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Biomedical subjects

A S Rudolph

Publications and source records attributed to A S Rudolph.

At least 19 recordsLinked to original sources
PubMed1992

Calorimetric studies of lipid tubule formation from ethanol-water solutions.

We have used differential scanning calorimetry to systematically investigate the thermal formation of hollow cylindrical crystalline microstructures or 'tubules' upon cooling a diacetylenic phosphatidylcholine (1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine) dispersed in varying volume fractions of ethanol/water. Tubule formation is characterized by a large exothermic event, observed upon cooling the lipid in 60-80% ethanol. The enthalpy of the transition was observed to be highest in this window of tubule formation (128-138 J/g) which is significantly higher than previously reported values for the enthalpy of tubule formation in water (90 -95 J/g). The enthalpy associated with the formation of tubules in 70% ethanol was also found to be strongly dependent on the efficiency of tubule formation and decreased as the number density of tubules decreased. A significant decrease in tubule number density could be brought about by increasing the lipid concentration of the 70% ethanol solution. Tubule number density was maximized at lipid concentrations between 0.5 and 2 mg/ml in 70% ethanol. Examination of the C-H stretch region from infrared spectra of the lipid below the phase transition, indicate that the intramolecular chain order-disorder is similar, regardless of the fraction of ethanol. The higher transition enthalpy for the melting of tubules in 60-80% ethanol (compared to water) implies that the high-temperature phase from which the tubules form in ethanol is more disordered than the lamellar liquid crystalline phase from which tubules form in water.

Calorimetry, Differential Scanning↗
PubMed1992

Effect of alcohol chain length on tubule formation in 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine.

Aqueous dispersions of 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine, on cooling below the chain melting temperature, form hollow cylindrical structures known as 'tubules'. We have studied the formation of tubules in methanol/water, ethanol/water and n-propanol/water. For each alcohol, there is a defined window of alcohol/water ratios in which the lipid precipitates with the tubule morphology. As the chain length of alcohol is increased, the window shifts towards lower alcohol fraction. Light scattering studies show that at very low lipid concentrations the tubules self-assemble directly from the isotropic phase where as for lipid concentrations greater than 4 mg/ml an intermediate L alpha phase is observed. These results indicate that the mechanism of tubule formation may be dependent on lipid concentration.

Alcohols↗
PubMed1992

Biocompatibility of lipid microcylinders: effect on cell growth and antigen presentation in culture.

The authors are developing a lipid-based microcylinder for the controlled release of biological response modifiers and as templates for cellular migration and differentiation. These structures are comprised of a photopolymerizable phosphatidylcholine (1,2-ditricosa-10,12-diynoyl-sn-glycero-3-phosphocholine) and form spontaneously as a result of a thermotropic phase transition in aqueous solution or in a cosolvent solution of 70:30 ethanol:water. The hollow cylinders are helically wrapped lipid bilayers, variable in length (50-250 microns, depending on conditions of formation) and are 0.5-1.0 microns in diameter. The interaction has been examined of three types of lipid microcylinders: (1) monomeric, (2) photopolymerized by exposure to 254 nm light, and (3) surface-modified by incorporation of 6 mol% gangliosides, with different human cell lines and peripheral blood leucocytes to evaluate the biocompatibility of these structures. The proliferative status of U937 (a histiocytic monocyte), K562 (an erythroleukaemic cell), and Jurkat's derivative (a T-lymphoblast) as measured by pulsed tritiated thymidine was unaffected by the presence of up to 100 micrograms/ml of lipid microcylinders after 3 d in culture. Adherent human peripheral blood monocytes were shown to form adhesive contacts with the lipid microcylinders. An 'association' index from this interaction shows that after 3 d in culture, the association was much lower for those microcylinders that had incorporated ganglioside compared with monomeric or polymerized structures. The lipid microcylinders do not activate T-cells isolated from human peripheral blood, nor do they inhibit the activation of T-cells by phorbol esters or other mitogens.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens↗
PubMed1992

A simple method for producing a technetium-99m-labeled liposome which is stable in vivo.

A new method for labelling preformed liposomes with technetium-99m (99mTc) has been developed which is simple to perform and stable in vivo. Previous 99mTc-liposome labels have had variable labeling efficiencies and stability. This method consistently achieves high labeling efficiencies (greater than 90%) with excellent stability. A commercially available radiopharmaceutical kit--hexamethylpropyleneamine oxime (HM-PAO)--is reconstituted with 99mTcO4- and then incubated with preformed liposomes that encapsulate glutathione. The incubation takes only 30 min at room temperature. Liposomes that co-encapsulate other proteins such as hemoglobin or albumin, in addition to glutathione, also label with high efficiency. Both in vitro and in vivo studies indicate good stability of this label. Rabbit images show significant spleen and liver uptake at 2 and 20 h after liposome infusion without visualization of thyroid, stomach or bladder activity. This labeling method can be used to study the biodistribution of a wide variety of liposome preparations that are being tested as novel drug delivery systems. This method of labeling liposomes with 99mTc may also have applications in diagnostic imaging.

Animals↗
PubMed1992

Liposome encapsulated hemoglobin: long-term storage stability and in vivo characterization.

Liposome Encapsulated Hemoglobin (LEH) has been the focus of research and development at the Naval Research Laboratory in an effort to find a viable oxygen-carrying resuscitative fluid. Previous reports from our laboratory have shown that LEH binds and releases oxygen in a manner similar to red blood cells, and that it can sustain life when red cell hematocrits are decreased to critical levels. We have also reported on LEH with regards to preparative methods, scale-up feasibility, toxicity, hemodynamics, hemoglobin P50 modification by coencapsulation of organic phosphates, liposomal surface modification, and storage strategies. In this report, the issue of LEH efficacy following long-term storage in the dry state will be addressed. We have shown that hemoglobin, liposomes, and LEH may be successfully lyophilized and rehydrated to viable states. The modification of the LEH formulation by addition of the carbohydrate trehalose results in the successful lyophilization and storage of LEH. In vitro characterization of LEH stored in the dry state for up to six months includes measurement of oxygen-carrying capacity, liposome size retention, methemoglobin production, and the intraliposomal hemoglobin concentration. The in vivo studies report on physiological parameters such as circulation persistence, blood chemistry, and pathological examination in mice.

Animals↗
PubMed1992

Biodistribution studies of liposome encapsulated hemoglobin (LEH) studied with a newly developed 99m-technetium liposome label.

A new method has been developed to label preformed liposomes with 99m-Technetium (99mTc) using hexamethylpropylenamine oxime (HMPAO). 99mTc is an ideal isotope for performing non-invasive dynamic biodistribution studies. This labeling method results in a high labeling efficiency (greater than 95%) and is stable as determined by both in vitro and in vivo studies. In vitro studies indicated that glutathione encapsulated in the LEH is important in the labeling process with 99mTc-HMPAO. In vivo studies with LEH were performed on 7 rabbits with dynamic scintigraphic 1 minute images performed from 1-120 minutes. Delayed images were performed at 20 hours followed by sacrifice and organ counting. Dynamic images reveal a gradual deposition of the LEH in the liver and spleen. Twenty hour biodistributions revealed 50% of the LEH remaining in the blood, 15% in the liver, 14% in the spleen, 3% in lungs, 3% in muscle with trace amounts in the brain, kidneys, and heart. Doses per gram were highest in the spleen with 12.5% of the injected dose per gram of spleen vs. 0.2% per gram of liver. This labeling technique is an effective method for non-invasively monitoring dynamic changes in liposome biodistribution and can be used to study the effects of various liposome modifications on biodistribution.

Animals↗
PubMed1992

Biological responses to exchange transfusion with liposome-encapsulated hemoglobin.

The present study evaluates the biological responses to 10% blood volume infusion ("top-loading," n = 7) or 50% blood volume exchange transfusion (n = 8) with a new preparation of liposome-encapsulated hemoglobin (LEH) in the conscious rat. Top-loading did not affect systemic hemodynamic variables but induced transient thrombocytopenia (-36% +/- 7%; P less than 0.05) and elevation of plasma thromboxane B2 (12.4 +/- 3.8 pg/100 microliters vs. undetectable levels at the control; P less than 0.05). Rats exchanged with LEH maintained mean arterial pressure (MAP) during the 3 hr observation period, whereas control (0.9% NaCl or lactated Ringer's solution) animals developed hypotension (-23 +/- 5 mm Hg, P less than 0.05). The maintenance of MAP in the LEH group resulted from the opposing effects of LEH-induced increase in total peripheral resistance index (TPRI; +0.06 +/- 0.01 mm Hg/ml/min/kg; P less than 0.05), and decreased cardiac index, (Cl, -25 +/- 6 ml/min/kg, P less than 0.05). In contrast, the decline in MAP in the control groups could be attributed to the lower Cl (-65 +/- 8 ml/min/kg; P less than 0.05). All rats developed metabolic acidosis (base excess of -9.7 +/- 0.6 mmol/liter; P less than 0.05), which was transient in the LEH group but progressive in the control groups. These data support the possible use of LEH as a blood substitute when massive transfusions are needed.

6-Ketoprostaglandin F1 alpha↗
PubMed1991

In vivo biodistribution of a radiolabeled blood substitute: 99mTc-labeled liposome-encapsulated hemoglobin in an anesthetized rabbit.

Liposome-encapsulated hemoglobin (LEH) is an erythrocyte substitute that is a potential resuscitative fluid for the in vivo delivery of oxygen. We have noninvasively imaged radiolabeled LEH in vivo with technetium-99m (99mTc) to study the biodistribution in an anesthetized rabbit. Rabbits (2.5 kg, n = 8) were infused with 30 ml of LEH (200 mg of phospholipid, 2.5 g of hemoglobin per kg of body weight) and imaged with a gamma camera continuously for 2 hr. At 20 hr postinfusion, the animals were imaged again and sacrificed; the organs were weighted and their radioactivity was determined for autopsy organ distribution. Organ uptake from the images was corrected for organ-associated blood pool, which was determined by infusion of 99mTc-labeled rabbit erythrocytes. Blood pool and decay-corrected biodistribution data reveal the kinetics of LEH distribution, with an initial rapid uptake by the liver, 8% at 30 min and 15% at 2 hr. The spleen accumulates less LEH initially, 3% at 30 min and 7% at 2 hr, with an apparent linear uptake of LEH over this time period. Image biodistribution data was also validated at 20 hr by tissue sampling. At 20 hr postinfusion, autopsy biodistribution data reveals approximately 42.6% of the total counts remaining in the blood, 15.4% in the liver, 18.1% in spleen, 3.2% in the lungs, 2.4% in muscle, 1.6% in urine, and trace levels in the kidney, brain, and heart (less than 1%). There is no evidence of hemoglobin release from LEH or kidney dysfunction (normal creatinine and blood urea nitrogen) at any time over the course of the study.

Alanine Transaminase↗
PubMed1991

Self-assembling phospholipid filaments.

Aqueous dispersions of double-chain phospholipids spontaneously assemble into closed bilayers called vesicles (or liposomes). Although the vesicles are in general topologically spherical, cylindrical and helical liposomes have sometimes been observed. We present here video-enhanced microscopic studies of a diacetylenic phospholipid dispersed in ethanol/water, which reveal the existence of unusual bilayer morphologies. On cooling the dispersion from the isotropic phase, we have observed the formation of long (of the order of hundreds of micrometres), thin (0.2-2 microns) filaments, which fluctuate strongly. When the temperature is decreased further, the filaments rapidly retract into a mass of lipid. At constant temperature, on the other hand, the filaments transform into torus or ring-like vesicles. Such non-spherical structures have been predicted theoretically but not previously observed experimentally.

Phosphatidylcholines↗
PubMed1991

The effect of hydration stress solutes on the phase behavior of hydrated dipalmitoylphosphatidylcholine.

We have investigated the interaction of solutes found to accumulate in biological systems during chilling, dehydration, and salt stress with fully hydrated multilamellar and unilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC). We have focused on a series of mono-, di-, and tri-substituted amines (glycine, 4-hydroxyproline, proline, and betaine) and contrasted the action of these solutes to trehalose, a protective disaccharide. Differential scanning calorimetry studies show that when DPPC is scanned in the presence of increasing concentrations of these solutes (up to 3 M), there is a moderate increase in the pre-transition temperature (1-6 degrees C) with a smaller increase (1-2 degrees C) in the main transition temperature of hydrated multilamellar vesicles of DPPC. Other calorimetric parameters (delta H, delta T1/2, Cpmax) determined for the pre-transition and main transition were similar independent of the solute. In each case, the main phase transition was broadened with increasing solute while the transition enthalpy was not significantly affected.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1991

Diacetylenic lipid microstructures: structural characterization by X-ray diffraction and comparison with the saturated phosphatidylcholine analogue.

Thermotropic and lyotropic mesomorphism in the polymerizable lecithin 1,2-ditricosa-10,12-diynoyl-sn-glycero-3-phosphocholine and its saturated analogue, 1,2-ditricosanoyl-sn-glycero-3-phosphocholine, has been investigated by wide- and low-angle X-ray diffraction of both powder and oriented samples and by differential scanning calorimetry. Previous studies have shown that the hydrated diacetylenic lipid forms novel microstructures (tubules and stacked bilayer sheets) in its low-temperature phase. The diffraction results indicate that at low temperatures fully hydrated tubules and sheets have an identical lamellar repeat size (d001 = 66.4 A) and crystalline-like packing of the acyl chains. Chain packing in the lamellar crystalline phase is hydration independent. A model for the polymerizable lecithin with (1) fully extended all-trans methylene segments, (2) a long-axis tilt of 32 degrees, and (3) minimal chain interdigitation seems most reasonable on energetic grounds, is consistent with the diffraction data (to 3.93-A resolution), and is likely to support facile polymerization. Above the chain "melting" transition the lamellar repeat of the polymerizable lipid increases to 74 A. The conformational similarity between tubules, sheets, and the dry powder is corroborated by calorimetry, which reveals a cooling exotherm at the same temperature where tubules form upon cooling hydrated sheets. The data suggest that although a high degree of conformational order is a pertinent feature of tubules, this character alone is not sufficient to account for tubule formation. The conformation of the corresponding saturated phosphatidylcholine appears to be similar to that of other saturated phosphatidylcholines in the lamellar gel phase. Furthermore, above the main transition temperature, the dry, saturated lipid shows evidence of a P delta phase (112 degrees C), whereas the diacetylenic lipid appears to exhibit a centered rectangular phase, R alpha (55 degrees C).

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1990

Dry storage of liposome-encapsulated hemoglobin: a blood substitute.

We have previously demonstrated the stabilization of liposome-encapsulated hemoglobin (LEH) by lyophilization (Cryobiology 25, 277-284, 1988). In the present report, we examine the structural and functional recovery of LEH after 3 months in the dry state. We have investigated the incorporation of the protective carbohydrate trehalose in the production and preservation of lyophilized LEH. Vesicle size, retention of entrapped hemoglobin, oxygen-carrying capacity, and percentage methemoglobin were measured as a function of time stored in the dry state under vacuum at room temperature. The results indicate that 150-300 mM trehalose maintains LEH dry preparations with little change in their size or functional characteristics after 3 months in the dry state. These results are compared to those of LEH that has been stored hydrated at 4 degrees C for the same time period.

Animals↗
PubMed1990

Liposome-encapsulated hemoglobin: an oxygen-carrying fluid.

From the original concept of encapsulating hemoglobin in an inert shell, LEH has evolved into a fluid proven to carry oxygen, capable of surviving for reasonable periods in the circulation, and amenable to large-scale production. The formula for the outer shell evolved from synthetic, nonlipid materials, to egg-lecithin-based lipid mixtures, to distearoyl-phosphatidylcholine-based blends. The fabrication technology started with the production of milliliter quantities and methods detrimental to the hemoglobin and developed into high-pressure extrusion systems producing multi-liter quantities without damaging the hemoglobin. The development of methods for analysis and quality control of LEH has been difficult: even techniques for measuring basic characteristics of size and methemoglobin are still being standardized. In vivo studies have established that LEH has a circulation half-life of 16-20 hr and can carry oxygen sufficient to sustain life, but safety has yet to be proven. In each of the general areas mentioned above, there are opportunities for further improvement and characterization. The source of the hemoglobin and the coencapsulation of hemoglobin modifiers needs to be reassessed now that human hemoglobin has been cloned and functional hemoglobin can be produced by using fermentation techniques. The development of routine methods for quality control and assurance must accompany the production of large quantities of LEH for preclinical studies. Whether or not the LEH can and should be manufactured as a lyophilized product must be assessed. Animal studies must done to prove safety as well as efficacy in a variety of clinical models, including hemorrhagic and septic shock as well as various levels of isovolemic exchange. One approach toward the improvement of the LEH is to alter the liposome surface to increase its biocompatibility. The evolution of biocompatible liposome surfaces has included carbohydrate moieties, as carbohydrates are expressed on the majority of biological membrane surfaces including the red cell. It has been demonstrated that inclusion of carbohydrate components such as gangliosides into the liposomal bilayer results in increased circulation times. As a result, these ganglioside-containing liposomes may exhibit a reduced impact on the RES system. Goins et al. have examined methods of introducing the ganglioside GM1 into LEH preparations. The long-term preservation of LEH is essential for its use by paramedics both in civilian and military trauma settings.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Improved biological properties of synthetic distearoyl phosphatidyl choline-based liposome in the conscious rat.

We have previously produced and tested a liposome preparation based on hydrogenated soy lecithin (HSL-L) for the purpose of designing blood replacement in the form of liposome encapsulated hemoglobin (LEH). While these liposomes had acceptable physicochemical properties which addressed many of the desirable characteristics of "artificial blood," they produced hypotension, hemoconcentration, and thrombocytopenia when administered to rats. The following studies present improved synthetic distearoyl phosphatidylcholine-based liposomes (sDSPC-L) which were compared to the HSL-L for their biological effects in the conscious normovolemic rat (n = 6 - 11). HSL-L induced hypotension (-25 +/- 3 mmHg, P less than 0.01), tachycardia (+88 +/- 11 beats/min, P less than 0.01), decrease in cardiac index (-33 +/- 4%, P less than 0.01), and elevation of the total peripheral resistance index (+0.450 +/- 0.003 mmHg/ml/min/kg, P less than 0.01). The hematologic responses to HSL-L were: leukocytosis (+6,070 +/- 1,064/microliters, P less than 0.01), hemoconcentration (+4.0 +/- 0.1%, P less than 0.01), 0.01), and thrombocytopenia (-160 +/- 18 X 10(3)/microliters, P less than 0.01). Plasma thromboxane B2 (TXB2) was elevated to 30.4 +/- 5.6 pg/100 microliters (P less than 0.01). In contrast, the only effects induced by sDSPC-L were slight tachycardia (+37 +/- 9 beats/min, P less than 0.05) and a marginal increase in plasma TXB2 to 9.7 +/- 3.3 pg/100 microliters (P less than 0.05). All effects, except for those related to cardiac output and peripheral resistance, were transient. These data underscore the importance of pure synthetic DSPC in improving the biological effects of liposomes and suggest sDSPC-L as a promising vehicle for encapsulating hemoglobin.

Animals↗
PubMed1990

Biological responses to liposome-encapsulated hemoglobin (LEH) are improved by a PAF antagonist.

Liposome-encapsulated hemoglobin (LEH) is an experimental oxygen-carrying blood substitute. Previous studies in our laboratory with hydrogenated soy lecithin-based LEH indicated that while this solution maintained some physicochemical and oxygen-carrying properties of red blood cells, it evoked several transient (30-120 min) untoward biological responses, such as hypertension, tachycardia, thrombocytopenia, hemoconcentration, and elevation of plasma thromboxane B2 (TXB2). Such biochemical, hematological, and hemodynamic derangements are also produced by platelet-activating factor (PAF). The purpose of this study was to test the biological responses to administration of a newly produced synthetic distearoyl phosphatidylcholine-based LEH (s-DSPC-LEH) in the normal conscious rat (n = 6-11) and to examine the role of PAF in these processes. Systemic (IV) administration of S-DSPC-LEH caused transient hypotension (-23 +/- 8 mmHg, P less than 0.05), bradycardia (-24 +/- 11 bpm, P less than 0.05) followed by tachycardia (+62 +/- 21 bpm, P less than 0.05), decreased cardiac index (217 +/- 21 ml/min/kg, P less than 0.01), increased peripheral resistance (0.570 +/- 0.003 mmHg/ml/min/kg, P less than 0.01), transient leukocytosis (+6,870 +/- 1,801/microliters, P less than 0.05), hemoconcentration (+5.2 +/- 0.4%, P less than 0.01), thrombocytopenia (-160 +/- 18 X 10(3)/microliters, P less than 0.01), and increase in plasma TXB2 (45.0 +/- 1.9 pg/100 microliters, P less than 0.01). Separate infusion of the liposome vehicle or free hemoglobin, the two components of s-DSPC-LEH, did not evoke any consistent responses. Administration of the PAF antagonist BN 50739 (10 mg/kg, i.p.) 30 min prior to LEH prevented the hemodynamic changes and hemoconcentration induced by s-DSPC-LEH. These data suggest that hemoglobin/phospholipid interactions might account for the transient side effects of s-DSPC-LEH, possibly through interactions with blood elements and the resultant production of PAF and TXA2. Furthermore, PAF antagonists incorporated into or co-administered with LEH might enhance its biological applications.

Animals↗
PubMed1989

Characterization of hemodynamic, hematologic, and biochemical responses to administration of liposome-encapsulated hemoglobin in the conscious, freely moving rat.

To improve the outcome of trauma victims and of patients undergoing high-blood-loss surgical procedures and to avoid the many serious complications of blood transfusion, there is a need for an oxygen-carrying blood substitute. Synthetic erythrocytes composed of liposome-encapsulated hemoglobin (LEH) represent one of the significant research efforts in this direction. The purpose of the present study was to examine some of the cardiovascular, hematologic, and biochemical effects of a recently developed LEH preparation in the conscious rat (n = 7). LEH increased mean arterial pressure (MAP) by +18.7 +/- 4.7 mm Hg (P less than 0.01) and heart rate (HR) by +117 +/- 18 beats/min (P less than 0.05). Platelet count dropped to 40% of basal value (P less than 0.01), while plasma thromboxane B2 (TXB2) increased by +25.1 +/- 5.4 pg/100 microliters (P less than 0.001). There was no effect on plasma 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha). Hemoglobin and hematocrit levels were elevated as well as the white blood cell count [( WBC] lymphocytosis). The platelet and TXB2 responses to LEH showed negative correlation (R = -0.56, P less than 0.01). The injection of the liposome vehicle (LIP) decreased MAP by -16.5 +/- 5.1 mm Hg (P less than 0.01) and platelets, but increased HR, WBC, and TXB2. All observed effects exerted by LEH and LIP were transient, and basal levels obtained 120 min after LEH injection. These data suggest that while LEH maintains some physicochemical properties of red blood cells, its biological properties at the present time indicate potential cardiovascular and hematological liabilities. Furthermore, it seems that the phospholipid bilayer alone or in combination with free Hb might be responsible for the biological effects of LEH.

6-Ketoprostaglandin F1 alpha↗
PubMed1989

Liposome encapsulated hemoglobin: stabilization, encapsulation, and storage.

Liposome encapsulated hemoglobin has numerous advantages as a red cell substitute. LEH has no blood type and can be made virus-free and sterile in large quantities. The technology is compatible with the use of either human or bovine hemoglobin and does not require chemical modification of the hemoglobin. Cofactors can be included in the liposome to modify the P50 and to prevent methemoglobin formation. The inclusion of dissacharides stabilizes the LEH during freezing and dehydration and provides a method for long-term storage. Most importantly, LEH can sustain life in animals after removal of red cells to lethal levels for periods commensurate with a 16-20 hour circulation half-life.

Animals↗