, whose bioactive conformation is not known. Actually, a few studies have reported on the conformations of enflurane in nonpolar solution and gas phase. The present computational and spectroscopic (infrared and NMR) work shows that three pairs of isoenergetic conformers take place in the gas phase, neat liquid, polar, and nonpolar solutions. According to docking studies, a single conformation is largely preferred over its isoenergetic isomers to complex with the active site of Integrin LFA-1 enzyme (PDB code: 3F78 ), where the widely used anesthetic isoflurane (a constitutional isomer of enflurane) is known to bind. Weak hydrogen bonding from an electrostatic interaction between the CHF2 hydrogen and the central CF2 fluorines was not found to rule the conformational isomerism of enflurane. Moreover, intramolecular interactions based on steric, electrostatic, and hyperconjugative effects usually invoked to describe the anomeric effect are not responsible for the possible bioactive conformation of enflurane, which is rather governed by the enzyme induced fit.

Salient features of enantioselective gas chromatography: the enantiomeric differentiation of chiral inhalation anesthetics as a representative methodological case in point.

The enantiomeric differentiation of the volatile chiral inhalation anesthetics enflurane, isoflurane, and desflurane by analytical and preparative gas chromatography on various modified cyclodextrins is described. Very large enantioseparation factors α are obtained on the chiral selector octakis(3-O-butanoyl-2,6-di-O-pentyl)-γ-cyclodextrin (Lipodex E). The gas-chromatographically observed enantioselectivities are corroborated by NMR-spectroscopy using Lipodex E as chiral solvating agent and by various sensor devices using Lipodex E as sensitive chiral coating layer. The assignment of the absolute configuration of desflurane is clarified. Methods are described for the determination of the enantiomeric distribution of chiral inhalation anesthetics during narcosis in clinical trials. The quantitation of enantiomers in a sample by the method of enantiomeric labeling is outlined. Reliable thermodynamic parameters of enantioselectivity are determined by using the retention-increment R' approach for the enantiomeric differentiation of various chiral halocarbon selectands on diluted cyclodextrin selectors.

Blue shifts and unusual intensity changes in the infrared spectra of the enflurane···acetone complexes: spectroscopic and theoretical studies.

Blue-shifting C-H···O hydrogen-bonded complexes between enflurane (CHFCl-CF(2)-O-CHF(2)) and deuterated acetone have been identified in CCl(4) solution by FT-IR spectroscopy. For the two ν(C-H) stretching vibrations of enflurane the observed blue shifts are +17 and +11 cm(-1). The corresponding two infrared ν(C-H) bands show the opposite changes of their intensity, one is decreasing, and the other is significantly increasing, upon formation of the hydrogen bonding. The structures, binding energies, and theoretical infrared spectra of the enflurane-acetone complexes were calculated by MP2 and B3LYP methods using the 6-311++G(d,p) basis set. The interaction energies were evaluated by the complete basis set limit (CBS) calculations at the HF, MP2, and CCSD(T) levels of theory. Although the MP2 method slightly overestimates the blue shifts, the MP2 predicted frequency difference and the relative IR intensities of two ν(C-H) stretching bands for the enflurane-acetone complexes show good agreement with experiment. Unfortunately, the B3LYP method predicts incorrect IR intensities of these hydrogen-bonded systems. The NBO analysis was performed to unravel the origin of the unusual intensity changes of two ν(C-H) stretching bands, in enflurane complexes.

Halogen bonded complexes between volatile anaesthetics (chloroform, halothane, enflurane, isoflurane) and formaldehyde: a theoretical study.

The structures and intermolecular interactions in the halogen bonded complexes of anaesthetics (chloroform, halothane, enflurane and isoflurane) with formaldehyde were studied by ab initio MP2 and CCSD(T) methods. The CCSD(T)/CBS calculated binding energies of these complexes are between -2.83 and -4.21 kcal mol(-1). The largest stabilization energy has been found for the C-Br···O bonded halothane···OCH(2) complex. In all complexes the C-X bond length (where X = Cl, Br) is slightly shortened, in comparison to a free compound, and an increase of the C-X stretching frequency is observed. The electrostatic interaction was excluded as being responsible for the C-X bond contraction. It is suggested that contraction of the C-X bond length can be explained in terms of the Pauli repulsion (the exchange overlap) between the electron pairs of oxygen and halogen atoms in the investigated complexes. This is supported by the DFT-SAPT results, which indicate that the repulsive exchange energy overcompensates the electrostatic one. Moreover, the dispersion and electrostatic contributions cover about 95% of the total attraction forces, in these complexes.

Weak polar interactions confer albumin binding site selectivity for haloether anesthetics.

BACKGROUND: Enflurane and isoflurane are structural isomers with different anesthetic potencies and side effects. It is not clear whether these differences are produced by differing occupancy of common protein binding sites or by occupancy of different sites, but the very similar molecular properties make the latter possibility unlikely. In this study, the authors examined binding site selectivity of these anesthetics in human serum albumin (HSA). METHODS: Binding of isoflurane and enflurane with HSA was determined with isothermal titration calorimetry. Competition with known ligands (propofol) allowed localization of binding sites within the HSA molecule. Molecular properties of isoflurane and enflurane were calculated. RESULTS: Isoflurane binds HSA with higher affinity but smaller total enthalpy than enflurane. Enthalpogram analysis suggested that isoflurane bound a single site, whereas enflurane bound two. Competition experiments indicated that enflurane and isoflurane share one binding site, which also binds propofol. The additional enflurane site binds propofol but not isoflurane. Increased salt concentration decreased the affinity for isoflurane but not for enflurane. The dipole moment of isoflurane is higher than that of enflurane, and the isoflurane binding site is more polar. CONCLUSION: These data indicate two binding sites of different character for the haloether anesthetics on HSA. One site is more polar and prefers isoflurane, presumably because of its larger dipole. The second site prefers the less polar enflurane. Therefore, weak polar interactions confer considerable selectivity, and differences in drug action may arise from occupancy of different protein sites.

Binding of volatile anesthetics to serum albumin: measurements of enthalpy and solvent contributions.

This study directly examines the enthalpic contributions to binding in aqueous solution of closely related anesthetic haloethers (desflurane, isoflurane, enflurane, and sevoflurane), a haloalkane (halothane), and an intravenous anesthetic (propofol) to bovine and human serum albumin (BSA and HSA) using isothermal titration calorimetry. Binding to serum albumin is exothermic, yielding enthalpies (DeltaH(obs)) of -3 to -6 kcal/mol for BSA with a rank order of apparent equilibrium association constants (K(a) values): desflurane > isoflurane approximately enflurane > halothane >or= sevoflurane, with the differences being largely ascribed to entropic contributions. Competition experiments indicate that volatile anesthetics, at low concentrations, share the same sites in albumin previously identified in crystallographic and photo-cross-linking studies. The magnitude of the observed DeltaH increased linearly with increased reaction temperature, reflecting negative changes in heat capacities (DeltaC(p)). These -DeltaC(p) values significantly exceed those calculated for burial of each anesthetic in a hydrophobic pocket. The enhanced stabilities of the albumin/anesthetic complexes and -DeltaC(p) are consistent with favorable solvent rearrangements that promote binding. This idea is supported by substitution of D(2)O for H(2)O that significantly reduces the favorable binding enthalpy observed for desflurane and isoflurane, with an opposing increase of DeltaS(obs). From these results, we infer that solvent restructuring, resulting from release of water weakly bound to anesthetic and anesthetic-binding sites, is a dominant and favorable contributor to the enthalpy and entropy of binding to proteins.

An isothermal titration calorimetry study on the binding of four volatile general anesthetics to the hydrophobic core of a four-alpha-helix bundle protein.

A molecular understanding of volatile anesthetic mechanisms of action will require structural descriptions of anesthetic-protein complexes. Previous work has demonstrated that the halogenated alkane volatile anesthetics halothane and chloroform bind to the hydrophobic core of the four-alpha-helix bundle (Aalpha(2)-L38M)(2) (Johansson et al., 2000, 2003). This study shows that the halogenated ether anesthetics isoflurane, sevoflurane, and enflurane are also bound to the hydrophobic core of the four-alpha-helix bundle, using isothermal titration calorimetry. Isoflurane and sevoflurane both bound to the four-alpha-helix bundle with K(d) values of 140 +/- 10 micro M, whereas enflurane bound with a K(d) value of 240 +/- 10 micro M. The DeltaH degrees values associated with isoflurane, sevoflurane, and enflurane binding were -7.7 +/- 0.1 kcal/mol, -8.2 +/- 0.2 kcal/mol, and -7.2 +/- 0.1 kcal/mol, respectively. The DeltaS degrees values accompanying isoflurane, sevoflurane, and enflurane binding were -8.5 cal/mol K, -10.4 cal/mol K, and -8.0 cal/mol K, respectively. The results indicate that the hydrophobic core of (Aalpha(2)-L38M)(2) is able to accommodate three modern ether anesthetics with K(d) values that approximate their clinical EC(50) values. The DeltaH degrees values point to the importance of polar interactions for volatile general anesthetic binding, and suggest that hydrogen bonding to the ether oxygens may be operative.

Interaction between artificial membranes and enflurane, a general volatile anesthetic: DPPC-enflurane interaction.

The structural modifications of the dipalmitoylphosphatidylcholine (DPPC) organization induced by increasing concentration of the volatile anesthetic enflurane have been studied by differential scanning calorimetry, small-angle, and wide-angle x-ray scattering. The interaction of enflurane with DPPC depends on at least two factors: the enflurane-to-lipid concentration ratio and the initial organization of the lipids. At 25 degrees C (gel state), the penetration of enflurane within the lipids induces the apparition of two different mixed lipid phases. At low anesthetic-to-lipid molar ratio, the smectic distance increases whereas the direction of the chain tilt changes from a tilt toward next-neighbors to a tilt between next-neighbors creating a new gel phase called L(beta')(2NNN). At high ratio, the smectic distance is much smaller than for the pure L(beta') DPPC phase, i.e., 50 A compared to 65 A, the aliphatic chains are perpendicular to the membrane and the fusion temperature of the phase is 33 degrees C. The electron profile of this phase that has been called L(beta)(i), indicates that the lipids are fully interdigitated. At 45 degrees C (fluid state), a new melted phase, called L(alpha)(2), was found, in which the smectic distance decreased compared to the initial pure L(alpha)(1) DPPC phase. The thermotropic behavior of the mixed phases has also been characterized by simultaneous x-ray scattering and differential scanning calorimetry measurements using the Microcalix calorimeter of our own. Finally, titration curves of enflurane effect in the mixed lipidic phase has been obtained by using the fluorescent lipid probe Laurdan. Measurements as a function of temperature or at constant temperature, i.e., 25 degrees C and 45 degrees C give, for the maximal effect, an enflurane-to-lipid ratio (M/M), within the membrane, of 1 and 2 for the L(alpha)(2) and the L(beta)(i) lamellar phase respectively. All the results taken together allowed to draw a pseudo-binary phase diagram of enflurane-dipalmitoylphosphatidylcholine in excess water.

Predictive performance of a physiological model for enflurane closed-circuit anaesthesia: effects of continuous cardiac output measurements and age-related solubility data.

BACKGROUND: The disposition of inhalation anaesthetics is governed by the factors described in the Fick principle. METHODS: We have recalibrated a previously validated physiological model for enflurane closed-circuit inhalation anaesthesia, using individual continuous cardiac output measurements as well as age-related enflurane solubility coefficients as inputs to the model. Two model versions using 'calculated' (Brody's formula) or 'measured' (thoracic electrical bioimpedance) cardiac output values, and two versions with 'standard' (fixed) or 'age-related' solubility coefficients were formulated. RESULTS: Data from 62 ophthalmic surgical patients were used to validate the predictive performance of the four model versions. The root mean squared errors (total error) and scatters (error variation) were similar with the extended model versions, but the group biases (systematic error component) were significantly less with the model versions that included age-related solubility compared with the versions using standard solubility coefficients (bias -0.76/-0.78% vs -3.44/-3.60%). CONCLUSION: The inclusion of age-related solubility coefficients but not of continuous cardiac output measurements improves the predictive performance of the physiological model for closed-circuit inhalation anaesthetic conditions in routine clinical practice.

Carbon monoxide production from desflurane, enflurane, halothane, isoflurane, and sevoflurane with dry soda lime.

BACKGROUND: Previous studies in which volatile anesthetics were exposed to small amounts of dry soda lime, generally controlled at or close to ambient temperatures, have demonstrated a large carbon monoxide (CO) production from desflurane and enflurane, less from isoflurane, and none from halothane and sevoflurane. However, there is a report of increased CO hemoglobin in children who had been induced with sevoflurane that had passed through dry soda lime. Because this clinical report appears to be inconsistent with existing laboratory work, the authors investigated CO production from volatile anesthetics more realistically simulating conditions in clinical absorbers. METHODS: Each agent, 2.5 or 5% in 2 l/min oxygen, were passed for 2 h through a Dräger absorber canister (bottom to top) filled with dried soda lime (Drägersorb 800). CO concentrations were continuously measured at the absorber outlet. CO production was calculated. Experiments were performed in ambient air (19-20 degrees C). The absorbent temperature was not controlled. RESULTS: Carbon monoxide production peaked initially and was highest with desflurane (507 +/- 70, 656 +/- 59 ml CO), followed by enflurane (460 +/- 41, 475 +/- 99 ml CO), isoflurane (176 +/- 2.8, 227 +/- 21 ml CO), sevoflurane (34 +/- 1, 104 +/- 4 ml CO), and halothane (22 +/- 3, 20 +/- 1 ml CO) (mean +/- SD at 2.5 and 5%, respectively). CONCLUSIONS: The absorbent temperature increased with all anesthetics but was highest for sevoflurane. The reported magnitude of CO formation from desflurane, enflurane, and isoflurane was confirmed. In contrast, a smaller but significant CO formation from sevoflurane was found, which may account for the CO hemoglobin concentrations reported in infants. With all agents, CO formation appears to be self-limited.

Characterization of thioether compounds formed from alkaline degradation products of enflurane.

BACKGROUND: Renal toxicity has occasionally been observed after enflurane anesthesia. Although originally attributed to its oxidative metabolism to inorganic fluoride, serum levels of inorganic fluoride appear to be small to explain these renal effects. Formation of potentially nephrotoxic halogenated alkenes during alkaline degradation in carbon dioxide absorbers and subsequent bioactivation via the glutathione conjugation pathway may be considered as an alternative mechanism for renal toxicity. The aim of this study was to characterize the thioethers formed chemically and biosynthetically. METHODS: Alkaline degradation of enflurane was achieved by stirring with pulverized potassium hydroxide. Volatile degradation products were analyzed by 19F nuclear magnetic resonance (NMR) analysis of head space gasses trapped in dimethyl sulfoxide (DMSO). Thioethers were generated chemically by trapping head space gasses in DMSO containing N-acetyl-L-cysteine or 2-mercaptoacetic acid as model thiol compounds. Glutathione conjugates were generated biosynthetically by passing head space through rat liver fractions in presence of glutathione. Products formed were analyzed by gas chromatography-mass spectroscopy and 19F-NMR. RESULTS: Direct analysis of head space gasses showed formation of 1-chloro-1,2-difluorovinyl difluoromethyl ether and two unidentified fluorine-containing products as alkaline degradation products of enflurane. When trapped in DMSO-N-acetyl-L-cysteine-triethylamine, N-acetyl-S-(2-chloro-1,2-difluoro-1-(difluoromethoxy)ethyl)-L-cysteine was identified as the major product. Another N-acetyl-L-cysteine S-conjugate formed was N-acetyl-S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine, a potent nephrotoxin in rats. 19F-NMR analysis of glutathione conjugates formed after incubation with rat liver fractions resulted in formation of corresponding S-conjugates. CONCLUSIONS: The current study demonstrates that alkaline degradation products of enflurane can be conjugated to thiol compounds, forming S-conjugates that could theoretically contribute to adverse renal effects observed occasionally with enflurane anesthesia. The N-acetyl-L-cysteine S-conjugates identified may be biomarkers to assess exposure of humans to alkaline degradation products of enflurane.

Amsorb: a new carbon dioxide absorbent for use in anesthetic breathing systems.

BACKGROUND: This article describes a carbon dioxide absorbent for use in anesthesia. The absorbent consists of calcium hydroxide with a compatible humectant, namely, calcium chloride. The absorbent mixture does not contain sodium or potassium hydroxide but includes two setting agents (calcium sulphate and polyvinylpyrrolidine) to improve hardness and porosity. METHODS: The resultant mixture was formulated and subjected to standardized tests for hardness, porosity, and carbon dioxide absorption. Additionally, the new absorbent was exposed in vitro to sevoflurane, desflurane, isoflurane, and enflurane to determine whether these anesthetics were degraded to either compound A or carbon monoxide. The performance data and inertness of the absorbent were compared with two currently available brands of soda lime: Intersorb (Intersurgical Ltd., Berkshire, United Kingdom) and Dragersorb (Drager, Lubeck, Germany). RESULTS: The new carbon dioxide absorbent conformed to United States Pharmacopeia specifications in terms of carbon dioxide absorption, granule hardness, and porosity. When the new material was exposed to sevoflurane (2%) in oxygen at a flow rate of 1 l/min, concentrations of compound A did not increase above those found in the parent drug (1.3-3.3 ppm). In the same experiment, mean +/-SD concentrations of compound A (32.5 +/- 4.5 ppm) were observed when both traditional brands of soda lime were used. After dehydration of the traditional soda limes, immediate exposure to desflurane (60%), enflurane (2%), and isoflurane (2%) produced concentrations of carbon monoxide of 600.0 +/- 10.0 ppm, 580.0 +/- 9.8 ppm, and 620.0 +/-10.1 ppm, respectively. In contrast, concentrations of carbon monoxide were negligible (1-3 ppm) when the anhydrous new absorbent was exposed to the same anesthetics. CONCLUSIONS: The new material is an effective carbon dioxide absorbent and is chemically unreactive with sevoflurane, enflurane, isoflurane, and desflurane.

Effect of temperature and some common metals on the stability of volatile anaesthetic-Entonox mixtures.

Thermal stability of pressurised ready-to-use volatile liquid anaesthetic mixtures (halothane, isoflurane and enflurane) in Entonox (commercially available premixed 50% N2O, 50% O2 mixture) were investigated at temperatures of 20, 258, 400, 503 and 602 degrees C on glass, stainless steel, copper and aluminium by gas chromatography and GC-MS. It was found that most of the decomposition products formed were halogenated compounds and the observed thermal stabilities in glass, stainless steel and copper allowed a thermal treatment up to 250 degrees C without any decomposition problem. Aluminium was found to be the most effective metal at causing decomposition of the anaesthetic mixtures even at lower temperatures.

Chiral discrimination of inhalation anesthetics and methyl propionates by thickness shear mode resonators: new insights into the mechanisms of enantioselectivity by cyclodextrins.

The discrimination of the enantiomers of methyl lactate, methyl 2-chloropropionate, and the inhalation anesthetics enflurane, isoflurane, and desflurane in the gas phase has been performed using thickness shear mode resonators. The selective coating was a modified perpentylated gamma-cyclodextrin derivative dissolved in a polysiloxane matrix. A new model for the sorption of the chiral compounds into the cyclodextrin cavities and into the polymer matrix was established for the purpose of characterizing the sensor responses. This characterization included the fitting of the sensor responses (preferential and nonpreferential sorption) according to the model and extracting the characteristic parameters. In particular we attempted to explain the observed variation of the chiral discrimination factor alpha with changing analyte or cyclodextrin concentrations and search for an invariable parameter, characteristic for a certain analyte-cyclodextrin combination. The process of chiral or "molecular" recognition was thoroughly investigated.

Reduction in the incidence of carbon monoxide exposures in humans undergoing general anesthesia.

BACKGROUND: Carbon monoxide forms via reaction of isoflurane, enflurane, and desflurane with dried CO2 absorbents. The authors hypothesize that interventions by nonphysician support personnel to decrease absorbent drying will decrease the exposure rate of patients to carbon monoxide from anesthetic breakdown. METHODS: In the control group, all anesthetizing personnel were made aware of the factors enabling CO generation from anesthetic breakdown, and prevention techniques were left to the anesthetizing personnel. After data collection was complete, the following interventions were initiated to reduce absorbent drying: Anesthesia technicians and housekeeping personnel were instructed to turn off all anesthesia machines after the last case of the day in each room, and the CO2 absorbent was changed each morning if fresh gas was found flowing. Baralyme was used in all phases of this study. RESULTS: Five cases of intraoperative carbon monoxide exposure occurred among 1,085 (0.46%) first cases in the control group. Postintervention, patient carbon monoxide exposures decreased (P < 0.05), with one exposure among 1,961 (0.051%) first cases in the main operating room. Two exposures among 68 (2.9%) first cases occurred in remote locations (P < 0.001) versus main operating room. Predisposing factors for absorbent drying include the prolonged use of anesthesia machines for monitored anesthesia care, inappropriate drying techniques for expiratory flowmeters, understaffing of support personnel, and anesthesia in remote locations. CONCLUSIONS: These interventions reduced patient exposure to carbon monoxide. Monitoring for carbon monoxide exposures during general anesthesia may be necessary to recognize and end patient exposures that occur despite preventative measures.

Preparative enantiomer separation of the inhalation anesthetics enflurane, isoflurane and desflurane by gas chromatography on a derivatized gamma-cyclodextrin stationary phase.

The preparative enantiomeric separation of the inhalation anesthetics enflurane (1) and isoflurane (2) in very high chemical (> 99.5%) and enantiomeric excess (ee > 99%) by gas chromatography (GC) on octakis(3-O-butanoyl-2,6-di-O-n-pentyl)-gamma-cyclodextrin (4), dissolved in the apolar polysiloxane SE-54 and coated on Chromosorb P AW DMCS, is described. Up to 1 g of each enantiomer of 1-2 can been obtained per diem. The enantiomers of the highly volatile desflurane (3) can also be separated, albeit with diminished ee. The enantiomeric excess of 1-3 was checked by analytical GC on 4 and the absolute configuration of 2 and 3 has been determined via anomalous X-ray diffraction.