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A S Rudolph

Publications and source records attributed to A S Rudolph.

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Interactions of sugars with membranes.

Water profoundly affects the stability of biological membranes, and its removal leads to destructive events including fusion and liquid crystalline to gel phase transitions. In heterogeneous mixtures such as those found in biological membranes the phase transitions can lead to increases in permeability and lateral phase separations that often are irreparable. Certain sugars are capable of preventing these deleterious events by inhibiting fusion during drying and by maintaining the lipid in a fluid state in the absence of water. As a result, the increased permeability and lateral phase separations that accompany dehydration are absent. The weight of the evidence suggests strongly that there is a direct interaction between the sugars and lipids in the dry state. Although the evidence is less clear about whether these sugars can interact directly with hydrated bilayers, there are strong suggestions in the literature that sugars free in solution or covalently linked to membrane constituents can also affect the physical properties and presumably the stability of bilayers. Finally, we have far less evidence concerning the mechanism by which they do so, but the same sugars are also capable of preserving the structure and function of both membrane-bound and soluble proteins in the absence of water. We believe these effects may be important in the survival of intact cells and organisms such as seeds in the absence of water. Furthermore, in view of the practical importance of preserving biological structures we suspect that the results described here will ultimately have important applications in biology and medicine.

Animals↗

Differential scanning calorimetric study of the thermotropic phase behavior of a polymerizable, tubule-forming lipid.

A comparative study of the polymorphism exhibited by the polymerizable, tubule-forming phospholipid 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3- phosphocholine (DC23PC) and its saturated analog 1,2-ditricosanoyl-sn-glycero-3-phosphocholine (DTPC) in aqueous suspension is reported. Differential scanning calorimetry (DSC), as well as freeze-fracture electron microscopy and Raman spectroscopy, have been used to study the influence on phase behavior of rigid diacetylene groups in the fatty acyl chains of a phosphatidylcholine. DTPC large multilamellar vesicle (MLV) and small unilamellar vesicle (SUV) suspensions were found to retain liposome morphology after chain crystallization had occurred. In marked contrast, diacetylenic DC23PC suspensions do not maintain liposomal morphology in converting to the low temperature phase. Large MLVs of DC23PC with outer diameters in excess of 1 micron convert to a gel phase with cylindrical or tubular morphology at 38 degrees C, just a few degrees below the lipid's chain melting temperature (TM(H), i.e. temperature of an endothermic event observed during a heating scan) of 43.1 degrees C. Unlike the large MLVs, small MLVs or SUVs of DC23PC, with diameters of 0.4 +/- 0.3 micron and 0.04 +/- 0.02 micron, respectively, exhibit metastability in the liquid-crystalline state for several tens of degrees below the chain melting temperature prior to converting to a gel phase which, by electron microscopy, manifests itself as extended multilamellar sheets. Raman data collected at TM(H) -40 degrees C demonstrate that the gel state formed by DC23PC is very highly ordered relative to that of DTPC, suggesting that special chain packing requirements are responsible for the novel phase behavior of DC23PC.

Calorimetry, Differential Scanning↗

The freeze-dried preservation of liposome encapsulated hemoglobin: a potential blood substitute.

In this report, the ability of carbohydrates (trehalose, sucrose, and glucose) to preserve the blood substitute liposome-encapsulated hemoglobin (LEH) in the freeze-dried state is examined. The water-free stabilization of individual components of this blood substitute and LEH is reported. Lyophilization of hemoglobin solutions in the absence of carbohydrates results in significant oxidative degradation of Hb as measured by a large increase (approximately 60%) in methemoglobin. Hb samples lyophilized in increasing carbohydrate concentrations show reduced levels of methemoglobin, and at 0.5 M trehalose, sucrose, or glucose, these levels are reduced to nearly the same levels as unlyophilized controls. Storage of lyophilized Hb samples following rehydration at 4 degrees C shows the same rate of methemoglobin formation regardless of whether carbohydrates are present. This suggests that carbohydrates prevent Hb oxidation in the dry state but are less effective at retarding oxidative damage to Hb in solution. The addition of 0.25 M trehalose or sucrose to LEH results in the maintenance of liposomal size following lyophilization. In these experiments, glucose was least effective at inhibiting dehydration-induced LEH fusion. Lyophilization of LEH in 0.25 M trehalose or sucrose also results in significantly greater retention of the encapsulated hemoglobin following lyophilization and rehydration. These results suggest that the long-term stabilization of LEH in the dry state is a realizable goal.

Blood Substitutes↗

The reduction of methemoglobin levels by antioxidants.

Preventing the oxidation of hemoglobin in solution is one of the major requirements for the successful production and long-term storage of hemoglobin-based blood substitutes. To this end we have studied the effects of antioxidants on the rate of methemoglobin formation and disappearance in solutions of human and bovine hemoglobin at 4 degrees C and 37 degrees C. Ascorbate and desferal (5 mM) were observed to act as prooxidants, increasing the rate of methemoglobin formation at 37 degrees C. Trehalose, mannitol, glucose, and EDTA (5 mM) had no significant effect. Glutathione and NADH (10 mM) were the most effective antioxidants tested, causing a significant decrease in the rate of methemoglobin formation at 37 degrees C for periods of up to 50 hours. The combination of these antioxidants in bovine hemoglobin at 4 degrees C resulted in the reduction of methemoglobin levels to nearly undetectable levels in approximately 150 hours. In addition, NADH and glutathione were found to reduce methemoglobin levels to 10% over a period of 100 hours in a sample of human hemoglobin that had been stored at 4 degrees C for one year and had 60% methemoglobin. These results suggest that the prevention and reversal of methemoglobin formation during the long-term storage of hemoglobin solutions and hemoglobin-based blood substitutes may now be possible.

Animals↗

Liposome-encapsulated hemoglobin: oxygen binding properties and respiratory function.

Lipid bilayer membranes have been used to encapsulate hemoglobin and organic phosphates, resulting in a synthetic red cell with oxygen-binding properties very similar to red blood cells (RBCs). In addition to the oxygen-binding parameters, we report here an improvement in the lipid formulation that confers upon the liposome-encapsulated hemoglobin (LEH) a circulation retention half-time of 15 to 20 hours in mice. Cooperative oxygen binding of the encapsulated hemoglobin is retained and the oxygen-binding affinity can be maintained at or below the level of fresh whole blood for many weeks by co-encapsulation of an appropriate concentration of pyridoxal-5-phosphate. A comparison of the kinetics of oxygen exchange across the membranes of the synthetic red cells and RBCs indicates that the increased surface to volume ratio of the much smaller synthetic red cells, coupled with their somewhat lower concentration of hemoglobin, results in more rapid rates of oxygen uptake and release than are seen with RBCs. Efficacy studies have begun and we report here the successful transfusion of rats with LEH. After greater than 90% exchange, LEH transfused rats were able to maintain activity with normal respiratory and cardiac function parameters.

Animals↗

A Fourier-transform infrared spectroscopic study of the polymorphic phase behavior of 1,2- bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine; a polymerizable lipid which forms novel microstructures.

We have investigated the phase characteristics of 1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC23PC), a phosphatidylcholine with diacetylenic groups in the acyl chains, and its saturated analog 1,2-ditricosanoyl-sn-glycero-3-phosphocholine (DTPC), using Fourier-transform infrared spectroscopy (FTIR). Previous studies on the phase behavior of DC23PC in H2O have shown that DC23PC exhibits: (1) formation of cylindrical structures ('tubules') by cooling fluid phase multilamellar vesicles (MLVs) through Tm (43 degrees C), and 2) metastability of small unilamellar vesicles (SUVs) in the liquid-crystalline state some 40 degrees C below Tm, with subsequent formation of a gel phase comprised of multilamellar sheets at 2 degrees C. The sheets form tubules when heated and cooled through Tm. FTIR results presented here indicate that as metastable SUVs are cooled toward the transition to bilayer sheets, spectroscopic changes occur before the calorimetric transition as measured by a reduction in the CH2 symmetric stretch frequency and bandwidth. In spite of the vastly different morphologies, the sheet gel phase formed from SUVs is spectroscopically similar to the tubule gel phase. The C-H stretch region of DC23PC gel phase shows bands at 2937 and 2810 cm-1 not observed in the saturated analog of DC23PC, which may be related to perturbations in the acyl chains introduced by the diacetylenic moiety. The narrow CH2 scissoring mode at 1470 cm-1 and the prominent CH2 wagging progression indicate that DC23PC gel phase was highly ordered acyl chains with extended regions of all-trans methylene segments. In addition, the 13 cm-1 reduction in the C = O stretch frequency (1733-1720 cm-1) during the induction of DC23PC gel phase indicates that the interfacial region is dehydrated and rigid in the gel phase.

Acetylene↗

Modes of interaction of cryoprotectants with membrane phospholipids during freezing.

The abilities of a variety of compounds to inhibit liposome fusion during freeze/thaw were assessed by resonance energy transfer. Small unilamellar vesicles have been frozen according to three different protocols. Membrane intermixing was seen to be relatively independent of freezing protocol except when glycerol, dimethyl sulfoxide (DMSO), or sarcosine was used as the cryoprotectant. Low concentrations of polyvinylpyrolidone or 4-hydroxyproline enhanced fusion of liposomes, whereas high concentrations of these compounds had no effect. Glycerol, DMSO, proline, betaine, and sarcosine reduced fusion, but only when their concentrations were greater than 1 M. The most effective cryoprotectants were trehalose and sucrose, which both reduced fusion to minimal levels at concentrations of only 0.2 M. We have also used europium to probe the modes of interaction of these compounds with phospholipids. Europium, which is known to bind to the phosphate headgroup, maximized fusion in liposomes subjected to freeze/thaw. This "europium-induced" fusion was progressively reduced by the presence of increasing sucrose, trehalose, or glycerol, suggesting a competition for the headgroup. However, the presence of proline, betaine, or sarcosine did not reduce europium-induced fusion, suggesting that these compounds do not compete for the headgroup. Substitution of polar side chains on the hydrophobic regions of proline or sarcosine eliminate their cryoprotective properties, suggesting that these compounds interact with the acyl chains of the bilayer.

Chemical Phenomena↗

Effects of three stabilizing agents--proline, betaine, and trehalose--on membrane phospholipids.

We have studied the interaction between three compounds which accumulate in organisms under hydration stress--proline, betaine, and trehalose--and the membrane phospholipids dimyristoylphosphatidylcholine (DMPC), palmitoyloleoylphosphatidylcholine (POPC), and dimyristoylphosphatidylethanolamine in bulk solution. Film balance studies reveal that these compounds increase the area/molecule of these lipids. Differential scanning calorimetry has been employed to investigate the effect these agents have on the gel-to-liquid crystalline phase transition of multilamellar and small unilamellar vesicles of DMPC, dipalmitoylphosphatidylcholine, and POPC:phosphatidylserine (90:10 mole ratio) in bulk solution. In the presence of 1 M proline, trehalose, or betaine, the midtransition temperature in small unilamellar vesicles is reduced (up to 7 degrees C in 1 M trehalose), and the transition broadened. In contrast, multilamellar vesicles of similar lipid composition show an increased transition temperature in the presence of the same concentration of these compounds. This result suggests that the inner lamellae in multilamellar vesicles may be dehydrated with only a few outer lamellae exposed to the protective compound. Finally, we have used stereomodels of phosphatidylcholine to investigate the mechanism of action of these agents. Hydrogen bonding of trehalose to the head group region results in an increase in the distance between head groups of 6.9 A. This amount of spreading compares well with data from the monolayer experiments which indicate that maximal spreading of DMPC monolayers by trehalose is 6.5 A. Molecular models of proline and betaine have also been constructed, and these models suggest potential interactions between these compounds and phosphatidylcholines. For the amphipath proline, this interaction may involve intercalation between phospholipid head groups.

Betaine↗

Inhibition of dehydration-induced fusion between liposomal membranes by carbohydrates as measured by fluorescence energy transfer.

The relative abilities of a number of naturally occurring carbohydrates to inhibit dehydration-induced fusion between palmitoyloleoylphosphatidylcholine:phosphatidylserine (85:15) large unilamellar vesicles have been studied. Fusion events were quantified using a fluorescence resonance energy transfer technique. Trehalose was most effective at inhibiting fusion (0.4 g/trehalose/g lipid showed 30% probe intermixing), followed by maltose (60% intermixing), fructose (60%), sucrose (70%), glucose (80%), cellobiose, glycerol, raffinose, and myo-inositol (90%). The relative abilities of these carbohydrates to inhibit fusion correlate directly with their abilities to interact with phospholipids, maintain bilayer fluidity, and preserve biological membranes. The results are discussed in relation to the crystalline structure of the carbohydrates and their possible influence on level of interaction with phosphate head groups.

Energy Transfer↗

A calorimetric and infrared spectroscopic study of the stabilizing solute proline.

We have studied the calorimetric and infrared spectroscopic properties of the amino acid proline which has been implicated in the stabilization of biomacromolecules during reduced water states. It has been suggested that the ability of this molecule to protect biomacromolecules during these stress states may be related to the formation of polymeric aggregates of proline monomers in solution. The structure of this aggregate is thought to be an alternates stack, forming a hydrophilic colloid-like polymer which is thought to interact with hydrophobic moieties of biomacromolecules, reducing the exposed hydrophobic area during reduced water conditions. Calorimetric data presented in this work show that in increasing concentration of proline in solution the enthalpy associated with the melting of bulk water is greatly reduced, indicating strong hydrogen bonding character of proline in aqueous solution. Proline shows two eutectic phase separations at moderate concentrations and one of these eutectics may be the proposed intermolecular state. A partial phase diagram for proline is presented. Fourier-transform infrared spectroscopic data indicate that the COO- asymmetric stretch of proline shows marked splitting with increasing proline concentration. This suggests that the carboxylate is in different environments, with the high energy vibrations representing COO- groups which are participating in the hydrogen bonding pattern associated with the formation of the intermolecular stack. Changes in the CH2 asymmetric and symmetric stretches of the pyrrolidine rings of proline are consistent with the proposed stack structure. We also suggest a possible mechanism by which these intermolecular associations may be important in the protection of biomacromolecules during reduced water states.

Calorimetry↗

Membrane stabilization during freezing: the role of two natural cryoprotectants, trehalose and proline.

The relative effectiveness of two natural cryoprotectants, proline and trehalose, in preserving membrane structure and function during freezing was studied. Isolated vesicles of sarcoplasmic reticulum (SR) from lobster muscle (Homarus americanus) were employed to study changes in structure and function during rapid freeze-thaw conditions. Both proline and trehalose were shown to effectively preserve the structure (assessed with freeze fracture) and function (assessed by the ability of the membranes to transport calcium) in the frozen vesicles. As a first step toward determining the mechanism of cryoprotection by these compounds, we have investigated their effectiveness in inhibiting freezing induced fusion between phospholipid vesicles. Pamiltoyloleoyl-phosphatidylcholine: phosphatidylserine (85:15 mole ratio) small unilamellar vesicles (SUVs) were made incorporating one of the following fluorescent probes, and energy donor, cholesteryl anthracene-9-carboxylate, or an energy acceptor, nitrobenzo-2-oxa-1,3-diazole phosphatidylethanolamine to investigate the amount of membrane mixing during rapid freeze-thaw cycles, and storage at -20 degrees C. Membrane mixing was measured as an energy transfer from donor to acceptor when donor vesicles and acceptor vesicles were mixed before a particular freezing treatment. Membrane mixing was correlated with structural changes in these membranes by freeze-fracture analysis. Both trehalose and proline were found to be more effective in preventing membrane mixing between SUVs than the standard protectants, glycerol and dimethylsulfoxide.

Animals↗

Modification of dry 1,2-dipalmitoylphosphatidylcholine phase behavior with synthetic membrane-bound stabilizing carbohydrates.

Carbohydrates, particularly disaccharides, have been shown to accumulate in organisms as protective solutes during periods of stress such as freezing and desiccation. Cholesterol and lipid derivatives containing the protective carbohydrates galactose or maltose, O-[11-(1-beta-D-galactosyloxy)-3,6,9-trioxaundecanyl]ol (TEC-GAL), O-[11-(1-beta-D-maltosyloxy)-3,6,9-trioxaundecanyl]ol (TEC-MAL), and 14-(galactosyloxy)-N,N-dimethyl-O-(dipalmitoylphosphatidyl)- 6,9,12-trioxa-3- azoniatetradecanol (DP-GAL), have been synthesized to investigate the interaction of a protective carbohydrate moiety tethered to the 1,2-dipalmitoylphosphatidylcholine (DPPC) bilayer surface. Toward this goal, we have investigated the calorimetric and infrared spectroscopic behavior of mixtures of DPPC codried with these glycolipids. The synthetic glycolipids are shown to decrease significantly the main transition temperature (max Cp) of dry DPPC with a concomitant reduction in the cooperativity of the transition, as evidenced by a decrease in the enthalpy with increasing glycolipid. The decrease in transition temperature is shown to be related to chain melting monitored by the CH2 symmetric stretch frequency through the transition using FTIR. We also present evidence that the glycolipids interact with the interfacial region of DPPC, as shown by the decrease in the phosphate symmetric stretch intensity with increasing concentration of glycolipid. These observed effects are similar to the action of bulk protective sugars with DPPC; however, the concentration of glycolipid and the associated carbohydrate concentration needed to effect the observed changes are reduced compared to the quantity of bulk carbohydrate previously shown to give similar results with DPPC.

1,2-Dipalmitoylphosphatidylcholine↗

Effects of free and liposome-encapsulated hemoglobin on choroidal vascular plexus blood flow, using the rabbit eye as a model system.

PURPOSE: This study investigated the vasoconstrictive effects of both stroma-free and liposome-encapsulated cross-linked hemoglobin (Hb) on vascular plexus hemodynamics, using the choroid of the rabbit eye as a model system. METHODS: Sequential subtraction of high-speed ICG fluorescence angiogram images facilitated visualization of the time-varying patterns of blood flow distribution in the choriocapillaris during the cardiac cycle. Differences between baseline and post-hemoglobin injection blood flow distributions were analyzed. Likewise, differences in the time-varying patterns of flow distribution between the particulate and liquid phases of blood during a cardiac cycle were investigated, since this may bear on differences in vasoactivity induced by circulating stroma-free vs. encapsulated Hb. RESULTS: Cross-linked Hb induced a transient, but marked, localized reduction in choriocapillaris blood flow. This effect was significantly attenuated when liposome encapsulated cross-linked hemoglobin was administered. Plexus blood flow distribution was different for particulate and liquid ICG. CONCLUSIONS: Differences in particulate and liquid ICG flow patterns suggest that one contribution to the different plexus blood flow patterns observed in the encapsulated and free Hb experiments may be due to differences in liquid and particle-bound Hb distribution within the plexus. The observed choriocapillaris blood flow reductions may be attributable to an aggregate endothelial cell contractility induced by presence of extra-cellular Hb in the choriocapillaris plexus.

Animals↗

Controlled release of transforming growth factor-beta from lipid-based microcylinders.

The release of transforming growth factor-beta (TGF-beta) from a lipid microstructure has been demonstrated. Lipid microcylinders, with dimensions of 100 x 0.5 microns and composed of a diacetylenic lipid, have been loaded with 25 ng TGF-beta/mg lipid. Physical and bioactive release characteristics of TGF-beta from these microcylinders and from microcylinders embedded in an agarose hydrogel are reported. Release of TGF-beta from lipid microcylinders follows typical diffusion-limited characteristics, where 10-12% of the TGF is released in the first 10 h at 37 degrees C. The release rate is shown to be temperature controlled and dependent on the integrity of the lipid microcylinder. Immobilization of the lipid microcylinder in a hydrogel matrix composed of agarose and gelatin does not impair the diffusion of TGF-beta from the lipid microcylinders. The utilization of microcylinders as release vehicles in wound repair is discussed.

Animals↗

Liposome-encapsulated alginate: controlled hydrogel particle formation and release.

Large unilamellar liposomes of dipalmitoyl phosphatidylcholine have been employed as reaction sites for calcium alginate gelation. Encapsulation of sodium alginate was accomplished by extrusion of phospholipid dispersions through polycarbonate filter of uniform pore size followed by incubation with high concentrations of calcium chloride. The diffusion of calcium into the liposome interior resulted in alginate gelatin within the liposome. Detergent treatment of the liposomes resulted in solubilization of the lipid bilayer with subsequent release of the alginate beads which were measured by laser light scattering and electron microscopy. The release profiles of both the liposomes with entrapped alginate beads and the alginate beads (released by detergent treatment of the liposomes) were determined using cytochrome-c as a marker for release. The release profiles show a rapid release of cytochrome-c over the first 2 for both preparations with slower subsequent release rates for the liposomes with encapsulated alginate beads. The similar early profile may be due to the release of cytochrome-c from bound calcium alginate adsorbed to the outer leaflet of the liposome. This is also supported by calorimetric results which indicate a marked reduction in the enthalpy of the main gel to liquid crystalline phase transition of multilamellar and large unilamellar dispersions of the lipid. This method results in the fabrication of easily controlled, unimodal submicron hydrogel particles which may be used for controlled-release applications.

1,2-Dipalmitoylphosphatidylcholine↗