Effects of lipid-soluble substances on the thermotropic properties of liposome filtration.

Filtration of dimyristoyl phosphatidylcholine or dipalmitoyl phosphatidylcholine liposomes at various temperatures from 3 to 60 degrees C revealed a discontinuous change in filtration behavior centered about the gel-to-liquid crystal transition temperature. This change was continuous at temperatures immediately above or below the transition temperature. Although pure dipalmitoyl phosphatidylcholine liposomes are in the gel state at 22 degrees C, passage of liposomes composed of dipalmitoyl phosphatidylcholine and cholesterol through the filters at 22 degrees C gave results similar to those obtained with liquid-crystal liposomes. Low cholesterol concentrations were nearly as effective as high concentrations in producing this behavior; this observation is consistent with a shear mechanism for reduction of liposome size, since the stress induced by passage of the otherwise rigid liposome through a small pore would be relieved by fracture at a lattice imperfection. Liposomes composed of egg phosphatidylcholine and cholesterol were retained by the filters to a slightly greater extent than pure egg phosphatidylcholine liposomes; these results are consistent with the known condensing effect of cholesterol on liquid-crystal lipsomes and a shear mechanism occurring with filtration. Liposomes composed of dipalmitoyl phosphatidylcholine and either dipalmitoyl phosphatidic acid or dicetyl phosphate were filtered at 22 degrees C; they showed a filtration characteristic similar to liquid-crystal liposomes. Inclusion of the water-soluble dyes eosin Y or Evans blue in dipalmitoyl phosphatidylcholine liposomes resulted in filtration at 22 degrees C which was similar to that observed for liquid-crystal liposomes. The dyes, sodium fluorescein, 6-carboxyfluorescein and fluoresceinisothiocyanate dextran, did not alter 22 degrees C liposome filtration.

Liposome filtration. Dependence on transition temperature.

Liposomes formed by vortexing and passed through polycarbonate surface retention membranes showed appreciable differences in filtration behavior depending on the temperature of filtration relation relative to the liposome gel-liquid crystal transition temperature. Below transition, liposomes were filterable and size distributions could be determined; the cumulative volume distributions were log-normal. Above transition, liposomes were not filterable: smaller liposomes were formed until a limiting size was reached. These results suggest that liquid crystal liposome size distributions cannot be determined by filtration. This filtration behavior is a physical property of liposomes, related to the gel-liquid crystal transition, not previously reported. This property could be exploited as a new method for controlling liposome size distributions, but the implications for lipid membranes, including biological membranes, are general.

Synthetic erythrocytes from lipid encapsulated hemoglobin.

Synthetic erythrocytes have been formed by encapsulating concentrated hemoglobin solutions in microcapsules consisting of phospholipid-cholesterol mixtures. The microcapsules (hemosomes) are of the order of one micron in diameter or smaller, are osmometers, are somewhat sturdier than erythrocytes (RBC), and are stable upon freezing. They have essentially the same oxygen and carbon dioxide carrying capacity as RBC, and can be tailored to have the same electrokinetic properties. They appear to evoke no immune response in the rat. In experiments involving complete replacement of natural blood hemosomes appear to sustain life. Rats can live and breathe normally without signs of hypoxia or acidosis for extended periods of time after the hematocrit of natural RBC drops well below the critical level. Synthetic erythrocytes do not appear to cause disseminated coagulation, microembolism, or any observable changes in internal organs.

Regulation of ciliary beat frequency by autonomic mechanisms: in vitro.

The ciliated epithelium of the mammalian trachea separates the neurohumoral milieu of the tissue from that of the environment of the airway lumen. To determine whether specific autonomic receptors regulating ciliary beat frequency (CBF) were located on mucosal or serosal sides, we measured CBF by heterodyne mode correlation analysis laser light scattering in bovine tracheal tissues mounted in a two-sided chamber. A beta 2-adrenergic agonist, fenoterol, at 10(-7) M, stimulated serosal CBF from 7.9 +/- 1.3 to 20.2 +/- 5.8 Hz (P less than 0.01) and mucosal CBF from 6.6 +/- 0.9 to 14.7 +/- 4.6 Hz (P less than 0.01). A muscarinic cholinergic agonist, methacholine, at 10(-7) M, increased mucosal CBF from 8.4 +/- 1.0 to 19.5 +/- 5.5 Hz (P less than 0.01) and serosal CBF from 8.0 +/- 0.9 to 15.4 +/- 5.0 Hz (P less than 0.01). The differences in stimulation of CBF on the mucosal and serosal sides between fenoterol and methacholine were significant (P less than 0.01). Studies in which these autonomic agonist stimulating effects were inhibited by their respective antagonists, propranolol and atropine sulfate, demonstrated that CBF can be regulated independently by mediators both in the submucosa and within the mucus lining.

Nature of the mammalian ciliary metachronal wave.

The temporal and spatial coordination of ciliary beat (metachronicity) is fundamental to effective mucociliary transport. Metachronal wave period (MWP) and ciliary beat frequency (CBF) of fresh excised sheep and canine tracheal epithelial tissues were measured with the use of a newly developed alternating focal spot laser light scattering system. MWP was determined from cross correlation of the heterodyne signals from the alternating focal spots. CBF was determined by autocorrelation of the heterodyne signals from each of the spots. MWP and CBF were measured in four sheep tracheal epithelial tissues with the use of longitudinal interfocal spot distances of 6 and 18 microns. In three canine tracheal epithelial tissues MWP and CBF were measured both longitudinally and circumferentially with interfocal spot distances of 5, 15, 65, 87, and 96 microns. For the sheep tracheal epithelial tissues the mean CBF was 5.9 +/- 0.4 Hz (mean of means; range 3.6 +/- 0.5 to 9.9 +/- 1.5 Hz), whereas the mean MWPs for 6- and 18-microns interfocal spot distances were 0.50 +/- 0.1 and 0.47 +/- 0.1 s, respectively. For the canine tracheal epithelial tissues the mean CBF was 4.0 +/- 0.2 Hz (2.0 +/- 0.8 to 7.2 +/- 3.2 Hz), whereas the mean longitudinal MWP was 1.5 s and the mean circumferential MWP was 2.1 s. Geometric combination of the MWP components leads to a derived MWP of 2.6 s with a propagation direction of 54 degrees with respect to the longitudinal axis of the trachea. MWP was found to be episode modulated with 12- to 20-min intervals in the longitudinal direction, but modulation was not as apparent in the circumferential direction. These data suggest that MWP and CBF are regulated by separate intracellular, intercellular, and intraciliary mechanisms.

Stimulation of ciliary beat frequency by autonomic agonists: in vivo.

beta 2-Adrenergic bronchodilator and muscarinic cholinergic bronchoconstrictor agonists both stimulate ciliary activity in vitro. To test the hypothesis that increases in autonomic activity would result in increases in ciliary beat frequency (CBF) in vivo, a correlation analysis heterodyne laser light-scattering system was developed and validated to measure the stimulating effects of sympathomimetic and parasympathomimetic agonists on tracheal CBF in intact, anesthetized beagles. The mean baseline CBF from 42 studies of 274 measurements in 9 (5 male and 4 female) adult beagles was 6.6 +/- 1.1 Hz. The stimulating effects of a beta 2-adrenergic agonist, fenoterol, and a muscarinic cholinergic agonist, methacholine, on CBF were studied on four and eight beagles, respectively. The studies were randomized and blinded. Aerosolized 10(-5) M fenoterol stimulated the CBF from the base line of 6.8 +/- 2.5 to 32.0 +/- 17.9 Hz in four dogs. Aerosolized methacholine stimulated the CBF from the base line of 5.8 +/- 0.7 to 9.4 +/- 3.0 Hz for 10(-8) M, and to 12.6 +/- 3.1 Hz for 10(-6) M in eight dogs. These are the first data obtained in intact animals that demonstrate CBF in the lower respiratory tract is regulated by autonomic agonists.

Pathways of substance P stimulation of canine tracheal ciliary beat frequency.

Substance P (SP), an inflammatory neuropeptide, may be released by intraepithelial nerves in response to an irritant or inflammatory stimulus. To investigate the neural and humoral pathways mediating the response of tracheal ciliary beat frequency (CBF) to topically applied SP, CBF was measured on the ventral midtracheal surface of anesthetized beagles by using heterodyne-mode correlation analysis laser light scattering. In the first study, aerosolized SP, delivered to the lungs of eight beagle dogs, stimulated CBF in a dose-dependent manner from a baseline of 4.9 +/- 0.4 Hz to a maximum of 14.9 +/- 1.5 Hz at dose of 10(-7) M. In the second study, the tracheal lumen was isolated from the bronchial airways by inflating the cuff of an endotracheal tube near the carina. Intravenous hexamethonium bromide (2 mg/kg), ipratropium bromide (0.5 micrograms/kg), and indomethacin (2 mg/kg) were used as blocking agents to inhibit the nicotinic, muscarinic, and cyclooxygenase pathways, respectively. Aerosolized 10(-9), 10(-8), or 10(-7) M SP was delivered sequentially to the tracheal lumen for 3 min at 30-min intervals. SP caused two distinct CBF stimulatory episodes at 4 min (mean time of the maximal response) and at 18 min (mean time of the maximal response) after onset of delivery and returned to baseline after 25 min. SP stimulated CBF from the baseline of 5.1 +/- 0.4 Hz to a maximum of 14.2 +/- 2.5 Hz during the first episode (P less than 0.01) and to 10.4 +/- 0.6 Hz during the second episode (P less than 0.01) at dose of 10(-8) M. These responses were inhibited by all the blocking agents. These data suggest that SP stimulates CBF via a cyclooxygenase-dependent parasympathetic reflex.

Stimulation of tracheal ciliary beat frequency by capsaicin.

To determine the possible involvement of neural and cyclooxygenase pathways whereby irritants might affect cilia activity in vivo, the temporal response of canine tracheal ciliary beat frequency (CBF) to the inhaled surrogate irritant capsaicin was studied. CBF was measured on the ventral midtracheal surface of barbiturate-anesthetized eucapnically ventilated beagle dogs by heterodyne-mode laser light scattering. After base-line CBF was established, hexamethonium bromide (2 mg/kg iv), ipratropium bromide (0.5 microgram/kg iv), indomethacin (2 mg/kg iv), or intravenous 0.9% saline was administered. Aerosolized 3 Z 10(-9) M capsaicin in 0.9% saline was delivered for 2 min, and CBF was measured for the following 60 min. Control experiments used 0.9% saline sham aerosol with a 0.9% saline sham block. Aerosolized capsaicin stimulated CBF from a base line of 6.2 +/- 1.4 (SD) Hz (n = 230) to a mean maximum of 17.7 +/- 7.3 Hz (n = 16) 23 min after aerosol delivery, and CBF returned to base line within 60 min. Neither hexamethonium bromide, ipratropium bromide, nor indomethacin changed CBF from base-line values. The episodic CBF stimulatory response to capsaicin after commencement of aerosol was completely inhibited by hexamethonium bromide. Ipratropium bromide partially inhibited the first 15 min and totally inhibited the following 45 min of stimulatory response. Indomethacin inhibited the initial 15 min but had less effect on the following 45 min of stimulatory response. These data indicate that multiple stimulatory mechanisms function over a prolonged period of time to affect the removal of irritants from the airways and that these mechanisms differ from those involved in the maintenance of basal CBF.

Automated measurement of ciliary beat frequency.

Measurements of ciliary beat frequency using video images are dependent on observer interpretation. To obtain objective estimates of ciliary beat frequency from video-image sequences, a computer-based method was developed. Regions of interest of video-image sequences were selected and digitized. Variations in numerical values representing light intensity resulting from cilia beating were extracted and analyzed using autocorrelation techniques. The ciliary beat frequencies obtained for 14 in vitro experiments on ciliated cells or epithelium from the frog palate (Rana catesbeiana) over the range of frequencies 2-25 Hz correlated well with independent observer measurements (r = 0.979). The addition of such computer-based methods to video observer-based systems allows more objective and efficient determinations of ciliary beat frequency.

Work of adhesion of respiratory tract mucus.

A method was devised to measure the work of adhesion (WA) to a substrate of mucus, a viscoelastic gel, from the measured contact angle of glycerol on a mucus substrate and the known physical properties of a Teflon surface. Fifteen sputum samples from cystic fibrosis (CF) patients were compared with 25 mucus samples from canine tracheal pouches (CP), studied in the hydrated and partially dehydrated states. Apparent viscosity (eta A) and recoverable shear strain (SR) were measured by fluxgate magnetometry, and water content was inferred from vapor pressure osmometry. Na+, K+, and Ca2+ concentrations were measured with specific ion electrodes and Cl- with a chloridimeter. The Cl- concentration of the CP mucus was inversely proportional to its osmolality, and the Cl- concentration of the CP mucus was 102.5 +/- 1.6 meq/l compared with 55.6 +/- 2.5 meq/l for CF sputum. When CP mucus osmolality was increased from 316.0 +/- 5.5 to 430.0 +/- 7.5 mosmol/kg, WA increased from 25.1 +/- 1.8 to 31.1 +/- 1.2 ergs/cm2 and eta A increased from 391 +/- 55 to 622 +/- 121 P, respectively. CF sputum WA was 30.2 +/- 0.6 ergs/cm2, eta A was 1,110 +/- 316 P, and osmolality was 466.0 +/- 14.0 mosmol/kg. The increased WA and eta A of mucus in CF patients may thus be dependent on the hydration of mucus, which is related to the documented Cl- transport defect.

Controlled dissolution from wax-coated aerosol particles in canine lungs.

Treatment of pulmonary and systemic diseases may be improved and toxicity reduced by pulmonary deposition of drug-containing aerosols exhibiting delayed dissolution. Aqueous disodium fluorescein and pentamidine aerosols were dried, concentrated, and condensation coated with paraffin wax. The apparent mass median aerodynamic diameters of the coated fluorescein particles were 2.8-4.0 microns. Wax-to-fluorescein ratios were 0.38-1.05. The dissolution half times determined using a single-pass flow system were 1.5 min for uncoated fluorescein and 0.8 min for uncoated pentamidine. These increased over threefold when the aerosols were coated with paraffin wax to maxima of 5.3 and 2.6 min, respectively. Wax-coated aerosols generated from fluorescein mixed with 99mTc-labeled iron oxide colloid delivered to the canine lungs demonstrated a 3.4-fold increase in the absorption half time of disodium fluorescein compared with uncoated fluorescein (11.2 vs. 38.4 min). The absence of changes in pulmonary function on inhalation of these wax-coated aerosols, together with a high drug load and delayed release, establishes a foundation for future therapeutic applications.

First pregnancies and livebirths from transfer of sodium alginate encapsulated embryos in a rodent model.

OBJECTIVE: To determine the effect of sodium alginate encapsulation of rodent embryos on in vitro embryonic cleavage rates, implantation rates, and livebirth rates, and to find the in vivo degradation time for the capsules. DESIGN: Studies were conducted using both CB6F1 mice and Golden Syrian hamsters. RESULTS: Capsules made with 3.0% sodium alginate degraded in vivo within 24 to 48 hours after transfer. In vitro embryonic cleavage of encapsulated embryos was not impaired, nor were implantation rates in CB6F1 mice. Finally, 8.6% of transferred encapsulated embryos resulted in livebirths. CONCLUSIONS: Encapsulation of rodent embryos in 3.0% sodium alginate is not detrimental to embryonic development, implantation rates, or fetal development. Because the capsule degrades within 48 hours after transfer, encapsulating embryos may be beneficial for human in vitro fertilization and embryo transfer.

Development of a polymeric releasing device for 2'-carbomethoxyphenyl 4-guanidinobenzoate (a proteinase inhibitor): release rate, in vitro antifibrinolytic activity and in utero contraceptive effect.

A polymeric delivery system consisting of ethylene-vinyl acetate copolymer (EVAc) was developed for 2'-carbomethoxyphenyl 4-guanidinobenzoate (MSGB), a potent inhibitor of the sperm enzyme acrosin. The optimal device consists of copolymer with 40% vinyl acetate by weight (EVAc/40), 65% drug loading and MSGB with a particle size of 250-499 micron. This formulation yields a device that is highly flexible and can be shaped to many forms and sizes. Construction of the device does not alter the properties of MSGB. Well controlled release of MSGB from the device occurs in vitro and in the uteri of rats. The in vitro release rate under "infinite sink" conditions is essentially the same as the in vivo release rate. The contraceptive effect of the MSGB-releasing device was tested in rabbits by placing a blank (control) device in one uterine horn and an MSGB-releasing device in the contralateral horn. In contrast to blank devices, MSGB-releasing devices completely prevent pregnancy, not only by inhibiting fertilization but also by decreasing implantation. MSGB possesses high in vitro antifibrinolytic activity. These results indicate that a very flexible device can be constructed for uterine application which retains its contraceptive effect by release of MSGB. The antifibrinolytic activity of MSGB may further decrease the menorrhagia that can be associated with IUD use.

Development of the polyurethane sponge as a delivery system for aryl 4-guanidinobenzoates.

The Today Contraceptive Sponge was evaluated as a vehicle for the delivery of aryl 4-guanidinobenzoates (AGs) which are highly active sperm acrosin inhibitors. Studies in animals have shown that several AGs are more potent vaginal contraceptives and less irritating to the vagina than nonoxynol-9 (N-9), the most frequently used active ingredient in commercial vaginal contraceptive formulations. Neither nonoxynol-9 nor the material that could be solubilized from the sponge matrix altered the enzyme-inhibitory activity of 4'-acetamidophenyl 4-guanidinobenzoate HCl (AGB), 4'-carboxyphenyl 4-guanidinobenzoate HCl (EGB) or 4'-carbomethoxyphenyl 4-guanidinobenzoate HCl (MSGB). Besides being acrosin inhibitors, all three AGs exhibited antimotility activity towards human spermatozoa, EGB being as potent as N-9. The antimotility effects of the AGs and N-9 were additive. For subsequent studies, AGB was used as the model compound. Manufacture of the AGB-containing sponges did not affect the chemical structure of AGB. Good release rates of AGB were obtained from the sponges over a 7-day period. The release rates were 20-50% higher when the sponges also contained N-9. These results indicate that certain AGs exert a dual contraceptive action on spermatozoa by inhibiting both the sperm enzyme acrosin and sperm motility. Furthermore, the polyurethane sponge appears to be a convenient and satisfactory long-term delivery system for the AGs. A mixture of N-9 and AG can be used clinically because these compounds have no adverse effects on each other.

Measurements of viscosity of synthetic erythrocyte suspensions.

Measurements were made of the viscosity of suspensions of synthetic erythrocytes composed of hemoglobin solutions encapsulated in liposomes, as a function of shear rate, temperature, suspension concentration, lipid membrane composition, and the viscosity of the suspending medium. It was found that the viscous behavior of the synthetic erythrocyte suspensions was non-Newtonian and nearly the same as that of suspensions of natural erythrocytes prepared similarly, with the major difference being that synthetic erythrocyte suspensions are somewhat more viscous. Suspensions of Fluosol FC-43 prepared similarly were found to be essentially Newtonian fluids, and substantially different and more viscous than either erythrocyte suspension. The higher viscosity of synthetic erythrocyte suspensions probably accounts for the ability of these suspensions to maintain normal systemic vascular resistance in transfusion experiments, in spite of the fact that synthetic erythrocytes are smaller than natural erythrocytes.

Ultrasonic evaluation of erythrocyte aggregation dynamics.

The dynamics of aggregation and disaggregation of blood of varying hematocrit in oscillatory flow in a distensible horizontal tube was determined by measuring the developing echo intensity of the blood samples with a 10 MHz B-mode ultrasonic scanner. Early aggregation could be detected within 10 sec. of stoppage of flow. The rate of echo intensity buildup and thus, presumably the rate of aggregation when flow was stopped was inversely related to hematocrit, as was the rate of echo intensity reduction when flow was resumed. Polycythemic blood of 60% hematocrit showed no echo intensity increase over 5 min. Increasing the shear stress when flow was resumed resulted in rapid decreases in aggregation. In all cases, disaggregation following flow resumption was faster than aggregation following flow stoppage.

Kinetic studies of a doubly bound red cell antigen-antibody system.

The Polybrene method for detection of red cell antibodies which utilizes continuous flow equipment was modified so that kinetic studies could be performed on red cell antibodies doubly bound between adjacent red cells. In the anti-Rh(o)-Rh(o) erythrocyte system, deaggregation by temperature was studied over an antibody concentration range of from approximately 1 to 500 antibody molecules per erythrocyte, a residence time range of approximately eightfold, and a temperature range of from 10 to 55 degrees C. The rate of dissociation of antigen-antibody complex, as determined from deaggregation of antibody-dependent red cell aggregates, was found to be of apparent zero order. The apparent activation energy for the antigen-antibody reaction under the experimental conditions was determined and found to be higher than would be expected for singly bound antigen-antibody systems. Possible explanations are considered for these findings in terms of an antigen-antibody bond-breaking model.